1 running title: nitrate allocation and nitrogen use ... · pdf file1 running title: nitrate...
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Running Title Nitrate allocation and nitrogen use efficiency 1
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Corresponding author Zhen-hua Zhang 3
Southern Regional Collaborative Innovation Center for Grain and Oil Crops in China 4
National Engineering Laboratory for Efficient Utilization of Soil and Fertilizer Resources 5
Hunan Provincial Key Laboratory of Farmland Pollution Control and Agricultural Resources 6
Use Hunan Provincial Key Laboratory of Plant Nutrition in Common University 7
College of Resources and Environmental Sciences Hunan Agricultural University PR China 8
zhzh1468163com 9
10
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Title Nitrogen use efficiency is mediated by vacuolar nitrate sequestration capacity in roots 12
of Brassica napus 13
14
Yong-Liang Han1 Hai-Xing Song1 Qiong Liao1 Yin Yu1 Shao-Fen Jian1 Joe Eugene Lepo3 Qiang 15
Liu1 Xiang-Min Rong1 Chang Tian1 Jing Zeng1 Chun-Yun Guan12 Abdelbagi M Ismail4 Zhen-Hua 16
Zhang1sect 17
18
1 Southern Regional Collaborative Innovation Center for Grain and Oil Crops in China Hunan Agricultural 19
University Changsha China 20
2 National Center of Oilseed Crops Improvement Hunan Branch Changsha China 21
3 Center for Environmental Diagnostics and Bioremediation University of West Florida Pensacola 22
Florida 32514 United States of America 23
4 Crop and Environment Sciences Division International Rice Research Institute DAPO 7777 Metro 24
Manila Philippines 25
26
27
Decrease in VSC of NO3- in roots will enhance transport to shoot and essentially contribute to 28
higher NUE by promoting NO3- allocation to aerial parts 29
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Plant Physiology Preview Published on January 12 2016 as DOI101104pp1501377
Copyright 2016 by the American Society of Plant Biologists
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Footnotes 33
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 34
Shanghai Institutes for Biological Sciences) for providing nrt15-3and nrt18-2 seeds This 35
study was supported by the National Natural Science Foundation of China (Grant 36
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 37
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 38
The Ministry of Education in China Open Novel Science Foundation of Hunan province 39
(13K062) National Key Laboratory of Plant Molecular Genetics and the Twelfth Five-Year 40
National Science and technology support program (2012BAD15BO4) 41
42
These authors have contributed equally 43
sect Corresponding author e-mail zhzh1468163com 44
45
46
Author contributions 47
ZHZ conceived the original screening and research plans ZHZ supervised the experiments 48
YLH HXS QL YY SFJ and ZHZ performed most of the experiments CT and JZ provided 49
technical assistance to YLH HXS YY and ZHZ YLH and ZHZ designed the experiments 50
and analyzed the data QL XMR CYG and ZHZ interpreted the results and JEL AI and 51
ZHZ conceived the project and wrote the article with contributions of all the authors JEL AI 52
and ZHZ supervised and complemented the writing All authors read and approved the final 53
manuscript 54
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67
Abstract 68
Enhancing nitrogen use efficiency (NUE) in crop plants is an important breeding target 69
to reduce excessive use of chemical fertilizers with substantial benefits to farmers and the 70
environment In Arabidopsis thaliana allocation of more NO3- to shoots was associated with 71
higher NUE however the commonality of this process across plant species have not been 72
sufficiently studied Two Brassica napus genotypes were identified with high and low NUE 73
We found that activities of V-ATPase and V-PPase the two tonoplast proton-pumps were 74
significantly lower in roots of the high-NUE genotype (Xiangyou15) than in the low-NUE 75
genotype (814) and consequently less vacuolar NO3- was retained in roots of Xiangyou15 76
Moreover NO3- concentration in xylem sap [15N] shootroot (SR) and [NO3
-] SR ratios 77
were significantly higher in Xiangyou15 BnNRT15 expression was higher in roots of 78
Xiangyou15 compared with 814 while BnNRT18 expression was lower In both B napus 79
treated with proton pump inhibitors or Arabidopsis mutants impaired in proton pump activity 80
vacuolar sequestration capacity (VSC) of NO3- in roots substantially decreased Expression of 81
NRT15 was up-regulated but NRT18 was down-regulated driving greater NO3- 82
long-distance transport from roots to shoots NUE in Arabidopsis mutants impaired in proton 83
pumps was also significantly higher than in the wild type col-0 Taken together these data 84
suggest that decrease in VSC of NO3- in roots will enhance transport to shoot and essentially 85
contribute to higher NUE by promoting NO3- allocation to aerial parts likely through 86
coordinated regulation of NRT15 and NRT18 87
88
Key words Nitrogen use efficiency Nitrate allocation Proton-pumps Vacuoles 89
90
91
92
93
94
95
96
97
98
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4
Introduction 99
China is the largest consumer of nitrogen (N) fertilizer in the world however the 100
average N-fertilizer use efficiency (NUE) is only around 35 suggesting considerable 101
potential for improvements (Shen et al 2003 Wang et al 2014) With the high amounts of 102
N-fertilizer being used crop yields are declining in some areas where application is 103
exceeding the optimum required for local field crops (Shen et al 2003 Miller et al 2008Xu 104
et al 2012) The extremely low NUE results in waste of resources and environmental 105
contamination and also presents serious hazards for human health (Xu et al 2012 Chen et 106
al 2014) Consequently exploiting the maximum potential for improving NUE in crop plants 107
will have practical significance for agriculture production and the environment (Zhang et al 108
2010 Schroeder et al 2013 Wang et al 2014) Elucidating the genetic and physiological 109
regulatory mechanisms governing NUE in plants will allow breeding crops and varieties with 110
higher NUE 111
Ammonium (NH4+) and nitrate (NO3
-) are the main N species absorbed and utilized by 112
crops and NO3- accumulation and utilization are of major emphasis for N nutrient studies in 113
dry land crops such as Brassica napus Several studies revealed the close relationship 114
between NO3- content and NUE in plant tissues (Shen et al 2003 Zhang et al 2012 Tang et 115
al 2013Han et al 2015a)When plants are sufficiently illuminated NO3- assimilation 116
efficiency significantly increase in shoots compared with roots (Smirnoff et al 1985 Tang et 117
al 2013) Consequently under daytime with optimal illumination higher proportion of NO3- 118
in plant tissue is transported from root to shoot as an advantageous physiological adaptation 119
that reduces the cost of energy for metabolism (Tang et al 2013) NO3- assimilation in plant 120
shoots can therefore take advantage of solar energy while improving NUE (Smirnoff et al 121
1985 Andrews et al 1986 Tang et al 2012 Tang et al 2013) 122
The NO3- long-distance transport and distribution between root and shoot is regulated by 123
two genes encoding long transport mechanisms NRT15 is responsible for xylem NO3- 124
loading while NRT18 is responsible for xylem NO3- unloading (Lin et al 2008 Li et al 125
2010) Expression of the two genes is influenced by NO3- concentration NRT15 is strongly 126
induced by NO3- (Lin et al 2008) while NRT18 expression is extremely up-regulated in 127
nrt15 mutants (Chen et al 2012) A negative correlation between the extents of expression of 128
the two genes was observed when plants are subjected to abiotic stresses (Chen et al 2012) 129
Moreover expression of NRT15 is strongly inhibited by 1-aminocyclopropane-1-carboxylic 130
acid (ACC) and methyl jasmonate (MeJA) whereas the expression of NRT18 is significantly 131
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5
up-regulated (Zhang et al 2014) Based on these studies we argue that the expression and 132
functioning of NO3- long-distance transport genes NRT15 and NRT18 are regulated by 133
cytosolic NO3- concentration In addition the vacuolar and cytosolic NO3
- distribution is 134
likely regulated by proton-pumps located within the tonoplast (V-ATPase and V-PPase) 135
(Granstedt et al 1982 Glass et al 2002 Krebs et al 2010) Therefore NO3- use efficiency 136
must be affected by NO3- long-distant transport (between shoot and root) and short-distant 137
transport (between vacuole and cytosol) However the physiological mechanisms controlling 138
this regulation are still obscure 139
Previous studies showed that the chloride channel protein (CLCa) is mainly responsible 140
for vacuole NO3- short-distance transport as it is the main channel for NO3
-movement 141
between the vacuoles and cytosol (Angeli et al 2006 Wege et al 2014) The vacuole 142
proton-pumps (V-ATPase and V-PPase) located in the tonoplast supply energy for active 143
transport of NO3- and accumulation within the vacuole (Gaxiola et al 2001 Brux et al 2008 144
Krebs et al 2010) Despite that about 90 of the volume of mature plant cells is occupied by 145
vacuoles vacuolar NO3- cannot be efficiently assimilated because the enzyme nitrate 146
reductase (NR) is cytosolic (Shen et al 2003 Han et al 2015a) However re-translocation of 147
NO3- from the vacuole to the cytosol will permit its immediate assimilation and utilization 148
Generally NO3- concentrations in plant cell vacuoles and the cytoplasm are in the range 149
of 30-50 mol m-3 and 3-5 mol m-3 respectively (Martinoia et al 1981 Martinoia et al 2000) 150
Because vacuoles are obviously the organelle for high NO3- accumulation and storage in plant 151
tissues their function in NO3- use efficiency cannot be ignored (Martinoia et al 1981 Zhang 152
et al 2012 Han et al 2015b) NO3- assimilatory system in the cytoplasm is sufficient for its 153
assimilation when it is transported out of the vacuoles Therefore NO3- use efficiency could in 154
part be dependent on vacuolar-cytosolic NO3- short-distance transport in plant tissues 155
(Martinoia et al 1981 Shen et al 2003 Zhang et al 2012 Han et al 2015a) 156
Evidently NO3- use efficiency is regulated by both NO3
- long-distance transport from 157
root to shoot and short-distance transport and distribution between vacuoles and cytoplasm 158
within cells(Glass et al 2002 Dechorgnat et al 2011 Han et al 2015a) Although vacuoles 159
compartment excess NO3- that accumulates in plant cells (Granstedt et al 1982 Krebs et al 160
2010) neither NO3- inducible NR genes (NIA1and NIA2) (Fan et al 2007 Han et al 2015a) 161
nor the NO3- long-distance transport gene NRT15 (Lin et al 2008) are regulated by vacuolar 162
NO3- even though they are essential for NO3
- assimilation Only NO3- transported from the 163
vacuole to the cytosol can play a role in regulating NO3- inducible genes Consequently we 164
argue that both NO3- assimilation in cells and its long-distance transport from root to shoot are 165
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6
regulated by cytosolic NO3- concentration However this hypothesis needs to be substantiated 166
The mechanisms underlying both NO3- short-distance (Gaxiola et al 2001 Angeli et al 167
2006 Brux et al 2008 Krebs et al 2010) and long-distance transport (Lin et al 2008 Li et 168
al 2010) have been previously investigated yet the underlying mechanisms regulating the 169
flux of NO3- and the obvious relationship between the two transport pathways as well as their 170
relation to NUE are not well understood 171
The NRT family of genes play a partial role in vacuolar NO3- accumulation in petioles 172
(Chiu et al 2004) and seed tissues (Chopin et al 2007) whereas the proton pumps and 173
CLCa system in the tonoplast play a major role in accumulating NO3- in vacuoles (Gaxiola et 174
al 2001 Angeli et al 2006 Brux et al 2008 Krebs et al 2010) The vacuolar NO3- 175
short-distance transport system is spread throughout the plant tissues and is the principal 176
means by which vacuolar NO3- short-distance transport and distribution is controlled (Angeli 177
et al 2006Krebs et al 2010) 178
The NRT genes seem to work synergistically to control NO3- long-distance transport 179
between roots and shoots NRT19 is responsible for NO3- loading into the phloem (Wang et 180
al 2011) whereas NO3- loading and unloading into xylem are regulated by NRT15 and 181
NRT18 respectively (Lin et al 2008 Li et al 2010) Phloem transport mainly involves 182
organic N the inorganic-N (NO3-) concentrations in the phloem sap are typically very low 183
ranging from one-tenth to one-hundredth of that of the inorganic-N in xylem sap (Lin et al 184
2008Fan et al 2009) Therefore this study focused on NO3- short-distance transport 185
mediated through the tonoplast proton-pumps and the CLCa system and the long-distant 186
transport mechanisms responsible for xylem NO3- loading and unloading via NRT15 and 187
NRT18 respectively 188
Questions related to how long and short-distance transport of NO3- are coupled in plant 189
tissues and their role in determining NUE were addressed using a pair of high- and low-NUE 190
B napus genotypes and Arabidopsis thaliana Application of proton pump inhibitors and 191
ACC in the former and use of mutants with defective proton pumps in the latter allowed 192
experimental distinction of the physiological mechanisms regulating these processes Data 193
presented here provide strong evidence from both model plants supporting this linkage and 194
strongly suggest that cytosolic NO3- concentration in roots regulates NO3
- long-distance 195
transport from roots to shoots We also investigated how NO3- concentration in plant tissues 196
would be affected by NO3- long-distance transport vacuolar NO3
- sequestration and the 197
ensuing relationship with NO3- use efficiency We also proposed the physiological 198
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7
mechanisms likely to be important for enhancing NO3- use efficiency in plants These findings 199
will provide scientific rationales for improving NUE in important industrial and food crops 200
201
202
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8
Results 203
B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for NO3- 204
in root tissues 205
Our previous work identified high and low NUE B napus genotypes (Han et al 2015a 206
Han et al 2015b) NUE of B napus whether based on biomass or on grain yield (Table 1) 207
was significantly higher for the high-NUE genotype (H Xiangyou15) than for the low-NUE 208
genotype (L 814) That the activities of the tonoplast proton-pumps (V-ATPase and V-PPase) 209
of root tissues in the H genotype were lower than the L genotype at both seedling (Fig 1A) 210
and flowering stages (Fig S1A) Given that proton pumps in the tonoplast supply energy for 211
vacuolar NO3- accumulation (Krebs et al 2010) NO3
- influx into the vacuole was 212
consequently much slower in the H genotype compared with the L genotype at both seedling 213
(Fig 1B) and flowering stages (Fig S1B) Moreover the percentage of vacuolar NO3- relative 214
to the total NO3- in protoplasts of root tissues in the H genotype was lower than in the L 215
genotype at seedling (Fig 1C) and flowering stages (Fig S1C) and NO3- accumulation in the 216
cytosol increased significantly in the H genotype compared with the L genotype at seedling 217
(Fig 1D) and flowering stages (Fig S1D) 218
219
B napus with higher NUE showed enhanced long-distance transport of NO3- from roots 220
to shoots 221
The relative expression of BnNRT15 in roots of the H genotype was significantly higher 222
than that in the L genotype at both seedling and flowering stages while the relative expression 223
of BnNRT18 in roots of the H genotype was lower than that in the L genotype at both 224
seedling (Fig 2-AB) and flowering stages (Fig S2-AB) As a consequence total N 225
concentration (traced by 15N) in roots of the H genotype was significantly lower than that of 226
the L genotype at both seedling (Fig 2C) and flowering stages (Fig S2C) while shoot N 227
concentration in the H genotype was significantly higher than the L genotype at seedling stage 228
(Fig 2C) This resulted in significantly higher [15N] SR ratio in the H genotype compared 229
with the L genotype (Fig 2D Fig S2D) 230
No significant differences in total N per plant between the H and L genotypes were 231
observed at both seedling and flowering stages (Fig S3) However NO3- concentration in 232
roots of the H genotype was significantly lower than in the L genotype at both seedling and 233
flowering stages (Fig 2E Fig S2E) while NO3- concentration in shoot tissues of this 234
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9
genotype was significantly higher at seedling stage (Fig 2E) resulting in higher [NO3-] SR 235
ratios at both stages (Fig 2F Fig S2F) 236
NO3- concentration in the xylem sap xylem sap volume and total NO3
- in xylem sap 237
were significantly higher in the H genotype than in the L genotype at seedling stage (Fig 238
S4ACE) the amount of NO3- in xylem sap significantly increased in the H genotype relative 239
to the L genotype at flowering stage (Fig S4F) Together these data suggests greater 240
mobilization of NO3- from root to shoot in the H genotype 241
242
NO3- up-regulates NRT15 but down-regulates NRT18 243
244
A previous study showed that increased NRT18 expression in nrt15 mutants (Chen et al 245
2012) and the expression of NRT15 was induced by NO3- (Lin et al 2008) We tested the 246
relationship between NRT15 and NRT18 expression in A thaliana and B napus No 247
significant differences were observed in AtNRT15 expression in roots between wild type 248
(col-0) and mutant plants (nrt18-2) (Fig 3A) However AtNRT15 expression was 249
significantly up-regulated by NO3- in roots of nrt18-2 mutants (Fig 3B) Expression of 250
AtNRT18 significantly increased in roots of nrt15-3 mutants but not in col-0 plants and 251
there were no significant differences in AtNRT18 expression with or without NO3- treatment 252
in roots of nrt15-3 mutants (Fig 3CD) 253
In contrast BnNRT15 expression in roots of the H genotype increased significantly with 254
NO3- treatment (Fig 3E) while the reverse was observed in expression of BnNRT18 which 255
showed lower expression under NO3- treatment than control (Fig 3F) This suggests that NO3
- 256
induces the expression of NRT15 but down-regulates NRT18 in A thaliana and B napus 257
Further studies are needed to elucidate the mechanisms regulating this reverse regulation 258
259
Reduced VSC of NO3- in roots drives its long-distance transport from roots to shoots 260
Our previous study showed that Bafi (Bafilomycin A1) inhibits V-ATPase DCCD 261
(DCCD + Na2SO3) inhibits V-PPase and a 11 combination of Bafi and DCCD (B+D) 262
inhibits both V-ATPase and V-PPase (Han et al 2015a) These inhibitors were used to 263
control activities of the tonoplast proton pumps in the H B napus plants V-ATPase activity 264
significantly decreased under Bafil and B+D treatments relative to the control and DCCD 265
treatments whereas V-PPase activity declined significantly under DCCD and B+D treatments 266
relative to that in the control and Bafil treatments (Fig 4A) 267
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The V-ATPase activity in roots of A thaliana was significantly lower in the V-ATPase 268
mutants (vha-a2 and vha-a3) than the wild type (col-0) and V-PPase mutants (avp1) (Fig 5A) 269
In contrast the V-PPase activity in roots of V-PPase mutant (avp1) were significantly lower 270
than in the wild type (col-0) and V-ATPase mutants (vha-a2 and vha-a3) (Fig 5A) 271
NO3- influxes into vacuoles of the H B napus plants significantly decreased when treated 272
with inhibitors of proton pumps (Bafil DCCD B+D) (Fig 4B) Similarly NO3- influxes into 273
the vacuole of the Arabidopsis mutants (vha-a2 vha-a3 and avp1) was significantly lower 274
than those found in the wild type (col-0) (Fig 5B) Previous studies showed that VSC of NO3- 275
decreased when the activities of the proton pumps decline (Li et al 2005 Krebs et al 2010 276
Han et al 2015a) 277
We further investigated NO3- distribution between the vacuole and cytosol as affected by 278
proton pump inhibition in the H genotype of B napus and the mutants of A thaliana 279
defective in vacuolar proton pumps The percentage of vacuolar NO3- relative to total NO3
- in 280
root protoplasts of the H genotype treated with inhibitors (Bafil DCCD B+D) was lower than 281
that observed in the control (Fig 4C) Results were also similar when using A thaliana 282
mutants where the percentage of vacuolar NO3- relative to the total NO3
- in protoplasts was 283
lower in the mutants (vha-a2 vha-a3 and avp1) than in the wild type (col-0) (Fig 5C) 284
Consequently NO3- accumulation in the cytosol showed a significant increase when energy 285
pumps were suppressed in roots of the H genotype (Fig 4D) and NO3- accumulation in the 286
cytosol of root tissues of A thaliana mutants (vha-a2 vha-a3 and avp1) similarly increased 287
compared with col-0 (Fig 5D) 288
Based on previous observations (Lin et al 2008) expression of NRT15 is strongly 289
induced by NO3- but expression of NRT18 is down-regulated (Fig3 Chen et al 2012) We 290
then hypothesized that expression of these two genes is contrastingly regulated by 291
concentration of NO3- in the cytosol Our results were congruent with this hypothesis the 292
expression of BnNRT15 in root tissues of the H genotype of B napus was significantly higher 293
in plants treated with Bafil DCCD or B+D compared with the control (Fig 4E) whereas the 294
expression of BnNRT18 decreased substantially (Fig 4F) Similar results were also observed 295
in A thaliana where expression of AtNRT15 in roots of the mutants (vha-a2 vha-a3 and 296
avp1) were significantly higher than in the wild type (col-0) but expression of AtNRT18 in 297
the same mutants were considerably lower (Fig 5EF) 298
NO3- concentrations in the xylem sap the N-distribution between shoot and root (SR 299
ratios based on [15N]) and the [NO3-] in shoots relative to roots of the H genotype treated with 300
energy pumpsrsquo inhibitors were significantly higher than in the control (Fig 4GH Fig S5A) 301
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11
Similar trends were also observed when using mutants of A thaliana deficient in the energy 302
pumpsrsquo activities NO3- concentration in the xylem sap the [15N] SR ratios and [NO3
-] SR 303
ratios were all significantly higher in the mutants than in the wild type (Fig 5GH Fig S5B) 304
These data clearly showed that prevention of N sequestration in vacuoles would enhance its 305
translocation to shoot 306
Additional evidence linking NO3- long-distance transport and NO3
- short-distance 307
distribution within cells were provided through experiments comparing A thaliana wild type 308
(Ws) and mutant clca-2 (Fig S6) The chloride channel (CLCa) is the main channel for 309
vacuolar anion accumulation Vacuolar sequestration capacity of NO3-significantly declines in 310
clca mutants (Angeli et al 2006) NO3- influx into the vacuolar space of Ws roots was 311
significantly higher than that observed in the clca-2 mutants (Fig S6A) This resulted in a 312
smaller proportion of vacuolar NO3- in clca-2 plants relative to the total NO3
- in root tissue 313
protoplasts as compared with Ws (Fig S6B) Consequently the accumulation of NO3- in the 314
cytosol was significantly higher in clca-2 than in Ws plants (Fig S6C) The higher 315
NO3-concentration in the cytosol together with the higher expression of AtNRT15 coupled 316
with lower expression of AtNRT18 in clca-2 roots (Fig S6DE) resulted in significant 317
increase in xylem sap NO3- concentration [15N] SR ratios and [NO3
-] SR ratios compared 318
with Ws plants (Fig S6FGH) 319
320
Increased NO3- translocation to shoots enhanced NUE 321
NO3- assimilation efficiency is known to be higher in shoots than in roots (Smirnoff et al 322
1985 Andrews et al 1986 Tang et al 2012 Tang et al 2013) We therefore hypothesize 323
that increased NO3- translocation to shoot and the consequent higher shootroot ratio will 324
contribute to higher NUE Our data support this hypothesis The H B napus genotype showed 325
enhanced long-distance transport of NO3- from roots to shoots (Fig 2 Fig S2) This enhanced 326
NO3- transport requires higher carbon skeleton provided through higher photosynthetic rate 327
(Tang et al 2012 Tang et al 2013) Our results showed that chlorophyll content 328
intercellular CO2 concentration and photosynthetic rate were significantly higher in the H than 329
the L genotype at both seedling and flowering stages (Table 2) Moreover the NO3- 330
assimilating enzymes were strongly induced by NO3- providing sufficient capacity for N 331
assimilation (Smirnoff et al 1985 Andrews et al 1986) Nitrate reductase (NR) and 332
glutamine synthetase (GS) activities in roots of the H genotype were significantly lower than 333
in the L genotype both at seedling and flowering stages (Fig S7) In contrast NR activities in 334
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12
shoots of the H genotype were significantly higher than in the L genotype at both stages (Fig 335
S7AB) and GS activity in the H genotype was also higher than those of the L genotype at 336
seedling stage (Fig S7C) 337
Experiments comparing A thaliana wild type (col-0 Ws) with mutants defective in 338
vacuolar proton-pumps (vha-a2 vha-a3avp1) and transport channel (clca-2) yielded similar 339
results that is increases in NO3- shootroot ratio essentially contributed to enhanced NUE 340
(Fig 6) The NO3- shootroot ratios in A thaliana mutants were higher than that in the wild 341
type (Fig 5GH) Generally the efficiency of inorganic N (NO3-) assimilation into organic N 342
is higher in shoots than in roots (Smirnoff et al 1985 Andrews et al 1986) therefore the 343
NO3- concentration in A thaliana mutants decreased (Fig 6CD) leading to higher NUE 344
compared with the wild type (Fig 6AB) 345
346
BnNRT15BnNRT18 in B napus affects NO3- long-distance transport from roots to 347
shoots 348
Both B napus and A thaliana are members of Cruciferae Family The amino acid 349
sequence identity between BnNRT15 and AtNRT15 was 90 (Fig S8A) the amino acid 350
sequence similarity between BnNRT18 and AtNRT18 was 908 (Fig S8B) Comparisons of 351
nucleotide and amino acid sequences (Harper et al2012) of these genes 352
(httpbrassicanbiacukcgi-binmicroarray_databasecgi) showed that BnNRT15 353
(EV220114) and BnNRT18 (EV116423) of B napus are respectively highly homologous 354
with AtNRT15 and AtNRT18 and the two genes are mainly expressed in roots of both species 355
showing similar organ-specificity (Lin et al 2008 Li et al 2010) 356
NO3- long-distance transport from roots to shoots is therefore regulated by NRT15 and 357
NRT18 as reported before (Lin et al 2008 Li et al 2010) The [15N-traced] SR ratios and 358
the [NO3-] SR ratios were significantly lower in nrt15-3 mutants relative to the wild type 359
(col-0) while [15N] SR and [NO3-] SR ratios showed significant increase in nrt18-2mutant 360
relative to col-0 (Fig S9CD) A previous study showed that expression of NRT15 is 361
down-regulated by ACC and MeJA treatments while the expression of NRT18 is strongly 362
up-regulated in A thaliana (Zhang et al 2014) Consequently NO3- accumulated in plant 363
roots probably as an adaptive measure for abiotic stresses (Chen et al 2012) Both the H- 364
and L- B napus genotypes showed significantly lower expression of BnNRT15 in roots when 365
treated with ACC (Fig S10A) but the expression of BnNRT18 remained higher than in the 366
control plants (Fig S10B) This resulted in significantly lower [15N] SR ratios following 367
ACC treatment (Fig S10CDE) These data indicate that BnNRT15 and BnNRT18 play 368
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13
similar functions for NO3- long-distance transport between root and shoot in both B napus 369
and A thaliana (Lin et al 2008 Li et al 2010) 370
371
372
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14
Discussion 373
Nitrate long-distance transport from root to shoot is regulated by cytosolic NO3- in roots 374
NO3- long-distance transport from root to shoot is controlled by NRT gene family For 375
instance xylem NO3- loading and unloading are mainly controlled by NRT15 and NRT18 376
respectively (Lin et al 2008 Li et al 2010) Whereas the NO3- short-distance transport 377
between the vacuole and cytosol is controlled by tonoplast proton-pumps (V-ATPase and 378
V-PPase) (Krebs et al 2010) and by activity of the CLCa channel in the tonoplast membrane 379
(Angeli et al 2006 Wege et al 2014) tonoplast proton-pumps provide the energy required 380
for NO3- accumulation into the vacuole through CLCa (Gaxiola et al 2001 Brux et al 2008 381
Krebs et al 2010) The expression of NRT15 is up-regulated by NO3- in plant culture 382
solution (Lin et al 2008) whereas regulation of NRT18 expression is contrary to that of 383
NRT15 under both control and abiotic stress conditions (Li et al 2010 Chen et al 2012 384
Zhang et al 2014) 385
Despite the progress made in characterizing the genes involved in transport and 386
metabolism of NO3- in plants the relationship between long- and short-distance transport and 387
how these two pathways are regulated have not been thoroughly investigated We hypothesize 388
that NRT15 expression and its role in long-distance transport is regulated by NO3- in plant 389
tissue as was observed in culture solution (Lin et al 2008) and our data support this 390
hypothesis Both BnNRT15 and AtNRT15 expression were up-regulated by increasing 391
cytosolic NO3- in roots (Fig 4 Fig 5) The increase in cytosolic NO3
- was achieved through (i) 392
use of A thaliana mutants defective in proton-pumps (V-ATPase and V-PPase) or CLCa 393
activities in the tonoplast and (ii) inhibition of B napus tonoplast proton-pumps In both 394
model plants the influx of NO3- into vacuoles was substantially reduced resulting in higher 395
concentrations of NO3- in the cytosol (Fig 4A-D Fig 5A-D Fig S6A-C) Under those 396
conditions expression of BnNRT15 and AtNRT15 was up-regulated by the enhanced 397
cytosolic NO3- while expression of BnNRT18 and AtNRT18 was down-regulated (Fig 4EF 398
Fig 5EF FigS6DE) As a consequence more NO3- was loaded into the xylem sap and 399
transported from root to shoot (Fig S5AB FigS6F) This is also reflected as increased 400
shootroot ratios of N traced by [15N] and [NO3-] (Fig 4GH Fig 5GH Fig S6GH) 401
Interestingly the expression of both BnNRT15 and AtNRT15 is up-regulated by 402
increased cytosolic NO3- concentration in roots but were not affected by NO3
- sequestered 403
into vacuoles (Fig 4 Fig 5 Fig S6) This is because vacuolar NO3- is functionally separated 404
by the tonoplast and cannot be assimilated by enzymes that are localized in the cytosol 405
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15
Therefore NO3--inducible genes such as NR and NRT15 were not influenced by vacuolar 406
NO3- concentration (Martinoia et al 1981 Martinoia et al 2000 Zhang et al 2012) In 407
addition NO3- concentration in the cytosol is controlled by its short-distance transport through 408
the tonoplast which is mediated through the tonoplast proton-pumps providing the required 409
energy for its sequestration into vacuoles against concentration gradient (Gaxiola et al 2001 410
Brux et al 2008 Krebs et al 2010) The gradient of H+ between the inside of the vacuole 411
and the cytosol is maintained by an 2NO3-1H+ antiporter mechanism that facilitatesNO3
- 412
transport through CLCa (Angeli et al 2006 Wege et al 2014) Our data provide clear 413
evidence that NO3- long-distance transport from root to shoot is regulated by cytosolic NO3
- 414
concentration in roots 415
Regulatory mechanisms that control the expression of NRT18 and NRT15 as mediated 416
by NO3- are still unknown however previous studies showed that their expression is also 417
affected by abiotic stresses whereby NRT15 is down-regulated and NRT18 is up-regulated 418
(Li et al 2010 Zhang et al 2014) AtNRT18 expression in A thaliana nrt15 mutants is 419
highly up-regulated (Chen et al 2012) as was also observed here (Fig 3C) Moreover 420
AtNRT15 expression was enhanced by NO3- in culture solution and was not affected in 421
nrt18-2 mutant (Fig 3AB) On the other hand AtNRT18 expression was not affected by 422
cytosolic NO3- but down-regulated when AtNRT15 expression was up-regulated by NO3
-(Fig 423
S6DE) Analogous results were also obtained with B napus genotypes (Fig 3EF) Based on 424
these results we argue that NRT18 expression is not directly influenced by cytosolic NO3- but 425
rather by NRT15 expression (Fig 4 Fig 5 and Fig S6) suggesting NRT18 is probably 426
acting downstream of NRT15 Apparently the regulatory mechanisms of this NRT gene 427
family still awaits further studies to be elucidated 428
429
Response of NUE to NO3- long-distance transport 430
NO3- assimilation efficiency is higher in shoot than in root tissues and can be further 431
enhanced by carbon assimilation (Smirnoff et al 1985 Tang et al 2013) Therefore 432
translocation of higher proportion of NO3- from roots to shoots likely contributes to better 433
crop growth and higher NUE (Andrews et al 1986 Tang et al 2012) The present study 434
provides direct evidence for these results using contrasting B napus genotype identified 435
before (Zhang et al 2009 Han et al 2015a) Our data confirmed the genetic variation in 436
NUE between these two genotypes (Table 1) 437
We measured NO3- distribution in root and shoot of these genotypes to elucidate the 438
physiological mechanisms contributing to variation in NUE The data were consistent with 439
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16
our hypothesis that concentration of NO3- in the xylem sap and shoot would be significantly 440
higher in the H genotype than in the L genotype (Fig 2EFFig S2EF Fig S4) The NO3- 441
long-distance transport from root to shoot was affected by cytosolic NO3- in roots of both A 442
thaliana and B napus genotypes (Fig 4-5 Fig S6) The activities of the tonoplast 443
proton-pumps in roots were significantly lower in the H genotype than in the L genotype (Fig 444
1AB) Consequently less NO3- was sequestered into the vacuole and more NO3
- was retained 445
in cytosol (Fig 1CD) BnNRT15 expression was then up-regulated resulting in greater 446
loading into the xylem while BnNRT18 was down-regulated (Fig 2AB) probably to 447
suppress NO3- unloading from xylem sap Consequently a higher proportion of NO3
- was 448
transported from root to shoots in the H genotype(Fig 2C-D Fig S4) 449
Moreover photosynthetic carbon fixation (Table 2) and activities of both NR and GS 450
were higher in shoots of the H genotype (Fig S7) both of which promote higher N 451
assimilation These results agreed with previous reports (Smirnoff et al 1985 Andrews et al 452
1986 Tang et al 2012 Tang et al 2013) where higher NO3- transport from root to shoot 453
higher photosynthetic rate and N assimilation efficiency in shoots were suggested as essential 454
mechanisms for high-NUE in crop plants These qualities endowed the B napus H genotype 455
Xiangyou15 with higher NUE as compared with the L genotype 814 456
NO3- long-distance transport in B napus is not only regulated by BnNRT15 and 457
BnNRT18 in root tissues (Fig 4 Fig 5 Fig S6) but also by stomatal conductance and 458
transpiration by indirectly controlling xylem sap flow and long-distance transport 459
(Dechorgnat et al 2011 Krapp et al 2014) Stomatal conductance and transpiration rates of 460
the H genotype were significantly higher than those of the L genotype (Table S1) a result 461
consistent with other studies describing characteristics of high-NUE plants (Daniel-Vedele et 462
al 1998 Wilkinson et al 2007 Dechorgnat et al 2011 Krapp et al 2014) 463
464
Response of NUE to NO3- short-distance transport 465
NUE is also strongly controlled by NO3- short-distance distribution between the vacuole 466
and cytosol (Han et al 2015a) A lower proportion of NO3- accumulating in vacuoles and a 467
higher proportion retained in the cytosol will contribute to better NUE in crop plants (Han et 468
al 2015b) The energy required for vacuolar NO3- sequestration is provided by the tonoplast 469
proton-pumps (Gaxiola et al 2001 Brux et al 2008 Krebs et al 2010) while the 470
distribution channel CLCa enables transport of NO3- across the vacuolar membrane (Angeli et 471
al 2006 Wege et al 2014) The activities of the tonoplast proton-pumps (V-ATPase and 472
V-PPase) in leaves of the H genotype were significantly lower than those of the L genotype at 473
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17
flowering stages (Han et al 2015aHan et al 2015b) thus we expected similar differences in 474
root tissues Our results confirmed that the activities of tonoplast proton-pumps in root tissues 475
of B napus H genotype and A thaliana mutants were lower than those in the L genotype and 476
A thaliana wild type leading to less NO3- influx into vacuoles and more in the cytosol (Fig 1 477
Fig 5A-D) Moreover the enzymes catalyzing N assimilation are located in the cytoplasm 478
where NO3- could be assimilated (Martinoia et al 1981 Han et al 2015a) or immediately 479
loaded into xylem sap for long-distance transport from roots to shoots (Lin et al 2008) 480
Consequently the NO3- concentration in root tissues of the H genotype and A thaliana 481
mutants defective in tonoplast proton-pumps was significantly lower than that in the L 482
genotype (Fig 2E) and in A thaliana wild type (Fig 6C) Therefore the higher proportion of 483
NO3- transported to shoot and the lower concentrations retained in root vacuoles contributed 484
to the higher NUE in Xiangyou15 and A thaliana mutants (vha-a2 vha-a3 and avp1) (Table 485
1Fig 6A) Further studies are needed to assess the impacts of regulating vacuolar 486
sequestration of NO3- and rates of long-distance transport for enhancing NUE in other crop 487
species and genotypes 488
These data suggested that NO3- distribution between vacuoles and cytosol within cells 489
regulates NO3- long-distance transport from root to shoot and ultimately affects NUE NO3
- 490
loading into the xylem sap is an active transport process (Lin et al 2008) therefore not 491
regulated by absolute concentration of NO3- in the cytosol but rather by genes involved in 492
NO3- long-distance transport (NRT15 and NRT18) Apparently the expression of NRT15 the 493
gene responsible for xylem loading is regulated by NO3-concentration in the cytosol 494
Therefore both NO3- short-distance transport across the tonoplast and its long-distance 495
transport from root to shoot are important determinants of NUE in both B napus and A 496
thaliana (Table 1 and Fig 6) 497
498
Roles of NRT15 and NRT18 in NO3- long-distance transport in B napus and A 499
thaliana 500
Our results (Figs S9 S10) showed that the proportion of NO3- distributed from root to 501
shoot is strongly controlled by NRT15 in accord with previous studies (Lin et al 2008 Li et 502
al 2010) The data suggest that the high-NUE genotypes possess lower activities of tonoplast 503
proton-pumps (V-ATPase and V-PPase) resulting in less NO3- accumulation in vacuoles and 504
more NO3- retention in cytosol (Fig 7) The higher NO3
- in the cytosol then up-regulated 505
NRT15 which then down-regulates NRT18 As a result more NO3- is loaded into the xylem 506
system by NRT15 mechanism and less NO3- is unloaded via NRT18 (Fig 7) Differential 507
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18
regulation of these NRT genes in high-NUE plants increased transport of NO3- from root to 508
shoot through the xylem vascular tissues (Fig 7) This coordinated NO3- long- and 509
short-distance transport likely fine-tunes NO3- allocation and modulates the balance of NO3
- 510
distribution between roots and shoots (Fig 7) 511
Our research also revealed differences between A thaliana and B napus in the 512
expression of the wild-type NRT genes in response to NO3- in the protoplast Moreover using 513
A thaliananrt15-3 mutants we uncovered possible regulatory interactions between a 514
functioning (wild-type) NRT15 gene and NRT18 expression in response to NO3- 515
concentration Because mutants defective in nrt15 are not available for B napus we could 516
not confirm that the respective NRT15 and NRT18 systems behave in an analogous way in 517
these plant species Based on Harper et al (2012) research database our homology 518
comparisons and gene function annotations provided strong evidence for the roles of 519
BnNRT15 and BnNRT18 in B napus Finally our studies were conducted using two model 520
dicotyledonous plant species for which we had ample data on responses to specific inhibitors 521
(B napus) or specific mutants were available with results forming the bases for the model 522
presented in Figure 7 Further research using genetically diverse plant species (eg 523
monocotyledonous crops such as rice wheat and corn) will help validate this model and its 524
broader application across species The information generated in this study could have future 525
application for improving NUE in commercial crops with consequent reduction in nitrogen 526
use and benefits to the environment 527
528
Materials and Methods 529
Plant material 530
The two oilseed rape (Bnapus) cultivars used in this study have been characterized 531
before as high- (Xiangyou15 referred to as H genotype hereafter) and low- (814 referred to 532
as L genotype) nitrogen use efficiency (NUE) genotypes (Zhang et al 2009 Han et al 2015b) 533
Here we define NUE as the total biomass per unit of N uptake by the plant with total 534
biomass includes roots shoots and grains The two genotypes were provided by the Hunan 535
Sub-Center of Improvement Center of National Oil Crops Hunan China 536
The Arabidopsis thaliana wild-type Columbia-0 (col-0) was used as control for 537
V-ATPase (vha-a2 and vha-a3) V-PPase (avp1) nrt15-3 and nrt18-2 mutants whereas the A 538
thaliana wild-type Wassilewskija (Ws) was used as control for clca-2 mutants A thaliana 539
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19
mutants and transgenic lines (nrt15-3 and nrt18-2) have been described previously (Chen et 540
al 2012 Zhang et al 2014) The mutant lines vha-a2 (Salk_142642) and vha-a3 541
(Salk_122135) described by Krebs et al (2010) were obtained from the Arabidopsis 542
Biological Resources Center (ABRC) The mutant line avp1 (GK-005004) described in Li et 543
al (2005) was obtained from the European Arabidopsis Stock Centre while the mutant line 544
clca-2 (FST 171A06) described in Angeli et al (2006) was from the Institute National de la 545
Recherche Agronomique (INRA) collection in France together with the T-DNA mutants used 546
for genotyping to select pure mutant lines 547
548
Growth conditions 549
B napus plants were grown hydroponically in ceramic pots(20cmtimes15cm) filled with a 550
nutrient solution in the greenhouse under natural light as described in Han et al (2015b) The 551
pots were arranged in a completely randomized design with six biological replications The 552
nutrient solution was replaced every 3 days and its pH adjusted to 55 daily Experiments were 553
conducted at the field station of Hunan Agricultural University Southern China 554
A thaliana plants were grown in a nutrient solution in plastic pots as described in Arteca 555
et al (2000) and Gong et al (2003) The solution was changed every 3 days with pH adjusted 556
daily to 58 Pots were arranged in a completely randomized design with six biological 557
replications The nutrient solution used for both species consisted of 125mM KNO3 558
0625mM KH2PO4 05mM MgSO4 05mM Ca (NO3)2middot4H2O 0025mM Fe-EDTA 025 559
mlL-1 micronutrients (stock solution concentrations 70mM B 14mM Mn 1mM Zn 05mM 560
Cu and 02mM Mo) The experiments were conducted at Hunan Agricultural University in a 561
phytotron set at 70 relative humidity16h-light8h-dark cycle and constant temperature of 562
22degC 563
564
Experimental treatments and phenotyping 565
All measurements made on B napus plants were conducted either at the seedling stage 566
(2 months after transplanting) or at flowering (5 months after transplanting) The whole root 567
tissue was harvested and used for analyses at either stage For shoot measurements the fourth 568
leaf from the bottom was used for measurements made at seedling stage whereas the 12th leaf 569
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20
from the bottom was harvested for measurements made at flowering stage For A thaliana 570
whole root and shoot of four-week-old plants were sampled and used for different assays 571
572
Vacuolar proton-pumps inhibitor treatments 573
Inhibition of vacuolar proton pumps of B napus (Xiangyou15) was conducted at the seedling 574
stage in hydroponic culture as described in Han et al (2015a) with minor modifications 575
Plants grown in hydroponic solution were used as control Inhibitor treatment was conducted 576
using several chemicals Bafi(25 nmol L-1 Bafilomycin A1) an inhibitor of V-ATPase 577
DCCD(10 μmol L-1DCCD + 50 mmolL-1 Na2SO3) specifically inhibits V-PPase and 578
Bafi+DCCD in a 11 ratio as inhibitor of both V-ATPase and V-PPase The inhibitors were 579
applied in the hydroponic solutions for 24 h Fresh plant tissue samples from all treatments 580
were collected for different assays 581
582
NO3- induced gene expression 583
Four-week old A thaliana mutants (nrt15-3nrt18-2) were grown in hydroponics with 584
225mM (NH4)2succinate for 3 days and shifted to hydroponics with 45mM NO3- for 12 h 585
then root tissues were collected to assess relative expression of AtNRT15 and AtNRT18 586
genes Seedlings of B napus (Xiangyou15) plants grown hydroponically were treated with 587
5mM (NH4)2succinate for 3 d and shifted to fresh hydroponic solution containing 15 mM 588
NO3- for 12 h Root tissue was then collected to assess the relative expression of BnNRT15 589
and BnNRT18 genes using quantitative RT-PCR 590
591
ACC treatment 592
Hydroponically grown seedlings of B napus were subjected to ACC treatment as described in 593
Zhang et al (2014) with minor modifications Seedlings of the two genotypes Xiangyou15 594
and 814 were transferred to a nutrient solution containing 002mM L-1 ACC for 6 h or grown 595
in normal hydroponic solution as control Root tissues were then collected to assay relative 596
expression of BnNRT15 and BnNRT18 using quantitative RT-PCR Another set of B napus 597
plants were grown in ACC-containing hydroponic solution but supplemented with 15NO3- (22 598
15N) replacing the unlabeled NO3- for 1 h Root and shoot tissues were collected separately 599
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21
for 15N measurement using a continuous-flow isotope ratio mass spectrometer coupled with a 600
C-N elemental analyzer (ANCA-MS PDZ Europa) 601
602
Biomass and N concentration 603
Grain yield dry biomass and N concentration were determined in plant samples at 604
harvest Plants were harvested (including senescing leaves) then dried in an oven for 30 min 605
at 105degC then at 70degC to a constant weight and weighed N concentration was determined 606
using the Kjeldahl method (Shi et al 2010 Han et al 2015b)02g of dried samples were 607
digested with H2SO4-H2O2 in a 100-ml Kjeldahl digestion flask after which N concentration 608
was determined using a Foss Auto Analyser Unit (Kjeldahl 8400) 609
610
V-ATPase and V-PPase activities 611
Roots (10 g) of B napus were collected at seedling and flowering stages and that of A 612
thaliana (05 g) sampled at seedling stage then used for determining the activities of 613
V-ATPase and V-PPase (Han et al 2015b) Activities of these two energy pumps within 614
microsomal membranes were followed colorimetrically based on released Pi as described by 615
Zhu et al (2001) and Krebs et al (2010) 616
617
Assay of NO3- flux in vacuoles 618
Roots of both species were collected similarly as that used for V-ATPase and V-PPase 619
activities then used for vacuole isolation and measurement of NO3- flux in vacuoles as 620
described by Robert et al (2007) with minor modification (Han et al 2015b) Net fluxes of 621
NO3- in vacuoles were measured non-invasively using SIET (scanning ion-selective electrode 622
technique SIET system BIO-003A Younger USA Science and Technology Corporation 623
USA) An NO3--selective microelectrode used for the assay of NO3
- flux was vibrated in the 624
measuring solution between two positions 1 μm and 11 μm from the vacuole surface 625
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles Background 626
signal was recorded by vibrating the electrode in the measuring solution in the absence of 627
vacuoles Ion flux was calculated by Fickrsquos law of diffusion J = -D(dcdx) where J represents 628
the ion flux (picomolescm-2s-1) dcdx is the ion concentration gradient and D is the ion 629
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22
diffusion constant in a particular medium The direction of the flux was derived from Fickrsquos 630
law of diffusion that relates the concentration gradient 631
632
Isolation of intact protoplasts and vacuoles for determining NO3- concentration 633
Similar to the above assays root tissues (10 g) were collected from B napus at seedling 634
and flowering stages and from A thaliana (05 g) seedlings and used to isolate intact 635
protoplasts and vacuoles as described by Robert et al (2007) with minor modifications as 636
outlined in Huang et al (2012) and Han et al(2015b) The purified protoplasts were divided 637
into two equal aliquots with one of them used for isolation of vacuoles The purified 638
protoplasts and vacuoles were then sub-sampled and used to determine NO3- concentrations 639
(Vogeli-Lange and Wagner 1990) and for enzyme activity assays (Ma et al 2005) 640
NO3-concentrations in protoplasts and vacuoles were measured by a continuous-flow 641
auto-analyzer (Auto Analyser 3 Bran and Luebbe Germany) as described previously (Han et 642
al 2015b)The activities of acid phosphatase (ACP) and cytochrome oxidase (COX) were 643
determined using plant ACP colorimetry- and COX-assay kits (GenMedSci Inc) following 644
the manufacturerrsquos instructions ACP activity specific to vacuoles was determined and used to 645
normalize NO3- accumulation We measured NO3
- in the protoplast outside the vacuole which 646
includes the cytosol and organelles eg mitochondria and Golgi Apparatus (Robert et 647
al2007) Since most of the NO3- in the protoplast outside the vacuole is located in the cytosol 648
(Krebs et al 2010) we refer to NO3- distribution between vacuoles and cytosol rather than 649
vacuole versus protoplast 650
651
Quantitative RT-PCR 652
Total RNA extracted from B napus roots was prepared using Trizol reagent (Invitrogen) 653
cDNAs were synthesized using M-MLV reverse transcriptase (Promega) following 654
manufacturerrsquos protocol The relative expression of BnNRT15 (EV220114) BnNRT18 655
(EV116423) and Bnactin (AF1118121) genes in plant roots were determined by quantitative 656
RT-PCR and run on a LightCycler instrument (Roche) with the SYBR Green Real-Time PCR 657
Master Mix Kit (TOYOBO Japan) under the following conditions 95degC for 2min then 45 658
cycles of 95degC for 10s 60degC for 10s and 72degC for 20s The primer sequences of BnNRT15 659
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23
BnNRT18 and Bnactin genes were obtained from httpbrassicanbiacuk as described in 660
Harper et al (2012) and expression levels were normalized to Bnactin as control 661
Total RNA extracted from the 4-week-old A thaliana roots under the treatments 662
conditions described above were prepared using Trizol reagent (Invitrogen) The cDNAs were 663
synthesized using M-MLV reverse transcriptase (Promega) based on the manufacturerrsquos 664
protocol and as described in Li et al (2010)The primer sequences of quantitative RT-PCR 665
used in these assays are listed in Supplemental Table 2 and were described previously (Chen 666
et al 2012 Zhang et al 2014) and their expression was normalized to Atactin2 as control 667
668
Determination of nitrate distribution using 15NO3- 669
Hydroponically grown B napus (seedling and flowering stages) and A thaliana 670
(seedling) were transferred to 01mM CaSO4 solution for 1min then to their respective 671
hydroponic nutrient solutions with15NO3- (22 excess) replacing unlabeled NO3
- for 1h 672
Plants were then transferred to 01mM CaSO4 solution for 1min after which roots were 673
washed with deionized water Root and shoot samples were separated and dried at 105degC for 674
30min followed by 70degC for 3 d then grounded and used for assaying 15N content using a 675
continuous-flow isotope ratio mass spectrometer coupled with a carbon-nitrogen elemental 676
analyzer (ANCA-MS PDZ Europa) 677
678
Xylem sap collection and assay of nitrate concentration 679
We used the method of Tang et al (2012) for collecting xylem sap Plants were cut 680
leaving1cm segments with intact roots at seedling and flowering stages for B napus and at 681
seedling stage for A thaliana Roots were immediately immersed in their respective nutrition 682
solutions Weighed cotton was put on the cut surface to absorb extruding xylem sap for 1 h 683
The cotton was then wrapped in plastic film and the volume of xylem sap was calculated as 684
the weight gain of the cotton Xylem sap was then squeezed from the cotton using a syringe 685
and used for subsequent assay of nitrate concentration using a continuous-flow auto-analyzer 686
(Auto Analyser 3 Bran and Luebbe Germany) 687
To determine nitrate concentration samples (10 g fresh root and shoot at seedling and 688
flowering of B napus and 05 g fresh root and shoot of 4-week-old A thaliana) were frozen 689
in liquid N2 and ground with a mortar and pestle the powder was then transferred to a beaker 690
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24
containing 70 ml deionized water and boiled for 30 min to extract nitrate then cooled and 691
made to 100 ml volume and 02 g activated carbon added to eliminate the effect of 692
chlorophyll The mixture was filtered and nitrate in the filtrate was determined using a 693
continuous-flow auto-analyser (Atuo-Analyser 3 Bran and Luebbe Germany) 694
695
Measurements of photosynthesis 696
All measurements of photosynthesis and related parameters were conducted at 1000 h 697
using intact leaves The 4th leaf from the bottom in B napus at seedling stage and the 12th leaf 698
from the bottom at flowering stage were used for determining chlorophyll concentration 699
photosynthetic rate intercellular CO2 concentration transpiration rate and stomatal 700
conductance Chlorophyll concentration was determined by SPAD-502 (Minolta Camera Co 701
Ltd Japan) (Luo et al 2006 Yandeau-Nelson et al 2011) Photosynthetic rate intercellular 702
CO2 concentration transpiration rate and stomatal conductance were measured using LI-6400 703
Portable Photosynthesis System (Li-Cor Biosciences Co Ltd USA) set at flow rate of 500 704
μmol s-1 photosynthetic photon flux density of 1000 μmol m-2 s-1 relative humidity of 65 705
CO2 concentration of 400ppm and ambient temperatures 706
707
NR and GS activities 708
Leaves with similar age as those used for photosynthesis in B napus were used for the 709
assay of nitrate reductase (NR) and glutamine synthetase (GS) activities NR activity was 710
measured by the modified method of Fan et al (2007) Fresh samples (05g) were frozen in 711
liquid N2 ground into powder in the presence of acid-washed sand and homogenized with 4 712
ml of extraction buffer (0025mol L-1phosphate buffer pH87 1211g L-1 cysteine 0372g L-1 713
EDTA) Homogenates were centrifuged at 30000timesg for 15 min at 4degC and the supernatants 714
were treated with sulfanilamide and α-naphthylamine reagents for colorimetric (540nm) 715
determination of nitrite as described by Fan et al (2007) 716
GS activity was determined using a modified reverseγ-glutamyltransferase method 717
(Wang et al2014) which measures the GS-catalyzed formation of glutamyl-γ-hydroxamate 718
from glutamine and hydroxylamine Fresh samples were frozen for 30 min at -20degC then 719
ground in 10ml Tris-HCl buffer and acid-washed sand The homogenates were filtered 720
through two layers of gauze and centrifuged at 8000timesg for 15 min at 4degC About 12ml of the 721
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
supernatant was added in a reaction mixture and treated as described (Wang et al2014) After 722
the GS-catalyzed reaction was stopped and the solution centrifuged glutamyl-γ723
-hydroxamate was quantified colorimetrically in the supernatant (at 485 nm) and the 724
concentration was determined using a standard curve 725
726
Statistical analyses 727
We used the SPSS software (Statistical Product and Service Solutions V130 USA) for 728
ANOVA and mean separation of main-effects and interactions using Duncanrsquos multiple range 729
test at Plt005Values are presented as means and SD of three or six replicates from three 730
independent experiments Different letters or an asterisk () associated with specific data (eg 731
at the top of histogram bars in figures or within tables) denote significant differences at 732
Plt005 733
734
Supplemental Data 735
Supplemental Figure 1 B napus with higher NUE showed lower vacuolar sequestration 736
capacity (VSC) for NO3-in roots at flowering stage 737
Supplemental Figure 2 B napus with higher NUE showed enhanced long-distance transport 738
of NO3- from roots to shoots at flowering stage 739
Supplemental Figure 3 The two B napus (H and L genotypes) showed the same total N per 740
plant at seedling stage (A) and flowering stage (B) 741
Supplemental Figure 4 B napus with higher NUE showed increased NO3- concentration in 742
the xylem sap at seedling and flowering stages 743
Supplemental Figure 5 NO3- concentration in the xylem sap as affected by inhibitor 744
treatments in B napus and in the energy pumpsrsquo mutants of A thaliana (col-0 vha-a2 vha-a3 745
avp1) 746
Supplemental Figure 6 Reduced VSC for NO3- in roots drives long-distance transport of 747
NO3- from roots to shoots in the A thaliana wild type (Ws) and mutant (clca-2) 748
Supplemental Figure 7 Differences of NR and GS activities between the two B napus (H 749
and L genotypes) at seedling and flowering stages 750
Supplemental Figure 8 Amino acid sequences of BnNRT15 and BnNRT18 751
Supplemental Figure 9 Functions of AtNRT15 and AtNRT18 genes in root tissues of A 752
thaliana in controlling NO3- long-distance transport from root to shoots 753
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26
Supplemental Figure 10 B napus BnNRT15 and BnNRT18 genes in roots were 754
coordinately modulated to facilitate NO3- long distance transport from roots to shoots 755
Supplemental Table 1 Differences in stomatal conductance and transpiration rate between 756
the two B napus genotypes 757
Supplemental Table 2 Sequences of primera used for qRT-PCR 758
759
Acknowledgements 760
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 761
Shanghai Institutes for Biological Sciences) for providing nrt15-3 and nrt18-2 seeds 762
This study was supported by the National Natural Science Foundation of China (Grant 763
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 764
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 765
The Ministry of Education in China Open Novel Science Foundation of Hunan province 766
(13K062) the National Key Laboratory of Plant Molecular Genetics and the Twelfth 767
Five-Year National Science and technology support program (2012BAD15BO4) 768
769
Figure 1 B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 770
NO3- in roots at the seedling stage 771
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 772
genotype 814 Specific activities of the tonoplast proton pumps are expressed as μmol Pi 773
released mg-1 protein h-1 NO3- fluxes are expressed as pmol NO3
- cm-2 S-1 Mature vacuoles 774
were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 775
measuring solution between the two positions 1 μm and 11 μm from the vacuole surface 776
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles recording the 777
stable reading data The background was recorded by vibrating the electrode in measuring 778
solution without vacuoles 779
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 780
for NO3- content and NO3
- accumulation normalized against the specific activity of the 781
vacuole acid phosphatase (ACP) as described in Materials and Methods and plotted as μmol 782
NO3- per μmol p-nitrophenol the end product of ACP assay 783
Proton pump activities in root tissues between H and L genotypes are shown at the seedling 784
stage (A) Different letters at the top of the histogram bars denote significant differences of 785
V-ATPase in root tissues between H and L genotypes (Plt005) an asterisk () at the top of the 786
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
histogram bars indicates significant difference in V-PPase activity in root tissues between H 787
and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788
Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789
genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790
bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791
bars on the figures indicate SD (n=6) 792
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793
seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794
to the total NO3- in protoplasts 795
NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796
total NO3-in the cytosol and was calculated as total NO3
- in protoplasts ndash total NO3- in 797
vacuole Different letters at the top of histogram bars denote significant differences of total 798
NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799
(n=6) 800
801
Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802
roots to shoots at the seedling stage 803
H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804
Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805
the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806
as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807
the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808
the histogram bars denote significant differences at Plt005 809
Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810
in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811
genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812
of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813
SD (n=3) different letters at the top of the histogram bars denote significant differences at 814
Plt005 level 815
The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816
at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817
genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818
different letters at the top of the histogram bars denote significant differences at Plt005 819
820
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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- Parsed Citations
- Reviewer PDF
- ONE
- TWO
- THREE
- FOUR
- FIVE
- SIX
- SEVEN
- T-ONE
- T-TWO
- Parsed Citations
-
2
Footnotes 33
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 34
Shanghai Institutes for Biological Sciences) for providing nrt15-3and nrt18-2 seeds This 35
study was supported by the National Natural Science Foundation of China (Grant 36
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 37
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 38
The Ministry of Education in China Open Novel Science Foundation of Hunan province 39
(13K062) National Key Laboratory of Plant Molecular Genetics and the Twelfth Five-Year 40
National Science and technology support program (2012BAD15BO4) 41
42
These authors have contributed equally 43
sect Corresponding author e-mail zhzh1468163com 44
45
46
Author contributions 47
ZHZ conceived the original screening and research plans ZHZ supervised the experiments 48
YLH HXS QL YY SFJ and ZHZ performed most of the experiments CT and JZ provided 49
technical assistance to YLH HXS YY and ZHZ YLH and ZHZ designed the experiments 50
and analyzed the data QL XMR CYG and ZHZ interpreted the results and JEL AI and 51
ZHZ conceived the project and wrote the article with contributions of all the authors JEL AI 52
and ZHZ supervised and complemented the writing All authors read and approved the final 53
manuscript 54
55
56
57
58
59
60
61
62
63
64
65
66
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
3
67
Abstract 68
Enhancing nitrogen use efficiency (NUE) in crop plants is an important breeding target 69
to reduce excessive use of chemical fertilizers with substantial benefits to farmers and the 70
environment In Arabidopsis thaliana allocation of more NO3- to shoots was associated with 71
higher NUE however the commonality of this process across plant species have not been 72
sufficiently studied Two Brassica napus genotypes were identified with high and low NUE 73
We found that activities of V-ATPase and V-PPase the two tonoplast proton-pumps were 74
significantly lower in roots of the high-NUE genotype (Xiangyou15) than in the low-NUE 75
genotype (814) and consequently less vacuolar NO3- was retained in roots of Xiangyou15 76
Moreover NO3- concentration in xylem sap [15N] shootroot (SR) and [NO3
-] SR ratios 77
were significantly higher in Xiangyou15 BnNRT15 expression was higher in roots of 78
Xiangyou15 compared with 814 while BnNRT18 expression was lower In both B napus 79
treated with proton pump inhibitors or Arabidopsis mutants impaired in proton pump activity 80
vacuolar sequestration capacity (VSC) of NO3- in roots substantially decreased Expression of 81
NRT15 was up-regulated but NRT18 was down-regulated driving greater NO3- 82
long-distance transport from roots to shoots NUE in Arabidopsis mutants impaired in proton 83
pumps was also significantly higher than in the wild type col-0 Taken together these data 84
suggest that decrease in VSC of NO3- in roots will enhance transport to shoot and essentially 85
contribute to higher NUE by promoting NO3- allocation to aerial parts likely through 86
coordinated regulation of NRT15 and NRT18 87
88
Key words Nitrogen use efficiency Nitrate allocation Proton-pumps Vacuoles 89
90
91
92
93
94
95
96
97
98
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
4
Introduction 99
China is the largest consumer of nitrogen (N) fertilizer in the world however the 100
average N-fertilizer use efficiency (NUE) is only around 35 suggesting considerable 101
potential for improvements (Shen et al 2003 Wang et al 2014) With the high amounts of 102
N-fertilizer being used crop yields are declining in some areas where application is 103
exceeding the optimum required for local field crops (Shen et al 2003 Miller et al 2008Xu 104
et al 2012) The extremely low NUE results in waste of resources and environmental 105
contamination and also presents serious hazards for human health (Xu et al 2012 Chen et 106
al 2014) Consequently exploiting the maximum potential for improving NUE in crop plants 107
will have practical significance for agriculture production and the environment (Zhang et al 108
2010 Schroeder et al 2013 Wang et al 2014) Elucidating the genetic and physiological 109
regulatory mechanisms governing NUE in plants will allow breeding crops and varieties with 110
higher NUE 111
Ammonium (NH4+) and nitrate (NO3
-) are the main N species absorbed and utilized by 112
crops and NO3- accumulation and utilization are of major emphasis for N nutrient studies in 113
dry land crops such as Brassica napus Several studies revealed the close relationship 114
between NO3- content and NUE in plant tissues (Shen et al 2003 Zhang et al 2012 Tang et 115
al 2013Han et al 2015a)When plants are sufficiently illuminated NO3- assimilation 116
efficiency significantly increase in shoots compared with roots (Smirnoff et al 1985 Tang et 117
al 2013) Consequently under daytime with optimal illumination higher proportion of NO3- 118
in plant tissue is transported from root to shoot as an advantageous physiological adaptation 119
that reduces the cost of energy for metabolism (Tang et al 2013) NO3- assimilation in plant 120
shoots can therefore take advantage of solar energy while improving NUE (Smirnoff et al 121
1985 Andrews et al 1986 Tang et al 2012 Tang et al 2013) 122
The NO3- long-distance transport and distribution between root and shoot is regulated by 123
two genes encoding long transport mechanisms NRT15 is responsible for xylem NO3- 124
loading while NRT18 is responsible for xylem NO3- unloading (Lin et al 2008 Li et al 125
2010) Expression of the two genes is influenced by NO3- concentration NRT15 is strongly 126
induced by NO3- (Lin et al 2008) while NRT18 expression is extremely up-regulated in 127
nrt15 mutants (Chen et al 2012) A negative correlation between the extents of expression of 128
the two genes was observed when plants are subjected to abiotic stresses (Chen et al 2012) 129
Moreover expression of NRT15 is strongly inhibited by 1-aminocyclopropane-1-carboxylic 130
acid (ACC) and methyl jasmonate (MeJA) whereas the expression of NRT18 is significantly 131
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5
up-regulated (Zhang et al 2014) Based on these studies we argue that the expression and 132
functioning of NO3- long-distance transport genes NRT15 and NRT18 are regulated by 133
cytosolic NO3- concentration In addition the vacuolar and cytosolic NO3
- distribution is 134
likely regulated by proton-pumps located within the tonoplast (V-ATPase and V-PPase) 135
(Granstedt et al 1982 Glass et al 2002 Krebs et al 2010) Therefore NO3- use efficiency 136
must be affected by NO3- long-distant transport (between shoot and root) and short-distant 137
transport (between vacuole and cytosol) However the physiological mechanisms controlling 138
this regulation are still obscure 139
Previous studies showed that the chloride channel protein (CLCa) is mainly responsible 140
for vacuole NO3- short-distance transport as it is the main channel for NO3
-movement 141
between the vacuoles and cytosol (Angeli et al 2006 Wege et al 2014) The vacuole 142
proton-pumps (V-ATPase and V-PPase) located in the tonoplast supply energy for active 143
transport of NO3- and accumulation within the vacuole (Gaxiola et al 2001 Brux et al 2008 144
Krebs et al 2010) Despite that about 90 of the volume of mature plant cells is occupied by 145
vacuoles vacuolar NO3- cannot be efficiently assimilated because the enzyme nitrate 146
reductase (NR) is cytosolic (Shen et al 2003 Han et al 2015a) However re-translocation of 147
NO3- from the vacuole to the cytosol will permit its immediate assimilation and utilization 148
Generally NO3- concentrations in plant cell vacuoles and the cytoplasm are in the range 149
of 30-50 mol m-3 and 3-5 mol m-3 respectively (Martinoia et al 1981 Martinoia et al 2000) 150
Because vacuoles are obviously the organelle for high NO3- accumulation and storage in plant 151
tissues their function in NO3- use efficiency cannot be ignored (Martinoia et al 1981 Zhang 152
et al 2012 Han et al 2015b) NO3- assimilatory system in the cytoplasm is sufficient for its 153
assimilation when it is transported out of the vacuoles Therefore NO3- use efficiency could in 154
part be dependent on vacuolar-cytosolic NO3- short-distance transport in plant tissues 155
(Martinoia et al 1981 Shen et al 2003 Zhang et al 2012 Han et al 2015a) 156
Evidently NO3- use efficiency is regulated by both NO3
- long-distance transport from 157
root to shoot and short-distance transport and distribution between vacuoles and cytoplasm 158
within cells(Glass et al 2002 Dechorgnat et al 2011 Han et al 2015a) Although vacuoles 159
compartment excess NO3- that accumulates in plant cells (Granstedt et al 1982 Krebs et al 160
2010) neither NO3- inducible NR genes (NIA1and NIA2) (Fan et al 2007 Han et al 2015a) 161
nor the NO3- long-distance transport gene NRT15 (Lin et al 2008) are regulated by vacuolar 162
NO3- even though they are essential for NO3
- assimilation Only NO3- transported from the 163
vacuole to the cytosol can play a role in regulating NO3- inducible genes Consequently we 164
argue that both NO3- assimilation in cells and its long-distance transport from root to shoot are 165
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regulated by cytosolic NO3- concentration However this hypothesis needs to be substantiated 166
The mechanisms underlying both NO3- short-distance (Gaxiola et al 2001 Angeli et al 167
2006 Brux et al 2008 Krebs et al 2010) and long-distance transport (Lin et al 2008 Li et 168
al 2010) have been previously investigated yet the underlying mechanisms regulating the 169
flux of NO3- and the obvious relationship between the two transport pathways as well as their 170
relation to NUE are not well understood 171
The NRT family of genes play a partial role in vacuolar NO3- accumulation in petioles 172
(Chiu et al 2004) and seed tissues (Chopin et al 2007) whereas the proton pumps and 173
CLCa system in the tonoplast play a major role in accumulating NO3- in vacuoles (Gaxiola et 174
al 2001 Angeli et al 2006 Brux et al 2008 Krebs et al 2010) The vacuolar NO3- 175
short-distance transport system is spread throughout the plant tissues and is the principal 176
means by which vacuolar NO3- short-distance transport and distribution is controlled (Angeli 177
et al 2006Krebs et al 2010) 178
The NRT genes seem to work synergistically to control NO3- long-distance transport 179
between roots and shoots NRT19 is responsible for NO3- loading into the phloem (Wang et 180
al 2011) whereas NO3- loading and unloading into xylem are regulated by NRT15 and 181
NRT18 respectively (Lin et al 2008 Li et al 2010) Phloem transport mainly involves 182
organic N the inorganic-N (NO3-) concentrations in the phloem sap are typically very low 183
ranging from one-tenth to one-hundredth of that of the inorganic-N in xylem sap (Lin et al 184
2008Fan et al 2009) Therefore this study focused on NO3- short-distance transport 185
mediated through the tonoplast proton-pumps and the CLCa system and the long-distant 186
transport mechanisms responsible for xylem NO3- loading and unloading via NRT15 and 187
NRT18 respectively 188
Questions related to how long and short-distance transport of NO3- are coupled in plant 189
tissues and their role in determining NUE were addressed using a pair of high- and low-NUE 190
B napus genotypes and Arabidopsis thaliana Application of proton pump inhibitors and 191
ACC in the former and use of mutants with defective proton pumps in the latter allowed 192
experimental distinction of the physiological mechanisms regulating these processes Data 193
presented here provide strong evidence from both model plants supporting this linkage and 194
strongly suggest that cytosolic NO3- concentration in roots regulates NO3
- long-distance 195
transport from roots to shoots We also investigated how NO3- concentration in plant tissues 196
would be affected by NO3- long-distance transport vacuolar NO3
- sequestration and the 197
ensuing relationship with NO3- use efficiency We also proposed the physiological 198
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mechanisms likely to be important for enhancing NO3- use efficiency in plants These findings 199
will provide scientific rationales for improving NUE in important industrial and food crops 200
201
202
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Results 203
B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for NO3- 204
in root tissues 205
Our previous work identified high and low NUE B napus genotypes (Han et al 2015a 206
Han et al 2015b) NUE of B napus whether based on biomass or on grain yield (Table 1) 207
was significantly higher for the high-NUE genotype (H Xiangyou15) than for the low-NUE 208
genotype (L 814) That the activities of the tonoplast proton-pumps (V-ATPase and V-PPase) 209
of root tissues in the H genotype were lower than the L genotype at both seedling (Fig 1A) 210
and flowering stages (Fig S1A) Given that proton pumps in the tonoplast supply energy for 211
vacuolar NO3- accumulation (Krebs et al 2010) NO3
- influx into the vacuole was 212
consequently much slower in the H genotype compared with the L genotype at both seedling 213
(Fig 1B) and flowering stages (Fig S1B) Moreover the percentage of vacuolar NO3- relative 214
to the total NO3- in protoplasts of root tissues in the H genotype was lower than in the L 215
genotype at seedling (Fig 1C) and flowering stages (Fig S1C) and NO3- accumulation in the 216
cytosol increased significantly in the H genotype compared with the L genotype at seedling 217
(Fig 1D) and flowering stages (Fig S1D) 218
219
B napus with higher NUE showed enhanced long-distance transport of NO3- from roots 220
to shoots 221
The relative expression of BnNRT15 in roots of the H genotype was significantly higher 222
than that in the L genotype at both seedling and flowering stages while the relative expression 223
of BnNRT18 in roots of the H genotype was lower than that in the L genotype at both 224
seedling (Fig 2-AB) and flowering stages (Fig S2-AB) As a consequence total N 225
concentration (traced by 15N) in roots of the H genotype was significantly lower than that of 226
the L genotype at both seedling (Fig 2C) and flowering stages (Fig S2C) while shoot N 227
concentration in the H genotype was significantly higher than the L genotype at seedling stage 228
(Fig 2C) This resulted in significantly higher [15N] SR ratio in the H genotype compared 229
with the L genotype (Fig 2D Fig S2D) 230
No significant differences in total N per plant between the H and L genotypes were 231
observed at both seedling and flowering stages (Fig S3) However NO3- concentration in 232
roots of the H genotype was significantly lower than in the L genotype at both seedling and 233
flowering stages (Fig 2E Fig S2E) while NO3- concentration in shoot tissues of this 234
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genotype was significantly higher at seedling stage (Fig 2E) resulting in higher [NO3-] SR 235
ratios at both stages (Fig 2F Fig S2F) 236
NO3- concentration in the xylem sap xylem sap volume and total NO3
- in xylem sap 237
were significantly higher in the H genotype than in the L genotype at seedling stage (Fig 238
S4ACE) the amount of NO3- in xylem sap significantly increased in the H genotype relative 239
to the L genotype at flowering stage (Fig S4F) Together these data suggests greater 240
mobilization of NO3- from root to shoot in the H genotype 241
242
NO3- up-regulates NRT15 but down-regulates NRT18 243
244
A previous study showed that increased NRT18 expression in nrt15 mutants (Chen et al 245
2012) and the expression of NRT15 was induced by NO3- (Lin et al 2008) We tested the 246
relationship between NRT15 and NRT18 expression in A thaliana and B napus No 247
significant differences were observed in AtNRT15 expression in roots between wild type 248
(col-0) and mutant plants (nrt18-2) (Fig 3A) However AtNRT15 expression was 249
significantly up-regulated by NO3- in roots of nrt18-2 mutants (Fig 3B) Expression of 250
AtNRT18 significantly increased in roots of nrt15-3 mutants but not in col-0 plants and 251
there were no significant differences in AtNRT18 expression with or without NO3- treatment 252
in roots of nrt15-3 mutants (Fig 3CD) 253
In contrast BnNRT15 expression in roots of the H genotype increased significantly with 254
NO3- treatment (Fig 3E) while the reverse was observed in expression of BnNRT18 which 255
showed lower expression under NO3- treatment than control (Fig 3F) This suggests that NO3
- 256
induces the expression of NRT15 but down-regulates NRT18 in A thaliana and B napus 257
Further studies are needed to elucidate the mechanisms regulating this reverse regulation 258
259
Reduced VSC of NO3- in roots drives its long-distance transport from roots to shoots 260
Our previous study showed that Bafi (Bafilomycin A1) inhibits V-ATPase DCCD 261
(DCCD + Na2SO3) inhibits V-PPase and a 11 combination of Bafi and DCCD (B+D) 262
inhibits both V-ATPase and V-PPase (Han et al 2015a) These inhibitors were used to 263
control activities of the tonoplast proton pumps in the H B napus plants V-ATPase activity 264
significantly decreased under Bafil and B+D treatments relative to the control and DCCD 265
treatments whereas V-PPase activity declined significantly under DCCD and B+D treatments 266
relative to that in the control and Bafil treatments (Fig 4A) 267
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The V-ATPase activity in roots of A thaliana was significantly lower in the V-ATPase 268
mutants (vha-a2 and vha-a3) than the wild type (col-0) and V-PPase mutants (avp1) (Fig 5A) 269
In contrast the V-PPase activity in roots of V-PPase mutant (avp1) were significantly lower 270
than in the wild type (col-0) and V-ATPase mutants (vha-a2 and vha-a3) (Fig 5A) 271
NO3- influxes into vacuoles of the H B napus plants significantly decreased when treated 272
with inhibitors of proton pumps (Bafil DCCD B+D) (Fig 4B) Similarly NO3- influxes into 273
the vacuole of the Arabidopsis mutants (vha-a2 vha-a3 and avp1) was significantly lower 274
than those found in the wild type (col-0) (Fig 5B) Previous studies showed that VSC of NO3- 275
decreased when the activities of the proton pumps decline (Li et al 2005 Krebs et al 2010 276
Han et al 2015a) 277
We further investigated NO3- distribution between the vacuole and cytosol as affected by 278
proton pump inhibition in the H genotype of B napus and the mutants of A thaliana 279
defective in vacuolar proton pumps The percentage of vacuolar NO3- relative to total NO3
- in 280
root protoplasts of the H genotype treated with inhibitors (Bafil DCCD B+D) was lower than 281
that observed in the control (Fig 4C) Results were also similar when using A thaliana 282
mutants where the percentage of vacuolar NO3- relative to the total NO3
- in protoplasts was 283
lower in the mutants (vha-a2 vha-a3 and avp1) than in the wild type (col-0) (Fig 5C) 284
Consequently NO3- accumulation in the cytosol showed a significant increase when energy 285
pumps were suppressed in roots of the H genotype (Fig 4D) and NO3- accumulation in the 286
cytosol of root tissues of A thaliana mutants (vha-a2 vha-a3 and avp1) similarly increased 287
compared with col-0 (Fig 5D) 288
Based on previous observations (Lin et al 2008) expression of NRT15 is strongly 289
induced by NO3- but expression of NRT18 is down-regulated (Fig3 Chen et al 2012) We 290
then hypothesized that expression of these two genes is contrastingly regulated by 291
concentration of NO3- in the cytosol Our results were congruent with this hypothesis the 292
expression of BnNRT15 in root tissues of the H genotype of B napus was significantly higher 293
in plants treated with Bafil DCCD or B+D compared with the control (Fig 4E) whereas the 294
expression of BnNRT18 decreased substantially (Fig 4F) Similar results were also observed 295
in A thaliana where expression of AtNRT15 in roots of the mutants (vha-a2 vha-a3 and 296
avp1) were significantly higher than in the wild type (col-0) but expression of AtNRT18 in 297
the same mutants were considerably lower (Fig 5EF) 298
NO3- concentrations in the xylem sap the N-distribution between shoot and root (SR 299
ratios based on [15N]) and the [NO3-] in shoots relative to roots of the H genotype treated with 300
energy pumpsrsquo inhibitors were significantly higher than in the control (Fig 4GH Fig S5A) 301
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11
Similar trends were also observed when using mutants of A thaliana deficient in the energy 302
pumpsrsquo activities NO3- concentration in the xylem sap the [15N] SR ratios and [NO3
-] SR 303
ratios were all significantly higher in the mutants than in the wild type (Fig 5GH Fig S5B) 304
These data clearly showed that prevention of N sequestration in vacuoles would enhance its 305
translocation to shoot 306
Additional evidence linking NO3- long-distance transport and NO3
- short-distance 307
distribution within cells were provided through experiments comparing A thaliana wild type 308
(Ws) and mutant clca-2 (Fig S6) The chloride channel (CLCa) is the main channel for 309
vacuolar anion accumulation Vacuolar sequestration capacity of NO3-significantly declines in 310
clca mutants (Angeli et al 2006) NO3- influx into the vacuolar space of Ws roots was 311
significantly higher than that observed in the clca-2 mutants (Fig S6A) This resulted in a 312
smaller proportion of vacuolar NO3- in clca-2 plants relative to the total NO3
- in root tissue 313
protoplasts as compared with Ws (Fig S6B) Consequently the accumulation of NO3- in the 314
cytosol was significantly higher in clca-2 than in Ws plants (Fig S6C) The higher 315
NO3-concentration in the cytosol together with the higher expression of AtNRT15 coupled 316
with lower expression of AtNRT18 in clca-2 roots (Fig S6DE) resulted in significant 317
increase in xylem sap NO3- concentration [15N] SR ratios and [NO3
-] SR ratios compared 318
with Ws plants (Fig S6FGH) 319
320
Increased NO3- translocation to shoots enhanced NUE 321
NO3- assimilation efficiency is known to be higher in shoots than in roots (Smirnoff et al 322
1985 Andrews et al 1986 Tang et al 2012 Tang et al 2013) We therefore hypothesize 323
that increased NO3- translocation to shoot and the consequent higher shootroot ratio will 324
contribute to higher NUE Our data support this hypothesis The H B napus genotype showed 325
enhanced long-distance transport of NO3- from roots to shoots (Fig 2 Fig S2) This enhanced 326
NO3- transport requires higher carbon skeleton provided through higher photosynthetic rate 327
(Tang et al 2012 Tang et al 2013) Our results showed that chlorophyll content 328
intercellular CO2 concentration and photosynthetic rate were significantly higher in the H than 329
the L genotype at both seedling and flowering stages (Table 2) Moreover the NO3- 330
assimilating enzymes were strongly induced by NO3- providing sufficient capacity for N 331
assimilation (Smirnoff et al 1985 Andrews et al 1986) Nitrate reductase (NR) and 332
glutamine synthetase (GS) activities in roots of the H genotype were significantly lower than 333
in the L genotype both at seedling and flowering stages (Fig S7) In contrast NR activities in 334
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12
shoots of the H genotype were significantly higher than in the L genotype at both stages (Fig 335
S7AB) and GS activity in the H genotype was also higher than those of the L genotype at 336
seedling stage (Fig S7C) 337
Experiments comparing A thaliana wild type (col-0 Ws) with mutants defective in 338
vacuolar proton-pumps (vha-a2 vha-a3avp1) and transport channel (clca-2) yielded similar 339
results that is increases in NO3- shootroot ratio essentially contributed to enhanced NUE 340
(Fig 6) The NO3- shootroot ratios in A thaliana mutants were higher than that in the wild 341
type (Fig 5GH) Generally the efficiency of inorganic N (NO3-) assimilation into organic N 342
is higher in shoots than in roots (Smirnoff et al 1985 Andrews et al 1986) therefore the 343
NO3- concentration in A thaliana mutants decreased (Fig 6CD) leading to higher NUE 344
compared with the wild type (Fig 6AB) 345
346
BnNRT15BnNRT18 in B napus affects NO3- long-distance transport from roots to 347
shoots 348
Both B napus and A thaliana are members of Cruciferae Family The amino acid 349
sequence identity between BnNRT15 and AtNRT15 was 90 (Fig S8A) the amino acid 350
sequence similarity between BnNRT18 and AtNRT18 was 908 (Fig S8B) Comparisons of 351
nucleotide and amino acid sequences (Harper et al2012) of these genes 352
(httpbrassicanbiacukcgi-binmicroarray_databasecgi) showed that BnNRT15 353
(EV220114) and BnNRT18 (EV116423) of B napus are respectively highly homologous 354
with AtNRT15 and AtNRT18 and the two genes are mainly expressed in roots of both species 355
showing similar organ-specificity (Lin et al 2008 Li et al 2010) 356
NO3- long-distance transport from roots to shoots is therefore regulated by NRT15 and 357
NRT18 as reported before (Lin et al 2008 Li et al 2010) The [15N-traced] SR ratios and 358
the [NO3-] SR ratios were significantly lower in nrt15-3 mutants relative to the wild type 359
(col-0) while [15N] SR and [NO3-] SR ratios showed significant increase in nrt18-2mutant 360
relative to col-0 (Fig S9CD) A previous study showed that expression of NRT15 is 361
down-regulated by ACC and MeJA treatments while the expression of NRT18 is strongly 362
up-regulated in A thaliana (Zhang et al 2014) Consequently NO3- accumulated in plant 363
roots probably as an adaptive measure for abiotic stresses (Chen et al 2012) Both the H- 364
and L- B napus genotypes showed significantly lower expression of BnNRT15 in roots when 365
treated with ACC (Fig S10A) but the expression of BnNRT18 remained higher than in the 366
control plants (Fig S10B) This resulted in significantly lower [15N] SR ratios following 367
ACC treatment (Fig S10CDE) These data indicate that BnNRT15 and BnNRT18 play 368
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13
similar functions for NO3- long-distance transport between root and shoot in both B napus 369
and A thaliana (Lin et al 2008 Li et al 2010) 370
371
372
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Discussion 373
Nitrate long-distance transport from root to shoot is regulated by cytosolic NO3- in roots 374
NO3- long-distance transport from root to shoot is controlled by NRT gene family For 375
instance xylem NO3- loading and unloading are mainly controlled by NRT15 and NRT18 376
respectively (Lin et al 2008 Li et al 2010) Whereas the NO3- short-distance transport 377
between the vacuole and cytosol is controlled by tonoplast proton-pumps (V-ATPase and 378
V-PPase) (Krebs et al 2010) and by activity of the CLCa channel in the tonoplast membrane 379
(Angeli et al 2006 Wege et al 2014) tonoplast proton-pumps provide the energy required 380
for NO3- accumulation into the vacuole through CLCa (Gaxiola et al 2001 Brux et al 2008 381
Krebs et al 2010) The expression of NRT15 is up-regulated by NO3- in plant culture 382
solution (Lin et al 2008) whereas regulation of NRT18 expression is contrary to that of 383
NRT15 under both control and abiotic stress conditions (Li et al 2010 Chen et al 2012 384
Zhang et al 2014) 385
Despite the progress made in characterizing the genes involved in transport and 386
metabolism of NO3- in plants the relationship between long- and short-distance transport and 387
how these two pathways are regulated have not been thoroughly investigated We hypothesize 388
that NRT15 expression and its role in long-distance transport is regulated by NO3- in plant 389
tissue as was observed in culture solution (Lin et al 2008) and our data support this 390
hypothesis Both BnNRT15 and AtNRT15 expression were up-regulated by increasing 391
cytosolic NO3- in roots (Fig 4 Fig 5) The increase in cytosolic NO3
- was achieved through (i) 392
use of A thaliana mutants defective in proton-pumps (V-ATPase and V-PPase) or CLCa 393
activities in the tonoplast and (ii) inhibition of B napus tonoplast proton-pumps In both 394
model plants the influx of NO3- into vacuoles was substantially reduced resulting in higher 395
concentrations of NO3- in the cytosol (Fig 4A-D Fig 5A-D Fig S6A-C) Under those 396
conditions expression of BnNRT15 and AtNRT15 was up-regulated by the enhanced 397
cytosolic NO3- while expression of BnNRT18 and AtNRT18 was down-regulated (Fig 4EF 398
Fig 5EF FigS6DE) As a consequence more NO3- was loaded into the xylem sap and 399
transported from root to shoot (Fig S5AB FigS6F) This is also reflected as increased 400
shootroot ratios of N traced by [15N] and [NO3-] (Fig 4GH Fig 5GH Fig S6GH) 401
Interestingly the expression of both BnNRT15 and AtNRT15 is up-regulated by 402
increased cytosolic NO3- concentration in roots but were not affected by NO3
- sequestered 403
into vacuoles (Fig 4 Fig 5 Fig S6) This is because vacuolar NO3- is functionally separated 404
by the tonoplast and cannot be assimilated by enzymes that are localized in the cytosol 405
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15
Therefore NO3--inducible genes such as NR and NRT15 were not influenced by vacuolar 406
NO3- concentration (Martinoia et al 1981 Martinoia et al 2000 Zhang et al 2012) In 407
addition NO3- concentration in the cytosol is controlled by its short-distance transport through 408
the tonoplast which is mediated through the tonoplast proton-pumps providing the required 409
energy for its sequestration into vacuoles against concentration gradient (Gaxiola et al 2001 410
Brux et al 2008 Krebs et al 2010) The gradient of H+ between the inside of the vacuole 411
and the cytosol is maintained by an 2NO3-1H+ antiporter mechanism that facilitatesNO3
- 412
transport through CLCa (Angeli et al 2006 Wege et al 2014) Our data provide clear 413
evidence that NO3- long-distance transport from root to shoot is regulated by cytosolic NO3
- 414
concentration in roots 415
Regulatory mechanisms that control the expression of NRT18 and NRT15 as mediated 416
by NO3- are still unknown however previous studies showed that their expression is also 417
affected by abiotic stresses whereby NRT15 is down-regulated and NRT18 is up-regulated 418
(Li et al 2010 Zhang et al 2014) AtNRT18 expression in A thaliana nrt15 mutants is 419
highly up-regulated (Chen et al 2012) as was also observed here (Fig 3C) Moreover 420
AtNRT15 expression was enhanced by NO3- in culture solution and was not affected in 421
nrt18-2 mutant (Fig 3AB) On the other hand AtNRT18 expression was not affected by 422
cytosolic NO3- but down-regulated when AtNRT15 expression was up-regulated by NO3
-(Fig 423
S6DE) Analogous results were also obtained with B napus genotypes (Fig 3EF) Based on 424
these results we argue that NRT18 expression is not directly influenced by cytosolic NO3- but 425
rather by NRT15 expression (Fig 4 Fig 5 and Fig S6) suggesting NRT18 is probably 426
acting downstream of NRT15 Apparently the regulatory mechanisms of this NRT gene 427
family still awaits further studies to be elucidated 428
429
Response of NUE to NO3- long-distance transport 430
NO3- assimilation efficiency is higher in shoot than in root tissues and can be further 431
enhanced by carbon assimilation (Smirnoff et al 1985 Tang et al 2013) Therefore 432
translocation of higher proportion of NO3- from roots to shoots likely contributes to better 433
crop growth and higher NUE (Andrews et al 1986 Tang et al 2012) The present study 434
provides direct evidence for these results using contrasting B napus genotype identified 435
before (Zhang et al 2009 Han et al 2015a) Our data confirmed the genetic variation in 436
NUE between these two genotypes (Table 1) 437
We measured NO3- distribution in root and shoot of these genotypes to elucidate the 438
physiological mechanisms contributing to variation in NUE The data were consistent with 439
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16
our hypothesis that concentration of NO3- in the xylem sap and shoot would be significantly 440
higher in the H genotype than in the L genotype (Fig 2EFFig S2EF Fig S4) The NO3- 441
long-distance transport from root to shoot was affected by cytosolic NO3- in roots of both A 442
thaliana and B napus genotypes (Fig 4-5 Fig S6) The activities of the tonoplast 443
proton-pumps in roots were significantly lower in the H genotype than in the L genotype (Fig 444
1AB) Consequently less NO3- was sequestered into the vacuole and more NO3
- was retained 445
in cytosol (Fig 1CD) BnNRT15 expression was then up-regulated resulting in greater 446
loading into the xylem while BnNRT18 was down-regulated (Fig 2AB) probably to 447
suppress NO3- unloading from xylem sap Consequently a higher proportion of NO3
- was 448
transported from root to shoots in the H genotype(Fig 2C-D Fig S4) 449
Moreover photosynthetic carbon fixation (Table 2) and activities of both NR and GS 450
were higher in shoots of the H genotype (Fig S7) both of which promote higher N 451
assimilation These results agreed with previous reports (Smirnoff et al 1985 Andrews et al 452
1986 Tang et al 2012 Tang et al 2013) where higher NO3- transport from root to shoot 453
higher photosynthetic rate and N assimilation efficiency in shoots were suggested as essential 454
mechanisms for high-NUE in crop plants These qualities endowed the B napus H genotype 455
Xiangyou15 with higher NUE as compared with the L genotype 814 456
NO3- long-distance transport in B napus is not only regulated by BnNRT15 and 457
BnNRT18 in root tissues (Fig 4 Fig 5 Fig S6) but also by stomatal conductance and 458
transpiration by indirectly controlling xylem sap flow and long-distance transport 459
(Dechorgnat et al 2011 Krapp et al 2014) Stomatal conductance and transpiration rates of 460
the H genotype were significantly higher than those of the L genotype (Table S1) a result 461
consistent with other studies describing characteristics of high-NUE plants (Daniel-Vedele et 462
al 1998 Wilkinson et al 2007 Dechorgnat et al 2011 Krapp et al 2014) 463
464
Response of NUE to NO3- short-distance transport 465
NUE is also strongly controlled by NO3- short-distance distribution between the vacuole 466
and cytosol (Han et al 2015a) A lower proportion of NO3- accumulating in vacuoles and a 467
higher proportion retained in the cytosol will contribute to better NUE in crop plants (Han et 468
al 2015b) The energy required for vacuolar NO3- sequestration is provided by the tonoplast 469
proton-pumps (Gaxiola et al 2001 Brux et al 2008 Krebs et al 2010) while the 470
distribution channel CLCa enables transport of NO3- across the vacuolar membrane (Angeli et 471
al 2006 Wege et al 2014) The activities of the tonoplast proton-pumps (V-ATPase and 472
V-PPase) in leaves of the H genotype were significantly lower than those of the L genotype at 473
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17
flowering stages (Han et al 2015aHan et al 2015b) thus we expected similar differences in 474
root tissues Our results confirmed that the activities of tonoplast proton-pumps in root tissues 475
of B napus H genotype and A thaliana mutants were lower than those in the L genotype and 476
A thaliana wild type leading to less NO3- influx into vacuoles and more in the cytosol (Fig 1 477
Fig 5A-D) Moreover the enzymes catalyzing N assimilation are located in the cytoplasm 478
where NO3- could be assimilated (Martinoia et al 1981 Han et al 2015a) or immediately 479
loaded into xylem sap for long-distance transport from roots to shoots (Lin et al 2008) 480
Consequently the NO3- concentration in root tissues of the H genotype and A thaliana 481
mutants defective in tonoplast proton-pumps was significantly lower than that in the L 482
genotype (Fig 2E) and in A thaliana wild type (Fig 6C) Therefore the higher proportion of 483
NO3- transported to shoot and the lower concentrations retained in root vacuoles contributed 484
to the higher NUE in Xiangyou15 and A thaliana mutants (vha-a2 vha-a3 and avp1) (Table 485
1Fig 6A) Further studies are needed to assess the impacts of regulating vacuolar 486
sequestration of NO3- and rates of long-distance transport for enhancing NUE in other crop 487
species and genotypes 488
These data suggested that NO3- distribution between vacuoles and cytosol within cells 489
regulates NO3- long-distance transport from root to shoot and ultimately affects NUE NO3
- 490
loading into the xylem sap is an active transport process (Lin et al 2008) therefore not 491
regulated by absolute concentration of NO3- in the cytosol but rather by genes involved in 492
NO3- long-distance transport (NRT15 and NRT18) Apparently the expression of NRT15 the 493
gene responsible for xylem loading is regulated by NO3-concentration in the cytosol 494
Therefore both NO3- short-distance transport across the tonoplast and its long-distance 495
transport from root to shoot are important determinants of NUE in both B napus and A 496
thaliana (Table 1 and Fig 6) 497
498
Roles of NRT15 and NRT18 in NO3- long-distance transport in B napus and A 499
thaliana 500
Our results (Figs S9 S10) showed that the proportion of NO3- distributed from root to 501
shoot is strongly controlled by NRT15 in accord with previous studies (Lin et al 2008 Li et 502
al 2010) The data suggest that the high-NUE genotypes possess lower activities of tonoplast 503
proton-pumps (V-ATPase and V-PPase) resulting in less NO3- accumulation in vacuoles and 504
more NO3- retention in cytosol (Fig 7) The higher NO3
- in the cytosol then up-regulated 505
NRT15 which then down-regulates NRT18 As a result more NO3- is loaded into the xylem 506
system by NRT15 mechanism and less NO3- is unloaded via NRT18 (Fig 7) Differential 507
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18
regulation of these NRT genes in high-NUE plants increased transport of NO3- from root to 508
shoot through the xylem vascular tissues (Fig 7) This coordinated NO3- long- and 509
short-distance transport likely fine-tunes NO3- allocation and modulates the balance of NO3
- 510
distribution between roots and shoots (Fig 7) 511
Our research also revealed differences between A thaliana and B napus in the 512
expression of the wild-type NRT genes in response to NO3- in the protoplast Moreover using 513
A thaliananrt15-3 mutants we uncovered possible regulatory interactions between a 514
functioning (wild-type) NRT15 gene and NRT18 expression in response to NO3- 515
concentration Because mutants defective in nrt15 are not available for B napus we could 516
not confirm that the respective NRT15 and NRT18 systems behave in an analogous way in 517
these plant species Based on Harper et al (2012) research database our homology 518
comparisons and gene function annotations provided strong evidence for the roles of 519
BnNRT15 and BnNRT18 in B napus Finally our studies were conducted using two model 520
dicotyledonous plant species for which we had ample data on responses to specific inhibitors 521
(B napus) or specific mutants were available with results forming the bases for the model 522
presented in Figure 7 Further research using genetically diverse plant species (eg 523
monocotyledonous crops such as rice wheat and corn) will help validate this model and its 524
broader application across species The information generated in this study could have future 525
application for improving NUE in commercial crops with consequent reduction in nitrogen 526
use and benefits to the environment 527
528
Materials and Methods 529
Plant material 530
The two oilseed rape (Bnapus) cultivars used in this study have been characterized 531
before as high- (Xiangyou15 referred to as H genotype hereafter) and low- (814 referred to 532
as L genotype) nitrogen use efficiency (NUE) genotypes (Zhang et al 2009 Han et al 2015b) 533
Here we define NUE as the total biomass per unit of N uptake by the plant with total 534
biomass includes roots shoots and grains The two genotypes were provided by the Hunan 535
Sub-Center of Improvement Center of National Oil Crops Hunan China 536
The Arabidopsis thaliana wild-type Columbia-0 (col-0) was used as control for 537
V-ATPase (vha-a2 and vha-a3) V-PPase (avp1) nrt15-3 and nrt18-2 mutants whereas the A 538
thaliana wild-type Wassilewskija (Ws) was used as control for clca-2 mutants A thaliana 539
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19
mutants and transgenic lines (nrt15-3 and nrt18-2) have been described previously (Chen et 540
al 2012 Zhang et al 2014) The mutant lines vha-a2 (Salk_142642) and vha-a3 541
(Salk_122135) described by Krebs et al (2010) were obtained from the Arabidopsis 542
Biological Resources Center (ABRC) The mutant line avp1 (GK-005004) described in Li et 543
al (2005) was obtained from the European Arabidopsis Stock Centre while the mutant line 544
clca-2 (FST 171A06) described in Angeli et al (2006) was from the Institute National de la 545
Recherche Agronomique (INRA) collection in France together with the T-DNA mutants used 546
for genotyping to select pure mutant lines 547
548
Growth conditions 549
B napus plants were grown hydroponically in ceramic pots(20cmtimes15cm) filled with a 550
nutrient solution in the greenhouse under natural light as described in Han et al (2015b) The 551
pots were arranged in a completely randomized design with six biological replications The 552
nutrient solution was replaced every 3 days and its pH adjusted to 55 daily Experiments were 553
conducted at the field station of Hunan Agricultural University Southern China 554
A thaliana plants were grown in a nutrient solution in plastic pots as described in Arteca 555
et al (2000) and Gong et al (2003) The solution was changed every 3 days with pH adjusted 556
daily to 58 Pots were arranged in a completely randomized design with six biological 557
replications The nutrient solution used for both species consisted of 125mM KNO3 558
0625mM KH2PO4 05mM MgSO4 05mM Ca (NO3)2middot4H2O 0025mM Fe-EDTA 025 559
mlL-1 micronutrients (stock solution concentrations 70mM B 14mM Mn 1mM Zn 05mM 560
Cu and 02mM Mo) The experiments were conducted at Hunan Agricultural University in a 561
phytotron set at 70 relative humidity16h-light8h-dark cycle and constant temperature of 562
22degC 563
564
Experimental treatments and phenotyping 565
All measurements made on B napus plants were conducted either at the seedling stage 566
(2 months after transplanting) or at flowering (5 months after transplanting) The whole root 567
tissue was harvested and used for analyses at either stage For shoot measurements the fourth 568
leaf from the bottom was used for measurements made at seedling stage whereas the 12th leaf 569
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
from the bottom was harvested for measurements made at flowering stage For A thaliana 570
whole root and shoot of four-week-old plants were sampled and used for different assays 571
572
Vacuolar proton-pumps inhibitor treatments 573
Inhibition of vacuolar proton pumps of B napus (Xiangyou15) was conducted at the seedling 574
stage in hydroponic culture as described in Han et al (2015a) with minor modifications 575
Plants grown in hydroponic solution were used as control Inhibitor treatment was conducted 576
using several chemicals Bafi(25 nmol L-1 Bafilomycin A1) an inhibitor of V-ATPase 577
DCCD(10 μmol L-1DCCD + 50 mmolL-1 Na2SO3) specifically inhibits V-PPase and 578
Bafi+DCCD in a 11 ratio as inhibitor of both V-ATPase and V-PPase The inhibitors were 579
applied in the hydroponic solutions for 24 h Fresh plant tissue samples from all treatments 580
were collected for different assays 581
582
NO3- induced gene expression 583
Four-week old A thaliana mutants (nrt15-3nrt18-2) were grown in hydroponics with 584
225mM (NH4)2succinate for 3 days and shifted to hydroponics with 45mM NO3- for 12 h 585
then root tissues were collected to assess relative expression of AtNRT15 and AtNRT18 586
genes Seedlings of B napus (Xiangyou15) plants grown hydroponically were treated with 587
5mM (NH4)2succinate for 3 d and shifted to fresh hydroponic solution containing 15 mM 588
NO3- for 12 h Root tissue was then collected to assess the relative expression of BnNRT15 589
and BnNRT18 genes using quantitative RT-PCR 590
591
ACC treatment 592
Hydroponically grown seedlings of B napus were subjected to ACC treatment as described in 593
Zhang et al (2014) with minor modifications Seedlings of the two genotypes Xiangyou15 594
and 814 were transferred to a nutrient solution containing 002mM L-1 ACC for 6 h or grown 595
in normal hydroponic solution as control Root tissues were then collected to assay relative 596
expression of BnNRT15 and BnNRT18 using quantitative RT-PCR Another set of B napus 597
plants were grown in ACC-containing hydroponic solution but supplemented with 15NO3- (22 598
15N) replacing the unlabeled NO3- for 1 h Root and shoot tissues were collected separately 599
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21
for 15N measurement using a continuous-flow isotope ratio mass spectrometer coupled with a 600
C-N elemental analyzer (ANCA-MS PDZ Europa) 601
602
Biomass and N concentration 603
Grain yield dry biomass and N concentration were determined in plant samples at 604
harvest Plants were harvested (including senescing leaves) then dried in an oven for 30 min 605
at 105degC then at 70degC to a constant weight and weighed N concentration was determined 606
using the Kjeldahl method (Shi et al 2010 Han et al 2015b)02g of dried samples were 607
digested with H2SO4-H2O2 in a 100-ml Kjeldahl digestion flask after which N concentration 608
was determined using a Foss Auto Analyser Unit (Kjeldahl 8400) 609
610
V-ATPase and V-PPase activities 611
Roots (10 g) of B napus were collected at seedling and flowering stages and that of A 612
thaliana (05 g) sampled at seedling stage then used for determining the activities of 613
V-ATPase and V-PPase (Han et al 2015b) Activities of these two energy pumps within 614
microsomal membranes were followed colorimetrically based on released Pi as described by 615
Zhu et al (2001) and Krebs et al (2010) 616
617
Assay of NO3- flux in vacuoles 618
Roots of both species were collected similarly as that used for V-ATPase and V-PPase 619
activities then used for vacuole isolation and measurement of NO3- flux in vacuoles as 620
described by Robert et al (2007) with minor modification (Han et al 2015b) Net fluxes of 621
NO3- in vacuoles were measured non-invasively using SIET (scanning ion-selective electrode 622
technique SIET system BIO-003A Younger USA Science and Technology Corporation 623
USA) An NO3--selective microelectrode used for the assay of NO3
- flux was vibrated in the 624
measuring solution between two positions 1 μm and 11 μm from the vacuole surface 625
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles Background 626
signal was recorded by vibrating the electrode in the measuring solution in the absence of 627
vacuoles Ion flux was calculated by Fickrsquos law of diffusion J = -D(dcdx) where J represents 628
the ion flux (picomolescm-2s-1) dcdx is the ion concentration gradient and D is the ion 629
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
diffusion constant in a particular medium The direction of the flux was derived from Fickrsquos 630
law of diffusion that relates the concentration gradient 631
632
Isolation of intact protoplasts and vacuoles for determining NO3- concentration 633
Similar to the above assays root tissues (10 g) were collected from B napus at seedling 634
and flowering stages and from A thaliana (05 g) seedlings and used to isolate intact 635
protoplasts and vacuoles as described by Robert et al (2007) with minor modifications as 636
outlined in Huang et al (2012) and Han et al(2015b) The purified protoplasts were divided 637
into two equal aliquots with one of them used for isolation of vacuoles The purified 638
protoplasts and vacuoles were then sub-sampled and used to determine NO3- concentrations 639
(Vogeli-Lange and Wagner 1990) and for enzyme activity assays (Ma et al 2005) 640
NO3-concentrations in protoplasts and vacuoles were measured by a continuous-flow 641
auto-analyzer (Auto Analyser 3 Bran and Luebbe Germany) as described previously (Han et 642
al 2015b)The activities of acid phosphatase (ACP) and cytochrome oxidase (COX) were 643
determined using plant ACP colorimetry- and COX-assay kits (GenMedSci Inc) following 644
the manufacturerrsquos instructions ACP activity specific to vacuoles was determined and used to 645
normalize NO3- accumulation We measured NO3
- in the protoplast outside the vacuole which 646
includes the cytosol and organelles eg mitochondria and Golgi Apparatus (Robert et 647
al2007) Since most of the NO3- in the protoplast outside the vacuole is located in the cytosol 648
(Krebs et al 2010) we refer to NO3- distribution between vacuoles and cytosol rather than 649
vacuole versus protoplast 650
651
Quantitative RT-PCR 652
Total RNA extracted from B napus roots was prepared using Trizol reagent (Invitrogen) 653
cDNAs were synthesized using M-MLV reverse transcriptase (Promega) following 654
manufacturerrsquos protocol The relative expression of BnNRT15 (EV220114) BnNRT18 655
(EV116423) and Bnactin (AF1118121) genes in plant roots were determined by quantitative 656
RT-PCR and run on a LightCycler instrument (Roche) with the SYBR Green Real-Time PCR 657
Master Mix Kit (TOYOBO Japan) under the following conditions 95degC for 2min then 45 658
cycles of 95degC for 10s 60degC for 10s and 72degC for 20s The primer sequences of BnNRT15 659
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
BnNRT18 and Bnactin genes were obtained from httpbrassicanbiacuk as described in 660
Harper et al (2012) and expression levels were normalized to Bnactin as control 661
Total RNA extracted from the 4-week-old A thaliana roots under the treatments 662
conditions described above were prepared using Trizol reagent (Invitrogen) The cDNAs were 663
synthesized using M-MLV reverse transcriptase (Promega) based on the manufacturerrsquos 664
protocol and as described in Li et al (2010)The primer sequences of quantitative RT-PCR 665
used in these assays are listed in Supplemental Table 2 and were described previously (Chen 666
et al 2012 Zhang et al 2014) and their expression was normalized to Atactin2 as control 667
668
Determination of nitrate distribution using 15NO3- 669
Hydroponically grown B napus (seedling and flowering stages) and A thaliana 670
(seedling) were transferred to 01mM CaSO4 solution for 1min then to their respective 671
hydroponic nutrient solutions with15NO3- (22 excess) replacing unlabeled NO3
- for 1h 672
Plants were then transferred to 01mM CaSO4 solution for 1min after which roots were 673
washed with deionized water Root and shoot samples were separated and dried at 105degC for 674
30min followed by 70degC for 3 d then grounded and used for assaying 15N content using a 675
continuous-flow isotope ratio mass spectrometer coupled with a carbon-nitrogen elemental 676
analyzer (ANCA-MS PDZ Europa) 677
678
Xylem sap collection and assay of nitrate concentration 679
We used the method of Tang et al (2012) for collecting xylem sap Plants were cut 680
leaving1cm segments with intact roots at seedling and flowering stages for B napus and at 681
seedling stage for A thaliana Roots were immediately immersed in their respective nutrition 682
solutions Weighed cotton was put on the cut surface to absorb extruding xylem sap for 1 h 683
The cotton was then wrapped in plastic film and the volume of xylem sap was calculated as 684
the weight gain of the cotton Xylem sap was then squeezed from the cotton using a syringe 685
and used for subsequent assay of nitrate concentration using a continuous-flow auto-analyzer 686
(Auto Analyser 3 Bran and Luebbe Germany) 687
To determine nitrate concentration samples (10 g fresh root and shoot at seedling and 688
flowering of B napus and 05 g fresh root and shoot of 4-week-old A thaliana) were frozen 689
in liquid N2 and ground with a mortar and pestle the powder was then transferred to a beaker 690
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24
containing 70 ml deionized water and boiled for 30 min to extract nitrate then cooled and 691
made to 100 ml volume and 02 g activated carbon added to eliminate the effect of 692
chlorophyll The mixture was filtered and nitrate in the filtrate was determined using a 693
continuous-flow auto-analyser (Atuo-Analyser 3 Bran and Luebbe Germany) 694
695
Measurements of photosynthesis 696
All measurements of photosynthesis and related parameters were conducted at 1000 h 697
using intact leaves The 4th leaf from the bottom in B napus at seedling stage and the 12th leaf 698
from the bottom at flowering stage were used for determining chlorophyll concentration 699
photosynthetic rate intercellular CO2 concentration transpiration rate and stomatal 700
conductance Chlorophyll concentration was determined by SPAD-502 (Minolta Camera Co 701
Ltd Japan) (Luo et al 2006 Yandeau-Nelson et al 2011) Photosynthetic rate intercellular 702
CO2 concentration transpiration rate and stomatal conductance were measured using LI-6400 703
Portable Photosynthesis System (Li-Cor Biosciences Co Ltd USA) set at flow rate of 500 704
μmol s-1 photosynthetic photon flux density of 1000 μmol m-2 s-1 relative humidity of 65 705
CO2 concentration of 400ppm and ambient temperatures 706
707
NR and GS activities 708
Leaves with similar age as those used for photosynthesis in B napus were used for the 709
assay of nitrate reductase (NR) and glutamine synthetase (GS) activities NR activity was 710
measured by the modified method of Fan et al (2007) Fresh samples (05g) were frozen in 711
liquid N2 ground into powder in the presence of acid-washed sand and homogenized with 4 712
ml of extraction buffer (0025mol L-1phosphate buffer pH87 1211g L-1 cysteine 0372g L-1 713
EDTA) Homogenates were centrifuged at 30000timesg for 15 min at 4degC and the supernatants 714
were treated with sulfanilamide and α-naphthylamine reagents for colorimetric (540nm) 715
determination of nitrite as described by Fan et al (2007) 716
GS activity was determined using a modified reverseγ-glutamyltransferase method 717
(Wang et al2014) which measures the GS-catalyzed formation of glutamyl-γ-hydroxamate 718
from glutamine and hydroxylamine Fresh samples were frozen for 30 min at -20degC then 719
ground in 10ml Tris-HCl buffer and acid-washed sand The homogenates were filtered 720
through two layers of gauze and centrifuged at 8000timesg for 15 min at 4degC About 12ml of the 721
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
supernatant was added in a reaction mixture and treated as described (Wang et al2014) After 722
the GS-catalyzed reaction was stopped and the solution centrifuged glutamyl-γ723
-hydroxamate was quantified colorimetrically in the supernatant (at 485 nm) and the 724
concentration was determined using a standard curve 725
726
Statistical analyses 727
We used the SPSS software (Statistical Product and Service Solutions V130 USA) for 728
ANOVA and mean separation of main-effects and interactions using Duncanrsquos multiple range 729
test at Plt005Values are presented as means and SD of three or six replicates from three 730
independent experiments Different letters or an asterisk () associated with specific data (eg 731
at the top of histogram bars in figures or within tables) denote significant differences at 732
Plt005 733
734
Supplemental Data 735
Supplemental Figure 1 B napus with higher NUE showed lower vacuolar sequestration 736
capacity (VSC) for NO3-in roots at flowering stage 737
Supplemental Figure 2 B napus with higher NUE showed enhanced long-distance transport 738
of NO3- from roots to shoots at flowering stage 739
Supplemental Figure 3 The two B napus (H and L genotypes) showed the same total N per 740
plant at seedling stage (A) and flowering stage (B) 741
Supplemental Figure 4 B napus with higher NUE showed increased NO3- concentration in 742
the xylem sap at seedling and flowering stages 743
Supplemental Figure 5 NO3- concentration in the xylem sap as affected by inhibitor 744
treatments in B napus and in the energy pumpsrsquo mutants of A thaliana (col-0 vha-a2 vha-a3 745
avp1) 746
Supplemental Figure 6 Reduced VSC for NO3- in roots drives long-distance transport of 747
NO3- from roots to shoots in the A thaliana wild type (Ws) and mutant (clca-2) 748
Supplemental Figure 7 Differences of NR and GS activities between the two B napus (H 749
and L genotypes) at seedling and flowering stages 750
Supplemental Figure 8 Amino acid sequences of BnNRT15 and BnNRT18 751
Supplemental Figure 9 Functions of AtNRT15 and AtNRT18 genes in root tissues of A 752
thaliana in controlling NO3- long-distance transport from root to shoots 753
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
Supplemental Figure 10 B napus BnNRT15 and BnNRT18 genes in roots were 754
coordinately modulated to facilitate NO3- long distance transport from roots to shoots 755
Supplemental Table 1 Differences in stomatal conductance and transpiration rate between 756
the two B napus genotypes 757
Supplemental Table 2 Sequences of primera used for qRT-PCR 758
759
Acknowledgements 760
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 761
Shanghai Institutes for Biological Sciences) for providing nrt15-3 and nrt18-2 seeds 762
This study was supported by the National Natural Science Foundation of China (Grant 763
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 764
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 765
The Ministry of Education in China Open Novel Science Foundation of Hunan province 766
(13K062) the National Key Laboratory of Plant Molecular Genetics and the Twelfth 767
Five-Year National Science and technology support program (2012BAD15BO4) 768
769
Figure 1 B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 770
NO3- in roots at the seedling stage 771
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 772
genotype 814 Specific activities of the tonoplast proton pumps are expressed as μmol Pi 773
released mg-1 protein h-1 NO3- fluxes are expressed as pmol NO3
- cm-2 S-1 Mature vacuoles 774
were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 775
measuring solution between the two positions 1 μm and 11 μm from the vacuole surface 776
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles recording the 777
stable reading data The background was recorded by vibrating the electrode in measuring 778
solution without vacuoles 779
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 780
for NO3- content and NO3
- accumulation normalized against the specific activity of the 781
vacuole acid phosphatase (ACP) as described in Materials and Methods and plotted as μmol 782
NO3- per μmol p-nitrophenol the end product of ACP assay 783
Proton pump activities in root tissues between H and L genotypes are shown at the seedling 784
stage (A) Different letters at the top of the histogram bars denote significant differences of 785
V-ATPase in root tissues between H and L genotypes (Plt005) an asterisk () at the top of the 786
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
histogram bars indicates significant difference in V-PPase activity in root tissues between H 787
and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788
Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789
genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790
bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791
bars on the figures indicate SD (n=6) 792
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793
seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794
to the total NO3- in protoplasts 795
NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796
total NO3-in the cytosol and was calculated as total NO3
- in protoplasts ndash total NO3- in 797
vacuole Different letters at the top of histogram bars denote significant differences of total 798
NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799
(n=6) 800
801
Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802
roots to shoots at the seedling stage 803
H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804
Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805
the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806
as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807
the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808
the histogram bars denote significant differences at Plt005 809
Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810
in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811
genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812
of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813
SD (n=3) different letters at the top of the histogram bars denote significant differences at 814
Plt005 level 815
The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816
at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817
genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818
different letters at the top of the histogram bars denote significant differences at Plt005 819
820
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
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Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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- Parsed Citations
- Reviewer PDF
- ONE
- TWO
- THREE
- FOUR
- FIVE
- SIX
- SEVEN
- T-ONE
- T-TWO
- Parsed Citations
-
3
67
Abstract 68
Enhancing nitrogen use efficiency (NUE) in crop plants is an important breeding target 69
to reduce excessive use of chemical fertilizers with substantial benefits to farmers and the 70
environment In Arabidopsis thaliana allocation of more NO3- to shoots was associated with 71
higher NUE however the commonality of this process across plant species have not been 72
sufficiently studied Two Brassica napus genotypes were identified with high and low NUE 73
We found that activities of V-ATPase and V-PPase the two tonoplast proton-pumps were 74
significantly lower in roots of the high-NUE genotype (Xiangyou15) than in the low-NUE 75
genotype (814) and consequently less vacuolar NO3- was retained in roots of Xiangyou15 76
Moreover NO3- concentration in xylem sap [15N] shootroot (SR) and [NO3
-] SR ratios 77
were significantly higher in Xiangyou15 BnNRT15 expression was higher in roots of 78
Xiangyou15 compared with 814 while BnNRT18 expression was lower In both B napus 79
treated with proton pump inhibitors or Arabidopsis mutants impaired in proton pump activity 80
vacuolar sequestration capacity (VSC) of NO3- in roots substantially decreased Expression of 81
NRT15 was up-regulated but NRT18 was down-regulated driving greater NO3- 82
long-distance transport from roots to shoots NUE in Arabidopsis mutants impaired in proton 83
pumps was also significantly higher than in the wild type col-0 Taken together these data 84
suggest that decrease in VSC of NO3- in roots will enhance transport to shoot and essentially 85
contribute to higher NUE by promoting NO3- allocation to aerial parts likely through 86
coordinated regulation of NRT15 and NRT18 87
88
Key words Nitrogen use efficiency Nitrate allocation Proton-pumps Vacuoles 89
90
91
92
93
94
95
96
97
98
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4
Introduction 99
China is the largest consumer of nitrogen (N) fertilizer in the world however the 100
average N-fertilizer use efficiency (NUE) is only around 35 suggesting considerable 101
potential for improvements (Shen et al 2003 Wang et al 2014) With the high amounts of 102
N-fertilizer being used crop yields are declining in some areas where application is 103
exceeding the optimum required for local field crops (Shen et al 2003 Miller et al 2008Xu 104
et al 2012) The extremely low NUE results in waste of resources and environmental 105
contamination and also presents serious hazards for human health (Xu et al 2012 Chen et 106
al 2014) Consequently exploiting the maximum potential for improving NUE in crop plants 107
will have practical significance for agriculture production and the environment (Zhang et al 108
2010 Schroeder et al 2013 Wang et al 2014) Elucidating the genetic and physiological 109
regulatory mechanisms governing NUE in plants will allow breeding crops and varieties with 110
higher NUE 111
Ammonium (NH4+) and nitrate (NO3
-) are the main N species absorbed and utilized by 112
crops and NO3- accumulation and utilization are of major emphasis for N nutrient studies in 113
dry land crops such as Brassica napus Several studies revealed the close relationship 114
between NO3- content and NUE in plant tissues (Shen et al 2003 Zhang et al 2012 Tang et 115
al 2013Han et al 2015a)When plants are sufficiently illuminated NO3- assimilation 116
efficiency significantly increase in shoots compared with roots (Smirnoff et al 1985 Tang et 117
al 2013) Consequently under daytime with optimal illumination higher proportion of NO3- 118
in plant tissue is transported from root to shoot as an advantageous physiological adaptation 119
that reduces the cost of energy for metabolism (Tang et al 2013) NO3- assimilation in plant 120
shoots can therefore take advantage of solar energy while improving NUE (Smirnoff et al 121
1985 Andrews et al 1986 Tang et al 2012 Tang et al 2013) 122
The NO3- long-distance transport and distribution between root and shoot is regulated by 123
two genes encoding long transport mechanisms NRT15 is responsible for xylem NO3- 124
loading while NRT18 is responsible for xylem NO3- unloading (Lin et al 2008 Li et al 125
2010) Expression of the two genes is influenced by NO3- concentration NRT15 is strongly 126
induced by NO3- (Lin et al 2008) while NRT18 expression is extremely up-regulated in 127
nrt15 mutants (Chen et al 2012) A negative correlation between the extents of expression of 128
the two genes was observed when plants are subjected to abiotic stresses (Chen et al 2012) 129
Moreover expression of NRT15 is strongly inhibited by 1-aminocyclopropane-1-carboxylic 130
acid (ACC) and methyl jasmonate (MeJA) whereas the expression of NRT18 is significantly 131
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5
up-regulated (Zhang et al 2014) Based on these studies we argue that the expression and 132
functioning of NO3- long-distance transport genes NRT15 and NRT18 are regulated by 133
cytosolic NO3- concentration In addition the vacuolar and cytosolic NO3
- distribution is 134
likely regulated by proton-pumps located within the tonoplast (V-ATPase and V-PPase) 135
(Granstedt et al 1982 Glass et al 2002 Krebs et al 2010) Therefore NO3- use efficiency 136
must be affected by NO3- long-distant transport (between shoot and root) and short-distant 137
transport (between vacuole and cytosol) However the physiological mechanisms controlling 138
this regulation are still obscure 139
Previous studies showed that the chloride channel protein (CLCa) is mainly responsible 140
for vacuole NO3- short-distance transport as it is the main channel for NO3
-movement 141
between the vacuoles and cytosol (Angeli et al 2006 Wege et al 2014) The vacuole 142
proton-pumps (V-ATPase and V-PPase) located in the tonoplast supply energy for active 143
transport of NO3- and accumulation within the vacuole (Gaxiola et al 2001 Brux et al 2008 144
Krebs et al 2010) Despite that about 90 of the volume of mature plant cells is occupied by 145
vacuoles vacuolar NO3- cannot be efficiently assimilated because the enzyme nitrate 146
reductase (NR) is cytosolic (Shen et al 2003 Han et al 2015a) However re-translocation of 147
NO3- from the vacuole to the cytosol will permit its immediate assimilation and utilization 148
Generally NO3- concentrations in plant cell vacuoles and the cytoplasm are in the range 149
of 30-50 mol m-3 and 3-5 mol m-3 respectively (Martinoia et al 1981 Martinoia et al 2000) 150
Because vacuoles are obviously the organelle for high NO3- accumulation and storage in plant 151
tissues their function in NO3- use efficiency cannot be ignored (Martinoia et al 1981 Zhang 152
et al 2012 Han et al 2015b) NO3- assimilatory system in the cytoplasm is sufficient for its 153
assimilation when it is transported out of the vacuoles Therefore NO3- use efficiency could in 154
part be dependent on vacuolar-cytosolic NO3- short-distance transport in plant tissues 155
(Martinoia et al 1981 Shen et al 2003 Zhang et al 2012 Han et al 2015a) 156
Evidently NO3- use efficiency is regulated by both NO3
- long-distance transport from 157
root to shoot and short-distance transport and distribution between vacuoles and cytoplasm 158
within cells(Glass et al 2002 Dechorgnat et al 2011 Han et al 2015a) Although vacuoles 159
compartment excess NO3- that accumulates in plant cells (Granstedt et al 1982 Krebs et al 160
2010) neither NO3- inducible NR genes (NIA1and NIA2) (Fan et al 2007 Han et al 2015a) 161
nor the NO3- long-distance transport gene NRT15 (Lin et al 2008) are regulated by vacuolar 162
NO3- even though they are essential for NO3
- assimilation Only NO3- transported from the 163
vacuole to the cytosol can play a role in regulating NO3- inducible genes Consequently we 164
argue that both NO3- assimilation in cells and its long-distance transport from root to shoot are 165
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6
regulated by cytosolic NO3- concentration However this hypothesis needs to be substantiated 166
The mechanisms underlying both NO3- short-distance (Gaxiola et al 2001 Angeli et al 167
2006 Brux et al 2008 Krebs et al 2010) and long-distance transport (Lin et al 2008 Li et 168
al 2010) have been previously investigated yet the underlying mechanisms regulating the 169
flux of NO3- and the obvious relationship between the two transport pathways as well as their 170
relation to NUE are not well understood 171
The NRT family of genes play a partial role in vacuolar NO3- accumulation in petioles 172
(Chiu et al 2004) and seed tissues (Chopin et al 2007) whereas the proton pumps and 173
CLCa system in the tonoplast play a major role in accumulating NO3- in vacuoles (Gaxiola et 174
al 2001 Angeli et al 2006 Brux et al 2008 Krebs et al 2010) The vacuolar NO3- 175
short-distance transport system is spread throughout the plant tissues and is the principal 176
means by which vacuolar NO3- short-distance transport and distribution is controlled (Angeli 177
et al 2006Krebs et al 2010) 178
The NRT genes seem to work synergistically to control NO3- long-distance transport 179
between roots and shoots NRT19 is responsible for NO3- loading into the phloem (Wang et 180
al 2011) whereas NO3- loading and unloading into xylem are regulated by NRT15 and 181
NRT18 respectively (Lin et al 2008 Li et al 2010) Phloem transport mainly involves 182
organic N the inorganic-N (NO3-) concentrations in the phloem sap are typically very low 183
ranging from one-tenth to one-hundredth of that of the inorganic-N in xylem sap (Lin et al 184
2008Fan et al 2009) Therefore this study focused on NO3- short-distance transport 185
mediated through the tonoplast proton-pumps and the CLCa system and the long-distant 186
transport mechanisms responsible for xylem NO3- loading and unloading via NRT15 and 187
NRT18 respectively 188
Questions related to how long and short-distance transport of NO3- are coupled in plant 189
tissues and their role in determining NUE were addressed using a pair of high- and low-NUE 190
B napus genotypes and Arabidopsis thaliana Application of proton pump inhibitors and 191
ACC in the former and use of mutants with defective proton pumps in the latter allowed 192
experimental distinction of the physiological mechanisms regulating these processes Data 193
presented here provide strong evidence from both model plants supporting this linkage and 194
strongly suggest that cytosolic NO3- concentration in roots regulates NO3
- long-distance 195
transport from roots to shoots We also investigated how NO3- concentration in plant tissues 196
would be affected by NO3- long-distance transport vacuolar NO3
- sequestration and the 197
ensuing relationship with NO3- use efficiency We also proposed the physiological 198
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7
mechanisms likely to be important for enhancing NO3- use efficiency in plants These findings 199
will provide scientific rationales for improving NUE in important industrial and food crops 200
201
202
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8
Results 203
B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for NO3- 204
in root tissues 205
Our previous work identified high and low NUE B napus genotypes (Han et al 2015a 206
Han et al 2015b) NUE of B napus whether based on biomass or on grain yield (Table 1) 207
was significantly higher for the high-NUE genotype (H Xiangyou15) than for the low-NUE 208
genotype (L 814) That the activities of the tonoplast proton-pumps (V-ATPase and V-PPase) 209
of root tissues in the H genotype were lower than the L genotype at both seedling (Fig 1A) 210
and flowering stages (Fig S1A) Given that proton pumps in the tonoplast supply energy for 211
vacuolar NO3- accumulation (Krebs et al 2010) NO3
- influx into the vacuole was 212
consequently much slower in the H genotype compared with the L genotype at both seedling 213
(Fig 1B) and flowering stages (Fig S1B) Moreover the percentage of vacuolar NO3- relative 214
to the total NO3- in protoplasts of root tissues in the H genotype was lower than in the L 215
genotype at seedling (Fig 1C) and flowering stages (Fig S1C) and NO3- accumulation in the 216
cytosol increased significantly in the H genotype compared with the L genotype at seedling 217
(Fig 1D) and flowering stages (Fig S1D) 218
219
B napus with higher NUE showed enhanced long-distance transport of NO3- from roots 220
to shoots 221
The relative expression of BnNRT15 in roots of the H genotype was significantly higher 222
than that in the L genotype at both seedling and flowering stages while the relative expression 223
of BnNRT18 in roots of the H genotype was lower than that in the L genotype at both 224
seedling (Fig 2-AB) and flowering stages (Fig S2-AB) As a consequence total N 225
concentration (traced by 15N) in roots of the H genotype was significantly lower than that of 226
the L genotype at both seedling (Fig 2C) and flowering stages (Fig S2C) while shoot N 227
concentration in the H genotype was significantly higher than the L genotype at seedling stage 228
(Fig 2C) This resulted in significantly higher [15N] SR ratio in the H genotype compared 229
with the L genotype (Fig 2D Fig S2D) 230
No significant differences in total N per plant between the H and L genotypes were 231
observed at both seedling and flowering stages (Fig S3) However NO3- concentration in 232
roots of the H genotype was significantly lower than in the L genotype at both seedling and 233
flowering stages (Fig 2E Fig S2E) while NO3- concentration in shoot tissues of this 234
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genotype was significantly higher at seedling stage (Fig 2E) resulting in higher [NO3-] SR 235
ratios at both stages (Fig 2F Fig S2F) 236
NO3- concentration in the xylem sap xylem sap volume and total NO3
- in xylem sap 237
were significantly higher in the H genotype than in the L genotype at seedling stage (Fig 238
S4ACE) the amount of NO3- in xylem sap significantly increased in the H genotype relative 239
to the L genotype at flowering stage (Fig S4F) Together these data suggests greater 240
mobilization of NO3- from root to shoot in the H genotype 241
242
NO3- up-regulates NRT15 but down-regulates NRT18 243
244
A previous study showed that increased NRT18 expression in nrt15 mutants (Chen et al 245
2012) and the expression of NRT15 was induced by NO3- (Lin et al 2008) We tested the 246
relationship between NRT15 and NRT18 expression in A thaliana and B napus No 247
significant differences were observed in AtNRT15 expression in roots between wild type 248
(col-0) and mutant plants (nrt18-2) (Fig 3A) However AtNRT15 expression was 249
significantly up-regulated by NO3- in roots of nrt18-2 mutants (Fig 3B) Expression of 250
AtNRT18 significantly increased in roots of nrt15-3 mutants but not in col-0 plants and 251
there were no significant differences in AtNRT18 expression with or without NO3- treatment 252
in roots of nrt15-3 mutants (Fig 3CD) 253
In contrast BnNRT15 expression in roots of the H genotype increased significantly with 254
NO3- treatment (Fig 3E) while the reverse was observed in expression of BnNRT18 which 255
showed lower expression under NO3- treatment than control (Fig 3F) This suggests that NO3
- 256
induces the expression of NRT15 but down-regulates NRT18 in A thaliana and B napus 257
Further studies are needed to elucidate the mechanisms regulating this reverse regulation 258
259
Reduced VSC of NO3- in roots drives its long-distance transport from roots to shoots 260
Our previous study showed that Bafi (Bafilomycin A1) inhibits V-ATPase DCCD 261
(DCCD + Na2SO3) inhibits V-PPase and a 11 combination of Bafi and DCCD (B+D) 262
inhibits both V-ATPase and V-PPase (Han et al 2015a) These inhibitors were used to 263
control activities of the tonoplast proton pumps in the H B napus plants V-ATPase activity 264
significantly decreased under Bafil and B+D treatments relative to the control and DCCD 265
treatments whereas V-PPase activity declined significantly under DCCD and B+D treatments 266
relative to that in the control and Bafil treatments (Fig 4A) 267
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The V-ATPase activity in roots of A thaliana was significantly lower in the V-ATPase 268
mutants (vha-a2 and vha-a3) than the wild type (col-0) and V-PPase mutants (avp1) (Fig 5A) 269
In contrast the V-PPase activity in roots of V-PPase mutant (avp1) were significantly lower 270
than in the wild type (col-0) and V-ATPase mutants (vha-a2 and vha-a3) (Fig 5A) 271
NO3- influxes into vacuoles of the H B napus plants significantly decreased when treated 272
with inhibitors of proton pumps (Bafil DCCD B+D) (Fig 4B) Similarly NO3- influxes into 273
the vacuole of the Arabidopsis mutants (vha-a2 vha-a3 and avp1) was significantly lower 274
than those found in the wild type (col-0) (Fig 5B) Previous studies showed that VSC of NO3- 275
decreased when the activities of the proton pumps decline (Li et al 2005 Krebs et al 2010 276
Han et al 2015a) 277
We further investigated NO3- distribution between the vacuole and cytosol as affected by 278
proton pump inhibition in the H genotype of B napus and the mutants of A thaliana 279
defective in vacuolar proton pumps The percentage of vacuolar NO3- relative to total NO3
- in 280
root protoplasts of the H genotype treated with inhibitors (Bafil DCCD B+D) was lower than 281
that observed in the control (Fig 4C) Results were also similar when using A thaliana 282
mutants where the percentage of vacuolar NO3- relative to the total NO3
- in protoplasts was 283
lower in the mutants (vha-a2 vha-a3 and avp1) than in the wild type (col-0) (Fig 5C) 284
Consequently NO3- accumulation in the cytosol showed a significant increase when energy 285
pumps were suppressed in roots of the H genotype (Fig 4D) and NO3- accumulation in the 286
cytosol of root tissues of A thaliana mutants (vha-a2 vha-a3 and avp1) similarly increased 287
compared with col-0 (Fig 5D) 288
Based on previous observations (Lin et al 2008) expression of NRT15 is strongly 289
induced by NO3- but expression of NRT18 is down-regulated (Fig3 Chen et al 2012) We 290
then hypothesized that expression of these two genes is contrastingly regulated by 291
concentration of NO3- in the cytosol Our results were congruent with this hypothesis the 292
expression of BnNRT15 in root tissues of the H genotype of B napus was significantly higher 293
in plants treated with Bafil DCCD or B+D compared with the control (Fig 4E) whereas the 294
expression of BnNRT18 decreased substantially (Fig 4F) Similar results were also observed 295
in A thaliana where expression of AtNRT15 in roots of the mutants (vha-a2 vha-a3 and 296
avp1) were significantly higher than in the wild type (col-0) but expression of AtNRT18 in 297
the same mutants were considerably lower (Fig 5EF) 298
NO3- concentrations in the xylem sap the N-distribution between shoot and root (SR 299
ratios based on [15N]) and the [NO3-] in shoots relative to roots of the H genotype treated with 300
energy pumpsrsquo inhibitors were significantly higher than in the control (Fig 4GH Fig S5A) 301
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Similar trends were also observed when using mutants of A thaliana deficient in the energy 302
pumpsrsquo activities NO3- concentration in the xylem sap the [15N] SR ratios and [NO3
-] SR 303
ratios were all significantly higher in the mutants than in the wild type (Fig 5GH Fig S5B) 304
These data clearly showed that prevention of N sequestration in vacuoles would enhance its 305
translocation to shoot 306
Additional evidence linking NO3- long-distance transport and NO3
- short-distance 307
distribution within cells were provided through experiments comparing A thaliana wild type 308
(Ws) and mutant clca-2 (Fig S6) The chloride channel (CLCa) is the main channel for 309
vacuolar anion accumulation Vacuolar sequestration capacity of NO3-significantly declines in 310
clca mutants (Angeli et al 2006) NO3- influx into the vacuolar space of Ws roots was 311
significantly higher than that observed in the clca-2 mutants (Fig S6A) This resulted in a 312
smaller proportion of vacuolar NO3- in clca-2 plants relative to the total NO3
- in root tissue 313
protoplasts as compared with Ws (Fig S6B) Consequently the accumulation of NO3- in the 314
cytosol was significantly higher in clca-2 than in Ws plants (Fig S6C) The higher 315
NO3-concentration in the cytosol together with the higher expression of AtNRT15 coupled 316
with lower expression of AtNRT18 in clca-2 roots (Fig S6DE) resulted in significant 317
increase in xylem sap NO3- concentration [15N] SR ratios and [NO3
-] SR ratios compared 318
with Ws plants (Fig S6FGH) 319
320
Increased NO3- translocation to shoots enhanced NUE 321
NO3- assimilation efficiency is known to be higher in shoots than in roots (Smirnoff et al 322
1985 Andrews et al 1986 Tang et al 2012 Tang et al 2013) We therefore hypothesize 323
that increased NO3- translocation to shoot and the consequent higher shootroot ratio will 324
contribute to higher NUE Our data support this hypothesis The H B napus genotype showed 325
enhanced long-distance transport of NO3- from roots to shoots (Fig 2 Fig S2) This enhanced 326
NO3- transport requires higher carbon skeleton provided through higher photosynthetic rate 327
(Tang et al 2012 Tang et al 2013) Our results showed that chlorophyll content 328
intercellular CO2 concentration and photosynthetic rate were significantly higher in the H than 329
the L genotype at both seedling and flowering stages (Table 2) Moreover the NO3- 330
assimilating enzymes were strongly induced by NO3- providing sufficient capacity for N 331
assimilation (Smirnoff et al 1985 Andrews et al 1986) Nitrate reductase (NR) and 332
glutamine synthetase (GS) activities in roots of the H genotype were significantly lower than 333
in the L genotype both at seedling and flowering stages (Fig S7) In contrast NR activities in 334
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shoots of the H genotype were significantly higher than in the L genotype at both stages (Fig 335
S7AB) and GS activity in the H genotype was also higher than those of the L genotype at 336
seedling stage (Fig S7C) 337
Experiments comparing A thaliana wild type (col-0 Ws) with mutants defective in 338
vacuolar proton-pumps (vha-a2 vha-a3avp1) and transport channel (clca-2) yielded similar 339
results that is increases in NO3- shootroot ratio essentially contributed to enhanced NUE 340
(Fig 6) The NO3- shootroot ratios in A thaliana mutants were higher than that in the wild 341
type (Fig 5GH) Generally the efficiency of inorganic N (NO3-) assimilation into organic N 342
is higher in shoots than in roots (Smirnoff et al 1985 Andrews et al 1986) therefore the 343
NO3- concentration in A thaliana mutants decreased (Fig 6CD) leading to higher NUE 344
compared with the wild type (Fig 6AB) 345
346
BnNRT15BnNRT18 in B napus affects NO3- long-distance transport from roots to 347
shoots 348
Both B napus and A thaliana are members of Cruciferae Family The amino acid 349
sequence identity between BnNRT15 and AtNRT15 was 90 (Fig S8A) the amino acid 350
sequence similarity between BnNRT18 and AtNRT18 was 908 (Fig S8B) Comparisons of 351
nucleotide and amino acid sequences (Harper et al2012) of these genes 352
(httpbrassicanbiacukcgi-binmicroarray_databasecgi) showed that BnNRT15 353
(EV220114) and BnNRT18 (EV116423) of B napus are respectively highly homologous 354
with AtNRT15 and AtNRT18 and the two genes are mainly expressed in roots of both species 355
showing similar organ-specificity (Lin et al 2008 Li et al 2010) 356
NO3- long-distance transport from roots to shoots is therefore regulated by NRT15 and 357
NRT18 as reported before (Lin et al 2008 Li et al 2010) The [15N-traced] SR ratios and 358
the [NO3-] SR ratios were significantly lower in nrt15-3 mutants relative to the wild type 359
(col-0) while [15N] SR and [NO3-] SR ratios showed significant increase in nrt18-2mutant 360
relative to col-0 (Fig S9CD) A previous study showed that expression of NRT15 is 361
down-regulated by ACC and MeJA treatments while the expression of NRT18 is strongly 362
up-regulated in A thaliana (Zhang et al 2014) Consequently NO3- accumulated in plant 363
roots probably as an adaptive measure for abiotic stresses (Chen et al 2012) Both the H- 364
and L- B napus genotypes showed significantly lower expression of BnNRT15 in roots when 365
treated with ACC (Fig S10A) but the expression of BnNRT18 remained higher than in the 366
control plants (Fig S10B) This resulted in significantly lower [15N] SR ratios following 367
ACC treatment (Fig S10CDE) These data indicate that BnNRT15 and BnNRT18 play 368
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13
similar functions for NO3- long-distance transport between root and shoot in both B napus 369
and A thaliana (Lin et al 2008 Li et al 2010) 370
371
372
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Discussion 373
Nitrate long-distance transport from root to shoot is regulated by cytosolic NO3- in roots 374
NO3- long-distance transport from root to shoot is controlled by NRT gene family For 375
instance xylem NO3- loading and unloading are mainly controlled by NRT15 and NRT18 376
respectively (Lin et al 2008 Li et al 2010) Whereas the NO3- short-distance transport 377
between the vacuole and cytosol is controlled by tonoplast proton-pumps (V-ATPase and 378
V-PPase) (Krebs et al 2010) and by activity of the CLCa channel in the tonoplast membrane 379
(Angeli et al 2006 Wege et al 2014) tonoplast proton-pumps provide the energy required 380
for NO3- accumulation into the vacuole through CLCa (Gaxiola et al 2001 Brux et al 2008 381
Krebs et al 2010) The expression of NRT15 is up-regulated by NO3- in plant culture 382
solution (Lin et al 2008) whereas regulation of NRT18 expression is contrary to that of 383
NRT15 under both control and abiotic stress conditions (Li et al 2010 Chen et al 2012 384
Zhang et al 2014) 385
Despite the progress made in characterizing the genes involved in transport and 386
metabolism of NO3- in plants the relationship between long- and short-distance transport and 387
how these two pathways are regulated have not been thoroughly investigated We hypothesize 388
that NRT15 expression and its role in long-distance transport is regulated by NO3- in plant 389
tissue as was observed in culture solution (Lin et al 2008) and our data support this 390
hypothesis Both BnNRT15 and AtNRT15 expression were up-regulated by increasing 391
cytosolic NO3- in roots (Fig 4 Fig 5) The increase in cytosolic NO3
- was achieved through (i) 392
use of A thaliana mutants defective in proton-pumps (V-ATPase and V-PPase) or CLCa 393
activities in the tonoplast and (ii) inhibition of B napus tonoplast proton-pumps In both 394
model plants the influx of NO3- into vacuoles was substantially reduced resulting in higher 395
concentrations of NO3- in the cytosol (Fig 4A-D Fig 5A-D Fig S6A-C) Under those 396
conditions expression of BnNRT15 and AtNRT15 was up-regulated by the enhanced 397
cytosolic NO3- while expression of BnNRT18 and AtNRT18 was down-regulated (Fig 4EF 398
Fig 5EF FigS6DE) As a consequence more NO3- was loaded into the xylem sap and 399
transported from root to shoot (Fig S5AB FigS6F) This is also reflected as increased 400
shootroot ratios of N traced by [15N] and [NO3-] (Fig 4GH Fig 5GH Fig S6GH) 401
Interestingly the expression of both BnNRT15 and AtNRT15 is up-regulated by 402
increased cytosolic NO3- concentration in roots but were not affected by NO3
- sequestered 403
into vacuoles (Fig 4 Fig 5 Fig S6) This is because vacuolar NO3- is functionally separated 404
by the tonoplast and cannot be assimilated by enzymes that are localized in the cytosol 405
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15
Therefore NO3--inducible genes such as NR and NRT15 were not influenced by vacuolar 406
NO3- concentration (Martinoia et al 1981 Martinoia et al 2000 Zhang et al 2012) In 407
addition NO3- concentration in the cytosol is controlled by its short-distance transport through 408
the tonoplast which is mediated through the tonoplast proton-pumps providing the required 409
energy for its sequestration into vacuoles against concentration gradient (Gaxiola et al 2001 410
Brux et al 2008 Krebs et al 2010) The gradient of H+ between the inside of the vacuole 411
and the cytosol is maintained by an 2NO3-1H+ antiporter mechanism that facilitatesNO3
- 412
transport through CLCa (Angeli et al 2006 Wege et al 2014) Our data provide clear 413
evidence that NO3- long-distance transport from root to shoot is regulated by cytosolic NO3
- 414
concentration in roots 415
Regulatory mechanisms that control the expression of NRT18 and NRT15 as mediated 416
by NO3- are still unknown however previous studies showed that their expression is also 417
affected by abiotic stresses whereby NRT15 is down-regulated and NRT18 is up-regulated 418
(Li et al 2010 Zhang et al 2014) AtNRT18 expression in A thaliana nrt15 mutants is 419
highly up-regulated (Chen et al 2012) as was also observed here (Fig 3C) Moreover 420
AtNRT15 expression was enhanced by NO3- in culture solution and was not affected in 421
nrt18-2 mutant (Fig 3AB) On the other hand AtNRT18 expression was not affected by 422
cytosolic NO3- but down-regulated when AtNRT15 expression was up-regulated by NO3
-(Fig 423
S6DE) Analogous results were also obtained with B napus genotypes (Fig 3EF) Based on 424
these results we argue that NRT18 expression is not directly influenced by cytosolic NO3- but 425
rather by NRT15 expression (Fig 4 Fig 5 and Fig S6) suggesting NRT18 is probably 426
acting downstream of NRT15 Apparently the regulatory mechanisms of this NRT gene 427
family still awaits further studies to be elucidated 428
429
Response of NUE to NO3- long-distance transport 430
NO3- assimilation efficiency is higher in shoot than in root tissues and can be further 431
enhanced by carbon assimilation (Smirnoff et al 1985 Tang et al 2013) Therefore 432
translocation of higher proportion of NO3- from roots to shoots likely contributes to better 433
crop growth and higher NUE (Andrews et al 1986 Tang et al 2012) The present study 434
provides direct evidence for these results using contrasting B napus genotype identified 435
before (Zhang et al 2009 Han et al 2015a) Our data confirmed the genetic variation in 436
NUE between these two genotypes (Table 1) 437
We measured NO3- distribution in root and shoot of these genotypes to elucidate the 438
physiological mechanisms contributing to variation in NUE The data were consistent with 439
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16
our hypothesis that concentration of NO3- in the xylem sap and shoot would be significantly 440
higher in the H genotype than in the L genotype (Fig 2EFFig S2EF Fig S4) The NO3- 441
long-distance transport from root to shoot was affected by cytosolic NO3- in roots of both A 442
thaliana and B napus genotypes (Fig 4-5 Fig S6) The activities of the tonoplast 443
proton-pumps in roots were significantly lower in the H genotype than in the L genotype (Fig 444
1AB) Consequently less NO3- was sequestered into the vacuole and more NO3
- was retained 445
in cytosol (Fig 1CD) BnNRT15 expression was then up-regulated resulting in greater 446
loading into the xylem while BnNRT18 was down-regulated (Fig 2AB) probably to 447
suppress NO3- unloading from xylem sap Consequently a higher proportion of NO3
- was 448
transported from root to shoots in the H genotype(Fig 2C-D Fig S4) 449
Moreover photosynthetic carbon fixation (Table 2) and activities of both NR and GS 450
were higher in shoots of the H genotype (Fig S7) both of which promote higher N 451
assimilation These results agreed with previous reports (Smirnoff et al 1985 Andrews et al 452
1986 Tang et al 2012 Tang et al 2013) where higher NO3- transport from root to shoot 453
higher photosynthetic rate and N assimilation efficiency in shoots were suggested as essential 454
mechanisms for high-NUE in crop plants These qualities endowed the B napus H genotype 455
Xiangyou15 with higher NUE as compared with the L genotype 814 456
NO3- long-distance transport in B napus is not only regulated by BnNRT15 and 457
BnNRT18 in root tissues (Fig 4 Fig 5 Fig S6) but also by stomatal conductance and 458
transpiration by indirectly controlling xylem sap flow and long-distance transport 459
(Dechorgnat et al 2011 Krapp et al 2014) Stomatal conductance and transpiration rates of 460
the H genotype were significantly higher than those of the L genotype (Table S1) a result 461
consistent with other studies describing characteristics of high-NUE plants (Daniel-Vedele et 462
al 1998 Wilkinson et al 2007 Dechorgnat et al 2011 Krapp et al 2014) 463
464
Response of NUE to NO3- short-distance transport 465
NUE is also strongly controlled by NO3- short-distance distribution between the vacuole 466
and cytosol (Han et al 2015a) A lower proportion of NO3- accumulating in vacuoles and a 467
higher proportion retained in the cytosol will contribute to better NUE in crop plants (Han et 468
al 2015b) The energy required for vacuolar NO3- sequestration is provided by the tonoplast 469
proton-pumps (Gaxiola et al 2001 Brux et al 2008 Krebs et al 2010) while the 470
distribution channel CLCa enables transport of NO3- across the vacuolar membrane (Angeli et 471
al 2006 Wege et al 2014) The activities of the tonoplast proton-pumps (V-ATPase and 472
V-PPase) in leaves of the H genotype were significantly lower than those of the L genotype at 473
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17
flowering stages (Han et al 2015aHan et al 2015b) thus we expected similar differences in 474
root tissues Our results confirmed that the activities of tonoplast proton-pumps in root tissues 475
of B napus H genotype and A thaliana mutants were lower than those in the L genotype and 476
A thaliana wild type leading to less NO3- influx into vacuoles and more in the cytosol (Fig 1 477
Fig 5A-D) Moreover the enzymes catalyzing N assimilation are located in the cytoplasm 478
where NO3- could be assimilated (Martinoia et al 1981 Han et al 2015a) or immediately 479
loaded into xylem sap for long-distance transport from roots to shoots (Lin et al 2008) 480
Consequently the NO3- concentration in root tissues of the H genotype and A thaliana 481
mutants defective in tonoplast proton-pumps was significantly lower than that in the L 482
genotype (Fig 2E) and in A thaliana wild type (Fig 6C) Therefore the higher proportion of 483
NO3- transported to shoot and the lower concentrations retained in root vacuoles contributed 484
to the higher NUE in Xiangyou15 and A thaliana mutants (vha-a2 vha-a3 and avp1) (Table 485
1Fig 6A) Further studies are needed to assess the impacts of regulating vacuolar 486
sequestration of NO3- and rates of long-distance transport for enhancing NUE in other crop 487
species and genotypes 488
These data suggested that NO3- distribution between vacuoles and cytosol within cells 489
regulates NO3- long-distance transport from root to shoot and ultimately affects NUE NO3
- 490
loading into the xylem sap is an active transport process (Lin et al 2008) therefore not 491
regulated by absolute concentration of NO3- in the cytosol but rather by genes involved in 492
NO3- long-distance transport (NRT15 and NRT18) Apparently the expression of NRT15 the 493
gene responsible for xylem loading is regulated by NO3-concentration in the cytosol 494
Therefore both NO3- short-distance transport across the tonoplast and its long-distance 495
transport from root to shoot are important determinants of NUE in both B napus and A 496
thaliana (Table 1 and Fig 6) 497
498
Roles of NRT15 and NRT18 in NO3- long-distance transport in B napus and A 499
thaliana 500
Our results (Figs S9 S10) showed that the proportion of NO3- distributed from root to 501
shoot is strongly controlled by NRT15 in accord with previous studies (Lin et al 2008 Li et 502
al 2010) The data suggest that the high-NUE genotypes possess lower activities of tonoplast 503
proton-pumps (V-ATPase and V-PPase) resulting in less NO3- accumulation in vacuoles and 504
more NO3- retention in cytosol (Fig 7) The higher NO3
- in the cytosol then up-regulated 505
NRT15 which then down-regulates NRT18 As a result more NO3- is loaded into the xylem 506
system by NRT15 mechanism and less NO3- is unloaded via NRT18 (Fig 7) Differential 507
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18
regulation of these NRT genes in high-NUE plants increased transport of NO3- from root to 508
shoot through the xylem vascular tissues (Fig 7) This coordinated NO3- long- and 509
short-distance transport likely fine-tunes NO3- allocation and modulates the balance of NO3
- 510
distribution between roots and shoots (Fig 7) 511
Our research also revealed differences between A thaliana and B napus in the 512
expression of the wild-type NRT genes in response to NO3- in the protoplast Moreover using 513
A thaliananrt15-3 mutants we uncovered possible regulatory interactions between a 514
functioning (wild-type) NRT15 gene and NRT18 expression in response to NO3- 515
concentration Because mutants defective in nrt15 are not available for B napus we could 516
not confirm that the respective NRT15 and NRT18 systems behave in an analogous way in 517
these plant species Based on Harper et al (2012) research database our homology 518
comparisons and gene function annotations provided strong evidence for the roles of 519
BnNRT15 and BnNRT18 in B napus Finally our studies were conducted using two model 520
dicotyledonous plant species for which we had ample data on responses to specific inhibitors 521
(B napus) or specific mutants were available with results forming the bases for the model 522
presented in Figure 7 Further research using genetically diverse plant species (eg 523
monocotyledonous crops such as rice wheat and corn) will help validate this model and its 524
broader application across species The information generated in this study could have future 525
application for improving NUE in commercial crops with consequent reduction in nitrogen 526
use and benefits to the environment 527
528
Materials and Methods 529
Plant material 530
The two oilseed rape (Bnapus) cultivars used in this study have been characterized 531
before as high- (Xiangyou15 referred to as H genotype hereafter) and low- (814 referred to 532
as L genotype) nitrogen use efficiency (NUE) genotypes (Zhang et al 2009 Han et al 2015b) 533
Here we define NUE as the total biomass per unit of N uptake by the plant with total 534
biomass includes roots shoots and grains The two genotypes were provided by the Hunan 535
Sub-Center of Improvement Center of National Oil Crops Hunan China 536
The Arabidopsis thaliana wild-type Columbia-0 (col-0) was used as control for 537
V-ATPase (vha-a2 and vha-a3) V-PPase (avp1) nrt15-3 and nrt18-2 mutants whereas the A 538
thaliana wild-type Wassilewskija (Ws) was used as control for clca-2 mutants A thaliana 539
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19
mutants and transgenic lines (nrt15-3 and nrt18-2) have been described previously (Chen et 540
al 2012 Zhang et al 2014) The mutant lines vha-a2 (Salk_142642) and vha-a3 541
(Salk_122135) described by Krebs et al (2010) were obtained from the Arabidopsis 542
Biological Resources Center (ABRC) The mutant line avp1 (GK-005004) described in Li et 543
al (2005) was obtained from the European Arabidopsis Stock Centre while the mutant line 544
clca-2 (FST 171A06) described in Angeli et al (2006) was from the Institute National de la 545
Recherche Agronomique (INRA) collection in France together with the T-DNA mutants used 546
for genotyping to select pure mutant lines 547
548
Growth conditions 549
B napus plants were grown hydroponically in ceramic pots(20cmtimes15cm) filled with a 550
nutrient solution in the greenhouse under natural light as described in Han et al (2015b) The 551
pots were arranged in a completely randomized design with six biological replications The 552
nutrient solution was replaced every 3 days and its pH adjusted to 55 daily Experiments were 553
conducted at the field station of Hunan Agricultural University Southern China 554
A thaliana plants were grown in a nutrient solution in plastic pots as described in Arteca 555
et al (2000) and Gong et al (2003) The solution was changed every 3 days with pH adjusted 556
daily to 58 Pots were arranged in a completely randomized design with six biological 557
replications The nutrient solution used for both species consisted of 125mM KNO3 558
0625mM KH2PO4 05mM MgSO4 05mM Ca (NO3)2middot4H2O 0025mM Fe-EDTA 025 559
mlL-1 micronutrients (stock solution concentrations 70mM B 14mM Mn 1mM Zn 05mM 560
Cu and 02mM Mo) The experiments were conducted at Hunan Agricultural University in a 561
phytotron set at 70 relative humidity16h-light8h-dark cycle and constant temperature of 562
22degC 563
564
Experimental treatments and phenotyping 565
All measurements made on B napus plants were conducted either at the seedling stage 566
(2 months after transplanting) or at flowering (5 months after transplanting) The whole root 567
tissue was harvested and used for analyses at either stage For shoot measurements the fourth 568
leaf from the bottom was used for measurements made at seedling stage whereas the 12th leaf 569
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
from the bottom was harvested for measurements made at flowering stage For A thaliana 570
whole root and shoot of four-week-old plants were sampled and used for different assays 571
572
Vacuolar proton-pumps inhibitor treatments 573
Inhibition of vacuolar proton pumps of B napus (Xiangyou15) was conducted at the seedling 574
stage in hydroponic culture as described in Han et al (2015a) with minor modifications 575
Plants grown in hydroponic solution were used as control Inhibitor treatment was conducted 576
using several chemicals Bafi(25 nmol L-1 Bafilomycin A1) an inhibitor of V-ATPase 577
DCCD(10 μmol L-1DCCD + 50 mmolL-1 Na2SO3) specifically inhibits V-PPase and 578
Bafi+DCCD in a 11 ratio as inhibitor of both V-ATPase and V-PPase The inhibitors were 579
applied in the hydroponic solutions for 24 h Fresh plant tissue samples from all treatments 580
were collected for different assays 581
582
NO3- induced gene expression 583
Four-week old A thaliana mutants (nrt15-3nrt18-2) were grown in hydroponics with 584
225mM (NH4)2succinate for 3 days and shifted to hydroponics with 45mM NO3- for 12 h 585
then root tissues were collected to assess relative expression of AtNRT15 and AtNRT18 586
genes Seedlings of B napus (Xiangyou15) plants grown hydroponically were treated with 587
5mM (NH4)2succinate for 3 d and shifted to fresh hydroponic solution containing 15 mM 588
NO3- for 12 h Root tissue was then collected to assess the relative expression of BnNRT15 589
and BnNRT18 genes using quantitative RT-PCR 590
591
ACC treatment 592
Hydroponically grown seedlings of B napus were subjected to ACC treatment as described in 593
Zhang et al (2014) with minor modifications Seedlings of the two genotypes Xiangyou15 594
and 814 were transferred to a nutrient solution containing 002mM L-1 ACC for 6 h or grown 595
in normal hydroponic solution as control Root tissues were then collected to assay relative 596
expression of BnNRT15 and BnNRT18 using quantitative RT-PCR Another set of B napus 597
plants were grown in ACC-containing hydroponic solution but supplemented with 15NO3- (22 598
15N) replacing the unlabeled NO3- for 1 h Root and shoot tissues were collected separately 599
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21
for 15N measurement using a continuous-flow isotope ratio mass spectrometer coupled with a 600
C-N elemental analyzer (ANCA-MS PDZ Europa) 601
602
Biomass and N concentration 603
Grain yield dry biomass and N concentration were determined in plant samples at 604
harvest Plants were harvested (including senescing leaves) then dried in an oven for 30 min 605
at 105degC then at 70degC to a constant weight and weighed N concentration was determined 606
using the Kjeldahl method (Shi et al 2010 Han et al 2015b)02g of dried samples were 607
digested with H2SO4-H2O2 in a 100-ml Kjeldahl digestion flask after which N concentration 608
was determined using a Foss Auto Analyser Unit (Kjeldahl 8400) 609
610
V-ATPase and V-PPase activities 611
Roots (10 g) of B napus were collected at seedling and flowering stages and that of A 612
thaliana (05 g) sampled at seedling stage then used for determining the activities of 613
V-ATPase and V-PPase (Han et al 2015b) Activities of these two energy pumps within 614
microsomal membranes were followed colorimetrically based on released Pi as described by 615
Zhu et al (2001) and Krebs et al (2010) 616
617
Assay of NO3- flux in vacuoles 618
Roots of both species were collected similarly as that used for V-ATPase and V-PPase 619
activities then used for vacuole isolation and measurement of NO3- flux in vacuoles as 620
described by Robert et al (2007) with minor modification (Han et al 2015b) Net fluxes of 621
NO3- in vacuoles were measured non-invasively using SIET (scanning ion-selective electrode 622
technique SIET system BIO-003A Younger USA Science and Technology Corporation 623
USA) An NO3--selective microelectrode used for the assay of NO3
- flux was vibrated in the 624
measuring solution between two positions 1 μm and 11 μm from the vacuole surface 625
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles Background 626
signal was recorded by vibrating the electrode in the measuring solution in the absence of 627
vacuoles Ion flux was calculated by Fickrsquos law of diffusion J = -D(dcdx) where J represents 628
the ion flux (picomolescm-2s-1) dcdx is the ion concentration gradient and D is the ion 629
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
diffusion constant in a particular medium The direction of the flux was derived from Fickrsquos 630
law of diffusion that relates the concentration gradient 631
632
Isolation of intact protoplasts and vacuoles for determining NO3- concentration 633
Similar to the above assays root tissues (10 g) were collected from B napus at seedling 634
and flowering stages and from A thaliana (05 g) seedlings and used to isolate intact 635
protoplasts and vacuoles as described by Robert et al (2007) with minor modifications as 636
outlined in Huang et al (2012) and Han et al(2015b) The purified protoplasts were divided 637
into two equal aliquots with one of them used for isolation of vacuoles The purified 638
protoplasts and vacuoles were then sub-sampled and used to determine NO3- concentrations 639
(Vogeli-Lange and Wagner 1990) and for enzyme activity assays (Ma et al 2005) 640
NO3-concentrations in protoplasts and vacuoles were measured by a continuous-flow 641
auto-analyzer (Auto Analyser 3 Bran and Luebbe Germany) as described previously (Han et 642
al 2015b)The activities of acid phosphatase (ACP) and cytochrome oxidase (COX) were 643
determined using plant ACP colorimetry- and COX-assay kits (GenMedSci Inc) following 644
the manufacturerrsquos instructions ACP activity specific to vacuoles was determined and used to 645
normalize NO3- accumulation We measured NO3
- in the protoplast outside the vacuole which 646
includes the cytosol and organelles eg mitochondria and Golgi Apparatus (Robert et 647
al2007) Since most of the NO3- in the protoplast outside the vacuole is located in the cytosol 648
(Krebs et al 2010) we refer to NO3- distribution between vacuoles and cytosol rather than 649
vacuole versus protoplast 650
651
Quantitative RT-PCR 652
Total RNA extracted from B napus roots was prepared using Trizol reagent (Invitrogen) 653
cDNAs were synthesized using M-MLV reverse transcriptase (Promega) following 654
manufacturerrsquos protocol The relative expression of BnNRT15 (EV220114) BnNRT18 655
(EV116423) and Bnactin (AF1118121) genes in plant roots were determined by quantitative 656
RT-PCR and run on a LightCycler instrument (Roche) with the SYBR Green Real-Time PCR 657
Master Mix Kit (TOYOBO Japan) under the following conditions 95degC for 2min then 45 658
cycles of 95degC for 10s 60degC for 10s and 72degC for 20s The primer sequences of BnNRT15 659
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
BnNRT18 and Bnactin genes were obtained from httpbrassicanbiacuk as described in 660
Harper et al (2012) and expression levels were normalized to Bnactin as control 661
Total RNA extracted from the 4-week-old A thaliana roots under the treatments 662
conditions described above were prepared using Trizol reagent (Invitrogen) The cDNAs were 663
synthesized using M-MLV reverse transcriptase (Promega) based on the manufacturerrsquos 664
protocol and as described in Li et al (2010)The primer sequences of quantitative RT-PCR 665
used in these assays are listed in Supplemental Table 2 and were described previously (Chen 666
et al 2012 Zhang et al 2014) and their expression was normalized to Atactin2 as control 667
668
Determination of nitrate distribution using 15NO3- 669
Hydroponically grown B napus (seedling and flowering stages) and A thaliana 670
(seedling) were transferred to 01mM CaSO4 solution for 1min then to their respective 671
hydroponic nutrient solutions with15NO3- (22 excess) replacing unlabeled NO3
- for 1h 672
Plants were then transferred to 01mM CaSO4 solution for 1min after which roots were 673
washed with deionized water Root and shoot samples were separated and dried at 105degC for 674
30min followed by 70degC for 3 d then grounded and used for assaying 15N content using a 675
continuous-flow isotope ratio mass spectrometer coupled with a carbon-nitrogen elemental 676
analyzer (ANCA-MS PDZ Europa) 677
678
Xylem sap collection and assay of nitrate concentration 679
We used the method of Tang et al (2012) for collecting xylem sap Plants were cut 680
leaving1cm segments with intact roots at seedling and flowering stages for B napus and at 681
seedling stage for A thaliana Roots were immediately immersed in their respective nutrition 682
solutions Weighed cotton was put on the cut surface to absorb extruding xylem sap for 1 h 683
The cotton was then wrapped in plastic film and the volume of xylem sap was calculated as 684
the weight gain of the cotton Xylem sap was then squeezed from the cotton using a syringe 685
and used for subsequent assay of nitrate concentration using a continuous-flow auto-analyzer 686
(Auto Analyser 3 Bran and Luebbe Germany) 687
To determine nitrate concentration samples (10 g fresh root and shoot at seedling and 688
flowering of B napus and 05 g fresh root and shoot of 4-week-old A thaliana) were frozen 689
in liquid N2 and ground with a mortar and pestle the powder was then transferred to a beaker 690
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
containing 70 ml deionized water and boiled for 30 min to extract nitrate then cooled and 691
made to 100 ml volume and 02 g activated carbon added to eliminate the effect of 692
chlorophyll The mixture was filtered and nitrate in the filtrate was determined using a 693
continuous-flow auto-analyser (Atuo-Analyser 3 Bran and Luebbe Germany) 694
695
Measurements of photosynthesis 696
All measurements of photosynthesis and related parameters were conducted at 1000 h 697
using intact leaves The 4th leaf from the bottom in B napus at seedling stage and the 12th leaf 698
from the bottom at flowering stage were used for determining chlorophyll concentration 699
photosynthetic rate intercellular CO2 concentration transpiration rate and stomatal 700
conductance Chlorophyll concentration was determined by SPAD-502 (Minolta Camera Co 701
Ltd Japan) (Luo et al 2006 Yandeau-Nelson et al 2011) Photosynthetic rate intercellular 702
CO2 concentration transpiration rate and stomatal conductance were measured using LI-6400 703
Portable Photosynthesis System (Li-Cor Biosciences Co Ltd USA) set at flow rate of 500 704
μmol s-1 photosynthetic photon flux density of 1000 μmol m-2 s-1 relative humidity of 65 705
CO2 concentration of 400ppm and ambient temperatures 706
707
NR and GS activities 708
Leaves with similar age as those used for photosynthesis in B napus were used for the 709
assay of nitrate reductase (NR) and glutamine synthetase (GS) activities NR activity was 710
measured by the modified method of Fan et al (2007) Fresh samples (05g) were frozen in 711
liquid N2 ground into powder in the presence of acid-washed sand and homogenized with 4 712
ml of extraction buffer (0025mol L-1phosphate buffer pH87 1211g L-1 cysteine 0372g L-1 713
EDTA) Homogenates were centrifuged at 30000timesg for 15 min at 4degC and the supernatants 714
were treated with sulfanilamide and α-naphthylamine reagents for colorimetric (540nm) 715
determination of nitrite as described by Fan et al (2007) 716
GS activity was determined using a modified reverseγ-glutamyltransferase method 717
(Wang et al2014) which measures the GS-catalyzed formation of glutamyl-γ-hydroxamate 718
from glutamine and hydroxylamine Fresh samples were frozen for 30 min at -20degC then 719
ground in 10ml Tris-HCl buffer and acid-washed sand The homogenates were filtered 720
through two layers of gauze and centrifuged at 8000timesg for 15 min at 4degC About 12ml of the 721
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
supernatant was added in a reaction mixture and treated as described (Wang et al2014) After 722
the GS-catalyzed reaction was stopped and the solution centrifuged glutamyl-γ723
-hydroxamate was quantified colorimetrically in the supernatant (at 485 nm) and the 724
concentration was determined using a standard curve 725
726
Statistical analyses 727
We used the SPSS software (Statistical Product and Service Solutions V130 USA) for 728
ANOVA and mean separation of main-effects and interactions using Duncanrsquos multiple range 729
test at Plt005Values are presented as means and SD of three or six replicates from three 730
independent experiments Different letters or an asterisk () associated with specific data (eg 731
at the top of histogram bars in figures or within tables) denote significant differences at 732
Plt005 733
734
Supplemental Data 735
Supplemental Figure 1 B napus with higher NUE showed lower vacuolar sequestration 736
capacity (VSC) for NO3-in roots at flowering stage 737
Supplemental Figure 2 B napus with higher NUE showed enhanced long-distance transport 738
of NO3- from roots to shoots at flowering stage 739
Supplemental Figure 3 The two B napus (H and L genotypes) showed the same total N per 740
plant at seedling stage (A) and flowering stage (B) 741
Supplemental Figure 4 B napus with higher NUE showed increased NO3- concentration in 742
the xylem sap at seedling and flowering stages 743
Supplemental Figure 5 NO3- concentration in the xylem sap as affected by inhibitor 744
treatments in B napus and in the energy pumpsrsquo mutants of A thaliana (col-0 vha-a2 vha-a3 745
avp1) 746
Supplemental Figure 6 Reduced VSC for NO3- in roots drives long-distance transport of 747
NO3- from roots to shoots in the A thaliana wild type (Ws) and mutant (clca-2) 748
Supplemental Figure 7 Differences of NR and GS activities between the two B napus (H 749
and L genotypes) at seedling and flowering stages 750
Supplemental Figure 8 Amino acid sequences of BnNRT15 and BnNRT18 751
Supplemental Figure 9 Functions of AtNRT15 and AtNRT18 genes in root tissues of A 752
thaliana in controlling NO3- long-distance transport from root to shoots 753
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
Supplemental Figure 10 B napus BnNRT15 and BnNRT18 genes in roots were 754
coordinately modulated to facilitate NO3- long distance transport from roots to shoots 755
Supplemental Table 1 Differences in stomatal conductance and transpiration rate between 756
the two B napus genotypes 757
Supplemental Table 2 Sequences of primera used for qRT-PCR 758
759
Acknowledgements 760
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 761
Shanghai Institutes for Biological Sciences) for providing nrt15-3 and nrt18-2 seeds 762
This study was supported by the National Natural Science Foundation of China (Grant 763
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 764
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 765
The Ministry of Education in China Open Novel Science Foundation of Hunan province 766
(13K062) the National Key Laboratory of Plant Molecular Genetics and the Twelfth 767
Five-Year National Science and technology support program (2012BAD15BO4) 768
769
Figure 1 B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 770
NO3- in roots at the seedling stage 771
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 772
genotype 814 Specific activities of the tonoplast proton pumps are expressed as μmol Pi 773
released mg-1 protein h-1 NO3- fluxes are expressed as pmol NO3
- cm-2 S-1 Mature vacuoles 774
were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 775
measuring solution between the two positions 1 μm and 11 μm from the vacuole surface 776
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles recording the 777
stable reading data The background was recorded by vibrating the electrode in measuring 778
solution without vacuoles 779
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 780
for NO3- content and NO3
- accumulation normalized against the specific activity of the 781
vacuole acid phosphatase (ACP) as described in Materials and Methods and plotted as μmol 782
NO3- per μmol p-nitrophenol the end product of ACP assay 783
Proton pump activities in root tissues between H and L genotypes are shown at the seedling 784
stage (A) Different letters at the top of the histogram bars denote significant differences of 785
V-ATPase in root tissues between H and L genotypes (Plt005) an asterisk () at the top of the 786
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
histogram bars indicates significant difference in V-PPase activity in root tissues between H 787
and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788
Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789
genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790
bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791
bars on the figures indicate SD (n=6) 792
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793
seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794
to the total NO3- in protoplasts 795
NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796
total NO3-in the cytosol and was calculated as total NO3
- in protoplasts ndash total NO3- in 797
vacuole Different letters at the top of histogram bars denote significant differences of total 798
NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799
(n=6) 800
801
Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802
roots to shoots at the seedling stage 803
H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804
Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805
the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806
as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807
the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808
the histogram bars denote significant differences at Plt005 809
Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810
in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811
genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812
of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813
SD (n=3) different letters at the top of the histogram bars denote significant differences at 814
Plt005 level 815
The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816
at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817
genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818
different letters at the top of the histogram bars denote significant differences at Plt005 819
820
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
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Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
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b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
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Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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4
Introduction 99
China is the largest consumer of nitrogen (N) fertilizer in the world however the 100
average N-fertilizer use efficiency (NUE) is only around 35 suggesting considerable 101
potential for improvements (Shen et al 2003 Wang et al 2014) With the high amounts of 102
N-fertilizer being used crop yields are declining in some areas where application is 103
exceeding the optimum required for local field crops (Shen et al 2003 Miller et al 2008Xu 104
et al 2012) The extremely low NUE results in waste of resources and environmental 105
contamination and also presents serious hazards for human health (Xu et al 2012 Chen et 106
al 2014) Consequently exploiting the maximum potential for improving NUE in crop plants 107
will have practical significance for agriculture production and the environment (Zhang et al 108
2010 Schroeder et al 2013 Wang et al 2014) Elucidating the genetic and physiological 109
regulatory mechanisms governing NUE in plants will allow breeding crops and varieties with 110
higher NUE 111
Ammonium (NH4+) and nitrate (NO3
-) are the main N species absorbed and utilized by 112
crops and NO3- accumulation and utilization are of major emphasis for N nutrient studies in 113
dry land crops such as Brassica napus Several studies revealed the close relationship 114
between NO3- content and NUE in plant tissues (Shen et al 2003 Zhang et al 2012 Tang et 115
al 2013Han et al 2015a)When plants are sufficiently illuminated NO3- assimilation 116
efficiency significantly increase in shoots compared with roots (Smirnoff et al 1985 Tang et 117
al 2013) Consequently under daytime with optimal illumination higher proportion of NO3- 118
in plant tissue is transported from root to shoot as an advantageous physiological adaptation 119
that reduces the cost of energy for metabolism (Tang et al 2013) NO3- assimilation in plant 120
shoots can therefore take advantage of solar energy while improving NUE (Smirnoff et al 121
1985 Andrews et al 1986 Tang et al 2012 Tang et al 2013) 122
The NO3- long-distance transport and distribution between root and shoot is regulated by 123
two genes encoding long transport mechanisms NRT15 is responsible for xylem NO3- 124
loading while NRT18 is responsible for xylem NO3- unloading (Lin et al 2008 Li et al 125
2010) Expression of the two genes is influenced by NO3- concentration NRT15 is strongly 126
induced by NO3- (Lin et al 2008) while NRT18 expression is extremely up-regulated in 127
nrt15 mutants (Chen et al 2012) A negative correlation between the extents of expression of 128
the two genes was observed when plants are subjected to abiotic stresses (Chen et al 2012) 129
Moreover expression of NRT15 is strongly inhibited by 1-aminocyclopropane-1-carboxylic 130
acid (ACC) and methyl jasmonate (MeJA) whereas the expression of NRT18 is significantly 131
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5
up-regulated (Zhang et al 2014) Based on these studies we argue that the expression and 132
functioning of NO3- long-distance transport genes NRT15 and NRT18 are regulated by 133
cytosolic NO3- concentration In addition the vacuolar and cytosolic NO3
- distribution is 134
likely regulated by proton-pumps located within the tonoplast (V-ATPase and V-PPase) 135
(Granstedt et al 1982 Glass et al 2002 Krebs et al 2010) Therefore NO3- use efficiency 136
must be affected by NO3- long-distant transport (between shoot and root) and short-distant 137
transport (between vacuole and cytosol) However the physiological mechanisms controlling 138
this regulation are still obscure 139
Previous studies showed that the chloride channel protein (CLCa) is mainly responsible 140
for vacuole NO3- short-distance transport as it is the main channel for NO3
-movement 141
between the vacuoles and cytosol (Angeli et al 2006 Wege et al 2014) The vacuole 142
proton-pumps (V-ATPase and V-PPase) located in the tonoplast supply energy for active 143
transport of NO3- and accumulation within the vacuole (Gaxiola et al 2001 Brux et al 2008 144
Krebs et al 2010) Despite that about 90 of the volume of mature plant cells is occupied by 145
vacuoles vacuolar NO3- cannot be efficiently assimilated because the enzyme nitrate 146
reductase (NR) is cytosolic (Shen et al 2003 Han et al 2015a) However re-translocation of 147
NO3- from the vacuole to the cytosol will permit its immediate assimilation and utilization 148
Generally NO3- concentrations in plant cell vacuoles and the cytoplasm are in the range 149
of 30-50 mol m-3 and 3-5 mol m-3 respectively (Martinoia et al 1981 Martinoia et al 2000) 150
Because vacuoles are obviously the organelle for high NO3- accumulation and storage in plant 151
tissues their function in NO3- use efficiency cannot be ignored (Martinoia et al 1981 Zhang 152
et al 2012 Han et al 2015b) NO3- assimilatory system in the cytoplasm is sufficient for its 153
assimilation when it is transported out of the vacuoles Therefore NO3- use efficiency could in 154
part be dependent on vacuolar-cytosolic NO3- short-distance transport in plant tissues 155
(Martinoia et al 1981 Shen et al 2003 Zhang et al 2012 Han et al 2015a) 156
Evidently NO3- use efficiency is regulated by both NO3
- long-distance transport from 157
root to shoot and short-distance transport and distribution between vacuoles and cytoplasm 158
within cells(Glass et al 2002 Dechorgnat et al 2011 Han et al 2015a) Although vacuoles 159
compartment excess NO3- that accumulates in plant cells (Granstedt et al 1982 Krebs et al 160
2010) neither NO3- inducible NR genes (NIA1and NIA2) (Fan et al 2007 Han et al 2015a) 161
nor the NO3- long-distance transport gene NRT15 (Lin et al 2008) are regulated by vacuolar 162
NO3- even though they are essential for NO3
- assimilation Only NO3- transported from the 163
vacuole to the cytosol can play a role in regulating NO3- inducible genes Consequently we 164
argue that both NO3- assimilation in cells and its long-distance transport from root to shoot are 165
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6
regulated by cytosolic NO3- concentration However this hypothesis needs to be substantiated 166
The mechanisms underlying both NO3- short-distance (Gaxiola et al 2001 Angeli et al 167
2006 Brux et al 2008 Krebs et al 2010) and long-distance transport (Lin et al 2008 Li et 168
al 2010) have been previously investigated yet the underlying mechanisms regulating the 169
flux of NO3- and the obvious relationship between the two transport pathways as well as their 170
relation to NUE are not well understood 171
The NRT family of genes play a partial role in vacuolar NO3- accumulation in petioles 172
(Chiu et al 2004) and seed tissues (Chopin et al 2007) whereas the proton pumps and 173
CLCa system in the tonoplast play a major role in accumulating NO3- in vacuoles (Gaxiola et 174
al 2001 Angeli et al 2006 Brux et al 2008 Krebs et al 2010) The vacuolar NO3- 175
short-distance transport system is spread throughout the plant tissues and is the principal 176
means by which vacuolar NO3- short-distance transport and distribution is controlled (Angeli 177
et al 2006Krebs et al 2010) 178
The NRT genes seem to work synergistically to control NO3- long-distance transport 179
between roots and shoots NRT19 is responsible for NO3- loading into the phloem (Wang et 180
al 2011) whereas NO3- loading and unloading into xylem are regulated by NRT15 and 181
NRT18 respectively (Lin et al 2008 Li et al 2010) Phloem transport mainly involves 182
organic N the inorganic-N (NO3-) concentrations in the phloem sap are typically very low 183
ranging from one-tenth to one-hundredth of that of the inorganic-N in xylem sap (Lin et al 184
2008Fan et al 2009) Therefore this study focused on NO3- short-distance transport 185
mediated through the tonoplast proton-pumps and the CLCa system and the long-distant 186
transport mechanisms responsible for xylem NO3- loading and unloading via NRT15 and 187
NRT18 respectively 188
Questions related to how long and short-distance transport of NO3- are coupled in plant 189
tissues and their role in determining NUE were addressed using a pair of high- and low-NUE 190
B napus genotypes and Arabidopsis thaliana Application of proton pump inhibitors and 191
ACC in the former and use of mutants with defective proton pumps in the latter allowed 192
experimental distinction of the physiological mechanisms regulating these processes Data 193
presented here provide strong evidence from both model plants supporting this linkage and 194
strongly suggest that cytosolic NO3- concentration in roots regulates NO3
- long-distance 195
transport from roots to shoots We also investigated how NO3- concentration in plant tissues 196
would be affected by NO3- long-distance transport vacuolar NO3
- sequestration and the 197
ensuing relationship with NO3- use efficiency We also proposed the physiological 198
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7
mechanisms likely to be important for enhancing NO3- use efficiency in plants These findings 199
will provide scientific rationales for improving NUE in important industrial and food crops 200
201
202
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Results 203
B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for NO3- 204
in root tissues 205
Our previous work identified high and low NUE B napus genotypes (Han et al 2015a 206
Han et al 2015b) NUE of B napus whether based on biomass or on grain yield (Table 1) 207
was significantly higher for the high-NUE genotype (H Xiangyou15) than for the low-NUE 208
genotype (L 814) That the activities of the tonoplast proton-pumps (V-ATPase and V-PPase) 209
of root tissues in the H genotype were lower than the L genotype at both seedling (Fig 1A) 210
and flowering stages (Fig S1A) Given that proton pumps in the tonoplast supply energy for 211
vacuolar NO3- accumulation (Krebs et al 2010) NO3
- influx into the vacuole was 212
consequently much slower in the H genotype compared with the L genotype at both seedling 213
(Fig 1B) and flowering stages (Fig S1B) Moreover the percentage of vacuolar NO3- relative 214
to the total NO3- in protoplasts of root tissues in the H genotype was lower than in the L 215
genotype at seedling (Fig 1C) and flowering stages (Fig S1C) and NO3- accumulation in the 216
cytosol increased significantly in the H genotype compared with the L genotype at seedling 217
(Fig 1D) and flowering stages (Fig S1D) 218
219
B napus with higher NUE showed enhanced long-distance transport of NO3- from roots 220
to shoots 221
The relative expression of BnNRT15 in roots of the H genotype was significantly higher 222
than that in the L genotype at both seedling and flowering stages while the relative expression 223
of BnNRT18 in roots of the H genotype was lower than that in the L genotype at both 224
seedling (Fig 2-AB) and flowering stages (Fig S2-AB) As a consequence total N 225
concentration (traced by 15N) in roots of the H genotype was significantly lower than that of 226
the L genotype at both seedling (Fig 2C) and flowering stages (Fig S2C) while shoot N 227
concentration in the H genotype was significantly higher than the L genotype at seedling stage 228
(Fig 2C) This resulted in significantly higher [15N] SR ratio in the H genotype compared 229
with the L genotype (Fig 2D Fig S2D) 230
No significant differences in total N per plant between the H and L genotypes were 231
observed at both seedling and flowering stages (Fig S3) However NO3- concentration in 232
roots of the H genotype was significantly lower than in the L genotype at both seedling and 233
flowering stages (Fig 2E Fig S2E) while NO3- concentration in shoot tissues of this 234
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9
genotype was significantly higher at seedling stage (Fig 2E) resulting in higher [NO3-] SR 235
ratios at both stages (Fig 2F Fig S2F) 236
NO3- concentration in the xylem sap xylem sap volume and total NO3
- in xylem sap 237
were significantly higher in the H genotype than in the L genotype at seedling stage (Fig 238
S4ACE) the amount of NO3- in xylem sap significantly increased in the H genotype relative 239
to the L genotype at flowering stage (Fig S4F) Together these data suggests greater 240
mobilization of NO3- from root to shoot in the H genotype 241
242
NO3- up-regulates NRT15 but down-regulates NRT18 243
244
A previous study showed that increased NRT18 expression in nrt15 mutants (Chen et al 245
2012) and the expression of NRT15 was induced by NO3- (Lin et al 2008) We tested the 246
relationship between NRT15 and NRT18 expression in A thaliana and B napus No 247
significant differences were observed in AtNRT15 expression in roots between wild type 248
(col-0) and mutant plants (nrt18-2) (Fig 3A) However AtNRT15 expression was 249
significantly up-regulated by NO3- in roots of nrt18-2 mutants (Fig 3B) Expression of 250
AtNRT18 significantly increased in roots of nrt15-3 mutants but not in col-0 plants and 251
there were no significant differences in AtNRT18 expression with or without NO3- treatment 252
in roots of nrt15-3 mutants (Fig 3CD) 253
In contrast BnNRT15 expression in roots of the H genotype increased significantly with 254
NO3- treatment (Fig 3E) while the reverse was observed in expression of BnNRT18 which 255
showed lower expression under NO3- treatment than control (Fig 3F) This suggests that NO3
- 256
induces the expression of NRT15 but down-regulates NRT18 in A thaliana and B napus 257
Further studies are needed to elucidate the mechanisms regulating this reverse regulation 258
259
Reduced VSC of NO3- in roots drives its long-distance transport from roots to shoots 260
Our previous study showed that Bafi (Bafilomycin A1) inhibits V-ATPase DCCD 261
(DCCD + Na2SO3) inhibits V-PPase and a 11 combination of Bafi and DCCD (B+D) 262
inhibits both V-ATPase and V-PPase (Han et al 2015a) These inhibitors were used to 263
control activities of the tonoplast proton pumps in the H B napus plants V-ATPase activity 264
significantly decreased under Bafil and B+D treatments relative to the control and DCCD 265
treatments whereas V-PPase activity declined significantly under DCCD and B+D treatments 266
relative to that in the control and Bafil treatments (Fig 4A) 267
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10
The V-ATPase activity in roots of A thaliana was significantly lower in the V-ATPase 268
mutants (vha-a2 and vha-a3) than the wild type (col-0) and V-PPase mutants (avp1) (Fig 5A) 269
In contrast the V-PPase activity in roots of V-PPase mutant (avp1) were significantly lower 270
than in the wild type (col-0) and V-ATPase mutants (vha-a2 and vha-a3) (Fig 5A) 271
NO3- influxes into vacuoles of the H B napus plants significantly decreased when treated 272
with inhibitors of proton pumps (Bafil DCCD B+D) (Fig 4B) Similarly NO3- influxes into 273
the vacuole of the Arabidopsis mutants (vha-a2 vha-a3 and avp1) was significantly lower 274
than those found in the wild type (col-0) (Fig 5B) Previous studies showed that VSC of NO3- 275
decreased when the activities of the proton pumps decline (Li et al 2005 Krebs et al 2010 276
Han et al 2015a) 277
We further investigated NO3- distribution between the vacuole and cytosol as affected by 278
proton pump inhibition in the H genotype of B napus and the mutants of A thaliana 279
defective in vacuolar proton pumps The percentage of vacuolar NO3- relative to total NO3
- in 280
root protoplasts of the H genotype treated with inhibitors (Bafil DCCD B+D) was lower than 281
that observed in the control (Fig 4C) Results were also similar when using A thaliana 282
mutants where the percentage of vacuolar NO3- relative to the total NO3
- in protoplasts was 283
lower in the mutants (vha-a2 vha-a3 and avp1) than in the wild type (col-0) (Fig 5C) 284
Consequently NO3- accumulation in the cytosol showed a significant increase when energy 285
pumps were suppressed in roots of the H genotype (Fig 4D) and NO3- accumulation in the 286
cytosol of root tissues of A thaliana mutants (vha-a2 vha-a3 and avp1) similarly increased 287
compared with col-0 (Fig 5D) 288
Based on previous observations (Lin et al 2008) expression of NRT15 is strongly 289
induced by NO3- but expression of NRT18 is down-regulated (Fig3 Chen et al 2012) We 290
then hypothesized that expression of these two genes is contrastingly regulated by 291
concentration of NO3- in the cytosol Our results were congruent with this hypothesis the 292
expression of BnNRT15 in root tissues of the H genotype of B napus was significantly higher 293
in plants treated with Bafil DCCD or B+D compared with the control (Fig 4E) whereas the 294
expression of BnNRT18 decreased substantially (Fig 4F) Similar results were also observed 295
in A thaliana where expression of AtNRT15 in roots of the mutants (vha-a2 vha-a3 and 296
avp1) were significantly higher than in the wild type (col-0) but expression of AtNRT18 in 297
the same mutants were considerably lower (Fig 5EF) 298
NO3- concentrations in the xylem sap the N-distribution between shoot and root (SR 299
ratios based on [15N]) and the [NO3-] in shoots relative to roots of the H genotype treated with 300
energy pumpsrsquo inhibitors were significantly higher than in the control (Fig 4GH Fig S5A) 301
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11
Similar trends were also observed when using mutants of A thaliana deficient in the energy 302
pumpsrsquo activities NO3- concentration in the xylem sap the [15N] SR ratios and [NO3
-] SR 303
ratios were all significantly higher in the mutants than in the wild type (Fig 5GH Fig S5B) 304
These data clearly showed that prevention of N sequestration in vacuoles would enhance its 305
translocation to shoot 306
Additional evidence linking NO3- long-distance transport and NO3
- short-distance 307
distribution within cells were provided through experiments comparing A thaliana wild type 308
(Ws) and mutant clca-2 (Fig S6) The chloride channel (CLCa) is the main channel for 309
vacuolar anion accumulation Vacuolar sequestration capacity of NO3-significantly declines in 310
clca mutants (Angeli et al 2006) NO3- influx into the vacuolar space of Ws roots was 311
significantly higher than that observed in the clca-2 mutants (Fig S6A) This resulted in a 312
smaller proportion of vacuolar NO3- in clca-2 plants relative to the total NO3
- in root tissue 313
protoplasts as compared with Ws (Fig S6B) Consequently the accumulation of NO3- in the 314
cytosol was significantly higher in clca-2 than in Ws plants (Fig S6C) The higher 315
NO3-concentration in the cytosol together with the higher expression of AtNRT15 coupled 316
with lower expression of AtNRT18 in clca-2 roots (Fig S6DE) resulted in significant 317
increase in xylem sap NO3- concentration [15N] SR ratios and [NO3
-] SR ratios compared 318
with Ws plants (Fig S6FGH) 319
320
Increased NO3- translocation to shoots enhanced NUE 321
NO3- assimilation efficiency is known to be higher in shoots than in roots (Smirnoff et al 322
1985 Andrews et al 1986 Tang et al 2012 Tang et al 2013) We therefore hypothesize 323
that increased NO3- translocation to shoot and the consequent higher shootroot ratio will 324
contribute to higher NUE Our data support this hypothesis The H B napus genotype showed 325
enhanced long-distance transport of NO3- from roots to shoots (Fig 2 Fig S2) This enhanced 326
NO3- transport requires higher carbon skeleton provided through higher photosynthetic rate 327
(Tang et al 2012 Tang et al 2013) Our results showed that chlorophyll content 328
intercellular CO2 concentration and photosynthetic rate were significantly higher in the H than 329
the L genotype at both seedling and flowering stages (Table 2) Moreover the NO3- 330
assimilating enzymes were strongly induced by NO3- providing sufficient capacity for N 331
assimilation (Smirnoff et al 1985 Andrews et al 1986) Nitrate reductase (NR) and 332
glutamine synthetase (GS) activities in roots of the H genotype were significantly lower than 333
in the L genotype both at seedling and flowering stages (Fig S7) In contrast NR activities in 334
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12
shoots of the H genotype were significantly higher than in the L genotype at both stages (Fig 335
S7AB) and GS activity in the H genotype was also higher than those of the L genotype at 336
seedling stage (Fig S7C) 337
Experiments comparing A thaliana wild type (col-0 Ws) with mutants defective in 338
vacuolar proton-pumps (vha-a2 vha-a3avp1) and transport channel (clca-2) yielded similar 339
results that is increases in NO3- shootroot ratio essentially contributed to enhanced NUE 340
(Fig 6) The NO3- shootroot ratios in A thaliana mutants were higher than that in the wild 341
type (Fig 5GH) Generally the efficiency of inorganic N (NO3-) assimilation into organic N 342
is higher in shoots than in roots (Smirnoff et al 1985 Andrews et al 1986) therefore the 343
NO3- concentration in A thaliana mutants decreased (Fig 6CD) leading to higher NUE 344
compared with the wild type (Fig 6AB) 345
346
BnNRT15BnNRT18 in B napus affects NO3- long-distance transport from roots to 347
shoots 348
Both B napus and A thaliana are members of Cruciferae Family The amino acid 349
sequence identity between BnNRT15 and AtNRT15 was 90 (Fig S8A) the amino acid 350
sequence similarity between BnNRT18 and AtNRT18 was 908 (Fig S8B) Comparisons of 351
nucleotide and amino acid sequences (Harper et al2012) of these genes 352
(httpbrassicanbiacukcgi-binmicroarray_databasecgi) showed that BnNRT15 353
(EV220114) and BnNRT18 (EV116423) of B napus are respectively highly homologous 354
with AtNRT15 and AtNRT18 and the two genes are mainly expressed in roots of both species 355
showing similar organ-specificity (Lin et al 2008 Li et al 2010) 356
NO3- long-distance transport from roots to shoots is therefore regulated by NRT15 and 357
NRT18 as reported before (Lin et al 2008 Li et al 2010) The [15N-traced] SR ratios and 358
the [NO3-] SR ratios were significantly lower in nrt15-3 mutants relative to the wild type 359
(col-0) while [15N] SR and [NO3-] SR ratios showed significant increase in nrt18-2mutant 360
relative to col-0 (Fig S9CD) A previous study showed that expression of NRT15 is 361
down-regulated by ACC and MeJA treatments while the expression of NRT18 is strongly 362
up-regulated in A thaliana (Zhang et al 2014) Consequently NO3- accumulated in plant 363
roots probably as an adaptive measure for abiotic stresses (Chen et al 2012) Both the H- 364
and L- B napus genotypes showed significantly lower expression of BnNRT15 in roots when 365
treated with ACC (Fig S10A) but the expression of BnNRT18 remained higher than in the 366
control plants (Fig S10B) This resulted in significantly lower [15N] SR ratios following 367
ACC treatment (Fig S10CDE) These data indicate that BnNRT15 and BnNRT18 play 368
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13
similar functions for NO3- long-distance transport between root and shoot in both B napus 369
and A thaliana (Lin et al 2008 Li et al 2010) 370
371
372
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14
Discussion 373
Nitrate long-distance transport from root to shoot is regulated by cytosolic NO3- in roots 374
NO3- long-distance transport from root to shoot is controlled by NRT gene family For 375
instance xylem NO3- loading and unloading are mainly controlled by NRT15 and NRT18 376
respectively (Lin et al 2008 Li et al 2010) Whereas the NO3- short-distance transport 377
between the vacuole and cytosol is controlled by tonoplast proton-pumps (V-ATPase and 378
V-PPase) (Krebs et al 2010) and by activity of the CLCa channel in the tonoplast membrane 379
(Angeli et al 2006 Wege et al 2014) tonoplast proton-pumps provide the energy required 380
for NO3- accumulation into the vacuole through CLCa (Gaxiola et al 2001 Brux et al 2008 381
Krebs et al 2010) The expression of NRT15 is up-regulated by NO3- in plant culture 382
solution (Lin et al 2008) whereas regulation of NRT18 expression is contrary to that of 383
NRT15 under both control and abiotic stress conditions (Li et al 2010 Chen et al 2012 384
Zhang et al 2014) 385
Despite the progress made in characterizing the genes involved in transport and 386
metabolism of NO3- in plants the relationship between long- and short-distance transport and 387
how these two pathways are regulated have not been thoroughly investigated We hypothesize 388
that NRT15 expression and its role in long-distance transport is regulated by NO3- in plant 389
tissue as was observed in culture solution (Lin et al 2008) and our data support this 390
hypothesis Both BnNRT15 and AtNRT15 expression were up-regulated by increasing 391
cytosolic NO3- in roots (Fig 4 Fig 5) The increase in cytosolic NO3
- was achieved through (i) 392
use of A thaliana mutants defective in proton-pumps (V-ATPase and V-PPase) or CLCa 393
activities in the tonoplast and (ii) inhibition of B napus tonoplast proton-pumps In both 394
model plants the influx of NO3- into vacuoles was substantially reduced resulting in higher 395
concentrations of NO3- in the cytosol (Fig 4A-D Fig 5A-D Fig S6A-C) Under those 396
conditions expression of BnNRT15 and AtNRT15 was up-regulated by the enhanced 397
cytosolic NO3- while expression of BnNRT18 and AtNRT18 was down-regulated (Fig 4EF 398
Fig 5EF FigS6DE) As a consequence more NO3- was loaded into the xylem sap and 399
transported from root to shoot (Fig S5AB FigS6F) This is also reflected as increased 400
shootroot ratios of N traced by [15N] and [NO3-] (Fig 4GH Fig 5GH Fig S6GH) 401
Interestingly the expression of both BnNRT15 and AtNRT15 is up-regulated by 402
increased cytosolic NO3- concentration in roots but were not affected by NO3
- sequestered 403
into vacuoles (Fig 4 Fig 5 Fig S6) This is because vacuolar NO3- is functionally separated 404
by the tonoplast and cannot be assimilated by enzymes that are localized in the cytosol 405
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15
Therefore NO3--inducible genes such as NR and NRT15 were not influenced by vacuolar 406
NO3- concentration (Martinoia et al 1981 Martinoia et al 2000 Zhang et al 2012) In 407
addition NO3- concentration in the cytosol is controlled by its short-distance transport through 408
the tonoplast which is mediated through the tonoplast proton-pumps providing the required 409
energy for its sequestration into vacuoles against concentration gradient (Gaxiola et al 2001 410
Brux et al 2008 Krebs et al 2010) The gradient of H+ between the inside of the vacuole 411
and the cytosol is maintained by an 2NO3-1H+ antiporter mechanism that facilitatesNO3
- 412
transport through CLCa (Angeli et al 2006 Wege et al 2014) Our data provide clear 413
evidence that NO3- long-distance transport from root to shoot is regulated by cytosolic NO3
- 414
concentration in roots 415
Regulatory mechanisms that control the expression of NRT18 and NRT15 as mediated 416
by NO3- are still unknown however previous studies showed that their expression is also 417
affected by abiotic stresses whereby NRT15 is down-regulated and NRT18 is up-regulated 418
(Li et al 2010 Zhang et al 2014) AtNRT18 expression in A thaliana nrt15 mutants is 419
highly up-regulated (Chen et al 2012) as was also observed here (Fig 3C) Moreover 420
AtNRT15 expression was enhanced by NO3- in culture solution and was not affected in 421
nrt18-2 mutant (Fig 3AB) On the other hand AtNRT18 expression was not affected by 422
cytosolic NO3- but down-regulated when AtNRT15 expression was up-regulated by NO3
-(Fig 423
S6DE) Analogous results were also obtained with B napus genotypes (Fig 3EF) Based on 424
these results we argue that NRT18 expression is not directly influenced by cytosolic NO3- but 425
rather by NRT15 expression (Fig 4 Fig 5 and Fig S6) suggesting NRT18 is probably 426
acting downstream of NRT15 Apparently the regulatory mechanisms of this NRT gene 427
family still awaits further studies to be elucidated 428
429
Response of NUE to NO3- long-distance transport 430
NO3- assimilation efficiency is higher in shoot than in root tissues and can be further 431
enhanced by carbon assimilation (Smirnoff et al 1985 Tang et al 2013) Therefore 432
translocation of higher proportion of NO3- from roots to shoots likely contributes to better 433
crop growth and higher NUE (Andrews et al 1986 Tang et al 2012) The present study 434
provides direct evidence for these results using contrasting B napus genotype identified 435
before (Zhang et al 2009 Han et al 2015a) Our data confirmed the genetic variation in 436
NUE between these two genotypes (Table 1) 437
We measured NO3- distribution in root and shoot of these genotypes to elucidate the 438
physiological mechanisms contributing to variation in NUE The data were consistent with 439
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16
our hypothesis that concentration of NO3- in the xylem sap and shoot would be significantly 440
higher in the H genotype than in the L genotype (Fig 2EFFig S2EF Fig S4) The NO3- 441
long-distance transport from root to shoot was affected by cytosolic NO3- in roots of both A 442
thaliana and B napus genotypes (Fig 4-5 Fig S6) The activities of the tonoplast 443
proton-pumps in roots were significantly lower in the H genotype than in the L genotype (Fig 444
1AB) Consequently less NO3- was sequestered into the vacuole and more NO3
- was retained 445
in cytosol (Fig 1CD) BnNRT15 expression was then up-regulated resulting in greater 446
loading into the xylem while BnNRT18 was down-regulated (Fig 2AB) probably to 447
suppress NO3- unloading from xylem sap Consequently a higher proportion of NO3
- was 448
transported from root to shoots in the H genotype(Fig 2C-D Fig S4) 449
Moreover photosynthetic carbon fixation (Table 2) and activities of both NR and GS 450
were higher in shoots of the H genotype (Fig S7) both of which promote higher N 451
assimilation These results agreed with previous reports (Smirnoff et al 1985 Andrews et al 452
1986 Tang et al 2012 Tang et al 2013) where higher NO3- transport from root to shoot 453
higher photosynthetic rate and N assimilation efficiency in shoots were suggested as essential 454
mechanisms for high-NUE in crop plants These qualities endowed the B napus H genotype 455
Xiangyou15 with higher NUE as compared with the L genotype 814 456
NO3- long-distance transport in B napus is not only regulated by BnNRT15 and 457
BnNRT18 in root tissues (Fig 4 Fig 5 Fig S6) but also by stomatal conductance and 458
transpiration by indirectly controlling xylem sap flow and long-distance transport 459
(Dechorgnat et al 2011 Krapp et al 2014) Stomatal conductance and transpiration rates of 460
the H genotype were significantly higher than those of the L genotype (Table S1) a result 461
consistent with other studies describing characteristics of high-NUE plants (Daniel-Vedele et 462
al 1998 Wilkinson et al 2007 Dechorgnat et al 2011 Krapp et al 2014) 463
464
Response of NUE to NO3- short-distance transport 465
NUE is also strongly controlled by NO3- short-distance distribution between the vacuole 466
and cytosol (Han et al 2015a) A lower proportion of NO3- accumulating in vacuoles and a 467
higher proportion retained in the cytosol will contribute to better NUE in crop plants (Han et 468
al 2015b) The energy required for vacuolar NO3- sequestration is provided by the tonoplast 469
proton-pumps (Gaxiola et al 2001 Brux et al 2008 Krebs et al 2010) while the 470
distribution channel CLCa enables transport of NO3- across the vacuolar membrane (Angeli et 471
al 2006 Wege et al 2014) The activities of the tonoplast proton-pumps (V-ATPase and 472
V-PPase) in leaves of the H genotype were significantly lower than those of the L genotype at 473
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17
flowering stages (Han et al 2015aHan et al 2015b) thus we expected similar differences in 474
root tissues Our results confirmed that the activities of tonoplast proton-pumps in root tissues 475
of B napus H genotype and A thaliana mutants were lower than those in the L genotype and 476
A thaliana wild type leading to less NO3- influx into vacuoles and more in the cytosol (Fig 1 477
Fig 5A-D) Moreover the enzymes catalyzing N assimilation are located in the cytoplasm 478
where NO3- could be assimilated (Martinoia et al 1981 Han et al 2015a) or immediately 479
loaded into xylem sap for long-distance transport from roots to shoots (Lin et al 2008) 480
Consequently the NO3- concentration in root tissues of the H genotype and A thaliana 481
mutants defective in tonoplast proton-pumps was significantly lower than that in the L 482
genotype (Fig 2E) and in A thaliana wild type (Fig 6C) Therefore the higher proportion of 483
NO3- transported to shoot and the lower concentrations retained in root vacuoles contributed 484
to the higher NUE in Xiangyou15 and A thaliana mutants (vha-a2 vha-a3 and avp1) (Table 485
1Fig 6A) Further studies are needed to assess the impacts of regulating vacuolar 486
sequestration of NO3- and rates of long-distance transport for enhancing NUE in other crop 487
species and genotypes 488
These data suggested that NO3- distribution between vacuoles and cytosol within cells 489
regulates NO3- long-distance transport from root to shoot and ultimately affects NUE NO3
- 490
loading into the xylem sap is an active transport process (Lin et al 2008) therefore not 491
regulated by absolute concentration of NO3- in the cytosol but rather by genes involved in 492
NO3- long-distance transport (NRT15 and NRT18) Apparently the expression of NRT15 the 493
gene responsible for xylem loading is regulated by NO3-concentration in the cytosol 494
Therefore both NO3- short-distance transport across the tonoplast and its long-distance 495
transport from root to shoot are important determinants of NUE in both B napus and A 496
thaliana (Table 1 and Fig 6) 497
498
Roles of NRT15 and NRT18 in NO3- long-distance transport in B napus and A 499
thaliana 500
Our results (Figs S9 S10) showed that the proportion of NO3- distributed from root to 501
shoot is strongly controlled by NRT15 in accord with previous studies (Lin et al 2008 Li et 502
al 2010) The data suggest that the high-NUE genotypes possess lower activities of tonoplast 503
proton-pumps (V-ATPase and V-PPase) resulting in less NO3- accumulation in vacuoles and 504
more NO3- retention in cytosol (Fig 7) The higher NO3
- in the cytosol then up-regulated 505
NRT15 which then down-regulates NRT18 As a result more NO3- is loaded into the xylem 506
system by NRT15 mechanism and less NO3- is unloaded via NRT18 (Fig 7) Differential 507
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18
regulation of these NRT genes in high-NUE plants increased transport of NO3- from root to 508
shoot through the xylem vascular tissues (Fig 7) This coordinated NO3- long- and 509
short-distance transport likely fine-tunes NO3- allocation and modulates the balance of NO3
- 510
distribution between roots and shoots (Fig 7) 511
Our research also revealed differences between A thaliana and B napus in the 512
expression of the wild-type NRT genes in response to NO3- in the protoplast Moreover using 513
A thaliananrt15-3 mutants we uncovered possible regulatory interactions between a 514
functioning (wild-type) NRT15 gene and NRT18 expression in response to NO3- 515
concentration Because mutants defective in nrt15 are not available for B napus we could 516
not confirm that the respective NRT15 and NRT18 systems behave in an analogous way in 517
these plant species Based on Harper et al (2012) research database our homology 518
comparisons and gene function annotations provided strong evidence for the roles of 519
BnNRT15 and BnNRT18 in B napus Finally our studies were conducted using two model 520
dicotyledonous plant species for which we had ample data on responses to specific inhibitors 521
(B napus) or specific mutants were available with results forming the bases for the model 522
presented in Figure 7 Further research using genetically diverse plant species (eg 523
monocotyledonous crops such as rice wheat and corn) will help validate this model and its 524
broader application across species The information generated in this study could have future 525
application for improving NUE in commercial crops with consequent reduction in nitrogen 526
use and benefits to the environment 527
528
Materials and Methods 529
Plant material 530
The two oilseed rape (Bnapus) cultivars used in this study have been characterized 531
before as high- (Xiangyou15 referred to as H genotype hereafter) and low- (814 referred to 532
as L genotype) nitrogen use efficiency (NUE) genotypes (Zhang et al 2009 Han et al 2015b) 533
Here we define NUE as the total biomass per unit of N uptake by the plant with total 534
biomass includes roots shoots and grains The two genotypes were provided by the Hunan 535
Sub-Center of Improvement Center of National Oil Crops Hunan China 536
The Arabidopsis thaliana wild-type Columbia-0 (col-0) was used as control for 537
V-ATPase (vha-a2 and vha-a3) V-PPase (avp1) nrt15-3 and nrt18-2 mutants whereas the A 538
thaliana wild-type Wassilewskija (Ws) was used as control for clca-2 mutants A thaliana 539
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19
mutants and transgenic lines (nrt15-3 and nrt18-2) have been described previously (Chen et 540
al 2012 Zhang et al 2014) The mutant lines vha-a2 (Salk_142642) and vha-a3 541
(Salk_122135) described by Krebs et al (2010) were obtained from the Arabidopsis 542
Biological Resources Center (ABRC) The mutant line avp1 (GK-005004) described in Li et 543
al (2005) was obtained from the European Arabidopsis Stock Centre while the mutant line 544
clca-2 (FST 171A06) described in Angeli et al (2006) was from the Institute National de la 545
Recherche Agronomique (INRA) collection in France together with the T-DNA mutants used 546
for genotyping to select pure mutant lines 547
548
Growth conditions 549
B napus plants were grown hydroponically in ceramic pots(20cmtimes15cm) filled with a 550
nutrient solution in the greenhouse under natural light as described in Han et al (2015b) The 551
pots were arranged in a completely randomized design with six biological replications The 552
nutrient solution was replaced every 3 days and its pH adjusted to 55 daily Experiments were 553
conducted at the field station of Hunan Agricultural University Southern China 554
A thaliana plants were grown in a nutrient solution in plastic pots as described in Arteca 555
et al (2000) and Gong et al (2003) The solution was changed every 3 days with pH adjusted 556
daily to 58 Pots were arranged in a completely randomized design with six biological 557
replications The nutrient solution used for both species consisted of 125mM KNO3 558
0625mM KH2PO4 05mM MgSO4 05mM Ca (NO3)2middot4H2O 0025mM Fe-EDTA 025 559
mlL-1 micronutrients (stock solution concentrations 70mM B 14mM Mn 1mM Zn 05mM 560
Cu and 02mM Mo) The experiments were conducted at Hunan Agricultural University in a 561
phytotron set at 70 relative humidity16h-light8h-dark cycle and constant temperature of 562
22degC 563
564
Experimental treatments and phenotyping 565
All measurements made on B napus plants were conducted either at the seedling stage 566
(2 months after transplanting) or at flowering (5 months after transplanting) The whole root 567
tissue was harvested and used for analyses at either stage For shoot measurements the fourth 568
leaf from the bottom was used for measurements made at seedling stage whereas the 12th leaf 569
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20
from the bottom was harvested for measurements made at flowering stage For A thaliana 570
whole root and shoot of four-week-old plants were sampled and used for different assays 571
572
Vacuolar proton-pumps inhibitor treatments 573
Inhibition of vacuolar proton pumps of B napus (Xiangyou15) was conducted at the seedling 574
stage in hydroponic culture as described in Han et al (2015a) with minor modifications 575
Plants grown in hydroponic solution were used as control Inhibitor treatment was conducted 576
using several chemicals Bafi(25 nmol L-1 Bafilomycin A1) an inhibitor of V-ATPase 577
DCCD(10 μmol L-1DCCD + 50 mmolL-1 Na2SO3) specifically inhibits V-PPase and 578
Bafi+DCCD in a 11 ratio as inhibitor of both V-ATPase and V-PPase The inhibitors were 579
applied in the hydroponic solutions for 24 h Fresh plant tissue samples from all treatments 580
were collected for different assays 581
582
NO3- induced gene expression 583
Four-week old A thaliana mutants (nrt15-3nrt18-2) were grown in hydroponics with 584
225mM (NH4)2succinate for 3 days and shifted to hydroponics with 45mM NO3- for 12 h 585
then root tissues were collected to assess relative expression of AtNRT15 and AtNRT18 586
genes Seedlings of B napus (Xiangyou15) plants grown hydroponically were treated with 587
5mM (NH4)2succinate for 3 d and shifted to fresh hydroponic solution containing 15 mM 588
NO3- for 12 h Root tissue was then collected to assess the relative expression of BnNRT15 589
and BnNRT18 genes using quantitative RT-PCR 590
591
ACC treatment 592
Hydroponically grown seedlings of B napus were subjected to ACC treatment as described in 593
Zhang et al (2014) with minor modifications Seedlings of the two genotypes Xiangyou15 594
and 814 were transferred to a nutrient solution containing 002mM L-1 ACC for 6 h or grown 595
in normal hydroponic solution as control Root tissues were then collected to assay relative 596
expression of BnNRT15 and BnNRT18 using quantitative RT-PCR Another set of B napus 597
plants were grown in ACC-containing hydroponic solution but supplemented with 15NO3- (22 598
15N) replacing the unlabeled NO3- for 1 h Root and shoot tissues were collected separately 599
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21
for 15N measurement using a continuous-flow isotope ratio mass spectrometer coupled with a 600
C-N elemental analyzer (ANCA-MS PDZ Europa) 601
602
Biomass and N concentration 603
Grain yield dry biomass and N concentration were determined in plant samples at 604
harvest Plants were harvested (including senescing leaves) then dried in an oven for 30 min 605
at 105degC then at 70degC to a constant weight and weighed N concentration was determined 606
using the Kjeldahl method (Shi et al 2010 Han et al 2015b)02g of dried samples were 607
digested with H2SO4-H2O2 in a 100-ml Kjeldahl digestion flask after which N concentration 608
was determined using a Foss Auto Analyser Unit (Kjeldahl 8400) 609
610
V-ATPase and V-PPase activities 611
Roots (10 g) of B napus were collected at seedling and flowering stages and that of A 612
thaliana (05 g) sampled at seedling stage then used for determining the activities of 613
V-ATPase and V-PPase (Han et al 2015b) Activities of these two energy pumps within 614
microsomal membranes were followed colorimetrically based on released Pi as described by 615
Zhu et al (2001) and Krebs et al (2010) 616
617
Assay of NO3- flux in vacuoles 618
Roots of both species were collected similarly as that used for V-ATPase and V-PPase 619
activities then used for vacuole isolation and measurement of NO3- flux in vacuoles as 620
described by Robert et al (2007) with minor modification (Han et al 2015b) Net fluxes of 621
NO3- in vacuoles were measured non-invasively using SIET (scanning ion-selective electrode 622
technique SIET system BIO-003A Younger USA Science and Technology Corporation 623
USA) An NO3--selective microelectrode used for the assay of NO3
- flux was vibrated in the 624
measuring solution between two positions 1 μm and 11 μm from the vacuole surface 625
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles Background 626
signal was recorded by vibrating the electrode in the measuring solution in the absence of 627
vacuoles Ion flux was calculated by Fickrsquos law of diffusion J = -D(dcdx) where J represents 628
the ion flux (picomolescm-2s-1) dcdx is the ion concentration gradient and D is the ion 629
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22
diffusion constant in a particular medium The direction of the flux was derived from Fickrsquos 630
law of diffusion that relates the concentration gradient 631
632
Isolation of intact protoplasts and vacuoles for determining NO3- concentration 633
Similar to the above assays root tissues (10 g) were collected from B napus at seedling 634
and flowering stages and from A thaliana (05 g) seedlings and used to isolate intact 635
protoplasts and vacuoles as described by Robert et al (2007) with minor modifications as 636
outlined in Huang et al (2012) and Han et al(2015b) The purified protoplasts were divided 637
into two equal aliquots with one of them used for isolation of vacuoles The purified 638
protoplasts and vacuoles were then sub-sampled and used to determine NO3- concentrations 639
(Vogeli-Lange and Wagner 1990) and for enzyme activity assays (Ma et al 2005) 640
NO3-concentrations in protoplasts and vacuoles were measured by a continuous-flow 641
auto-analyzer (Auto Analyser 3 Bran and Luebbe Germany) as described previously (Han et 642
al 2015b)The activities of acid phosphatase (ACP) and cytochrome oxidase (COX) were 643
determined using plant ACP colorimetry- and COX-assay kits (GenMedSci Inc) following 644
the manufacturerrsquos instructions ACP activity specific to vacuoles was determined and used to 645
normalize NO3- accumulation We measured NO3
- in the protoplast outside the vacuole which 646
includes the cytosol and organelles eg mitochondria and Golgi Apparatus (Robert et 647
al2007) Since most of the NO3- in the protoplast outside the vacuole is located in the cytosol 648
(Krebs et al 2010) we refer to NO3- distribution between vacuoles and cytosol rather than 649
vacuole versus protoplast 650
651
Quantitative RT-PCR 652
Total RNA extracted from B napus roots was prepared using Trizol reagent (Invitrogen) 653
cDNAs were synthesized using M-MLV reverse transcriptase (Promega) following 654
manufacturerrsquos protocol The relative expression of BnNRT15 (EV220114) BnNRT18 655
(EV116423) and Bnactin (AF1118121) genes in plant roots were determined by quantitative 656
RT-PCR and run on a LightCycler instrument (Roche) with the SYBR Green Real-Time PCR 657
Master Mix Kit (TOYOBO Japan) under the following conditions 95degC for 2min then 45 658
cycles of 95degC for 10s 60degC for 10s and 72degC for 20s The primer sequences of BnNRT15 659
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23
BnNRT18 and Bnactin genes were obtained from httpbrassicanbiacuk as described in 660
Harper et al (2012) and expression levels were normalized to Bnactin as control 661
Total RNA extracted from the 4-week-old A thaliana roots under the treatments 662
conditions described above were prepared using Trizol reagent (Invitrogen) The cDNAs were 663
synthesized using M-MLV reverse transcriptase (Promega) based on the manufacturerrsquos 664
protocol and as described in Li et al (2010)The primer sequences of quantitative RT-PCR 665
used in these assays are listed in Supplemental Table 2 and were described previously (Chen 666
et al 2012 Zhang et al 2014) and their expression was normalized to Atactin2 as control 667
668
Determination of nitrate distribution using 15NO3- 669
Hydroponically grown B napus (seedling and flowering stages) and A thaliana 670
(seedling) were transferred to 01mM CaSO4 solution for 1min then to their respective 671
hydroponic nutrient solutions with15NO3- (22 excess) replacing unlabeled NO3
- for 1h 672
Plants were then transferred to 01mM CaSO4 solution for 1min after which roots were 673
washed with deionized water Root and shoot samples were separated and dried at 105degC for 674
30min followed by 70degC for 3 d then grounded and used for assaying 15N content using a 675
continuous-flow isotope ratio mass spectrometer coupled with a carbon-nitrogen elemental 676
analyzer (ANCA-MS PDZ Europa) 677
678
Xylem sap collection and assay of nitrate concentration 679
We used the method of Tang et al (2012) for collecting xylem sap Plants were cut 680
leaving1cm segments with intact roots at seedling and flowering stages for B napus and at 681
seedling stage for A thaliana Roots were immediately immersed in their respective nutrition 682
solutions Weighed cotton was put on the cut surface to absorb extruding xylem sap for 1 h 683
The cotton was then wrapped in plastic film and the volume of xylem sap was calculated as 684
the weight gain of the cotton Xylem sap was then squeezed from the cotton using a syringe 685
and used for subsequent assay of nitrate concentration using a continuous-flow auto-analyzer 686
(Auto Analyser 3 Bran and Luebbe Germany) 687
To determine nitrate concentration samples (10 g fresh root and shoot at seedling and 688
flowering of B napus and 05 g fresh root and shoot of 4-week-old A thaliana) were frozen 689
in liquid N2 and ground with a mortar and pestle the powder was then transferred to a beaker 690
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
containing 70 ml deionized water and boiled for 30 min to extract nitrate then cooled and 691
made to 100 ml volume and 02 g activated carbon added to eliminate the effect of 692
chlorophyll The mixture was filtered and nitrate in the filtrate was determined using a 693
continuous-flow auto-analyser (Atuo-Analyser 3 Bran and Luebbe Germany) 694
695
Measurements of photosynthesis 696
All measurements of photosynthesis and related parameters were conducted at 1000 h 697
using intact leaves The 4th leaf from the bottom in B napus at seedling stage and the 12th leaf 698
from the bottom at flowering stage were used for determining chlorophyll concentration 699
photosynthetic rate intercellular CO2 concentration transpiration rate and stomatal 700
conductance Chlorophyll concentration was determined by SPAD-502 (Minolta Camera Co 701
Ltd Japan) (Luo et al 2006 Yandeau-Nelson et al 2011) Photosynthetic rate intercellular 702
CO2 concentration transpiration rate and stomatal conductance were measured using LI-6400 703
Portable Photosynthesis System (Li-Cor Biosciences Co Ltd USA) set at flow rate of 500 704
μmol s-1 photosynthetic photon flux density of 1000 μmol m-2 s-1 relative humidity of 65 705
CO2 concentration of 400ppm and ambient temperatures 706
707
NR and GS activities 708
Leaves with similar age as those used for photosynthesis in B napus were used for the 709
assay of nitrate reductase (NR) and glutamine synthetase (GS) activities NR activity was 710
measured by the modified method of Fan et al (2007) Fresh samples (05g) were frozen in 711
liquid N2 ground into powder in the presence of acid-washed sand and homogenized with 4 712
ml of extraction buffer (0025mol L-1phosphate buffer pH87 1211g L-1 cysteine 0372g L-1 713
EDTA) Homogenates were centrifuged at 30000timesg for 15 min at 4degC and the supernatants 714
were treated with sulfanilamide and α-naphthylamine reagents for colorimetric (540nm) 715
determination of nitrite as described by Fan et al (2007) 716
GS activity was determined using a modified reverseγ-glutamyltransferase method 717
(Wang et al2014) which measures the GS-catalyzed formation of glutamyl-γ-hydroxamate 718
from glutamine and hydroxylamine Fresh samples were frozen for 30 min at -20degC then 719
ground in 10ml Tris-HCl buffer and acid-washed sand The homogenates were filtered 720
through two layers of gauze and centrifuged at 8000timesg for 15 min at 4degC About 12ml of the 721
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
supernatant was added in a reaction mixture and treated as described (Wang et al2014) After 722
the GS-catalyzed reaction was stopped and the solution centrifuged glutamyl-γ723
-hydroxamate was quantified colorimetrically in the supernatant (at 485 nm) and the 724
concentration was determined using a standard curve 725
726
Statistical analyses 727
We used the SPSS software (Statistical Product and Service Solutions V130 USA) for 728
ANOVA and mean separation of main-effects and interactions using Duncanrsquos multiple range 729
test at Plt005Values are presented as means and SD of three or six replicates from three 730
independent experiments Different letters or an asterisk () associated with specific data (eg 731
at the top of histogram bars in figures or within tables) denote significant differences at 732
Plt005 733
734
Supplemental Data 735
Supplemental Figure 1 B napus with higher NUE showed lower vacuolar sequestration 736
capacity (VSC) for NO3-in roots at flowering stage 737
Supplemental Figure 2 B napus with higher NUE showed enhanced long-distance transport 738
of NO3- from roots to shoots at flowering stage 739
Supplemental Figure 3 The two B napus (H and L genotypes) showed the same total N per 740
plant at seedling stage (A) and flowering stage (B) 741
Supplemental Figure 4 B napus with higher NUE showed increased NO3- concentration in 742
the xylem sap at seedling and flowering stages 743
Supplemental Figure 5 NO3- concentration in the xylem sap as affected by inhibitor 744
treatments in B napus and in the energy pumpsrsquo mutants of A thaliana (col-0 vha-a2 vha-a3 745
avp1) 746
Supplemental Figure 6 Reduced VSC for NO3- in roots drives long-distance transport of 747
NO3- from roots to shoots in the A thaliana wild type (Ws) and mutant (clca-2) 748
Supplemental Figure 7 Differences of NR and GS activities between the two B napus (H 749
and L genotypes) at seedling and flowering stages 750
Supplemental Figure 8 Amino acid sequences of BnNRT15 and BnNRT18 751
Supplemental Figure 9 Functions of AtNRT15 and AtNRT18 genes in root tissues of A 752
thaliana in controlling NO3- long-distance transport from root to shoots 753
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
Supplemental Figure 10 B napus BnNRT15 and BnNRT18 genes in roots were 754
coordinately modulated to facilitate NO3- long distance transport from roots to shoots 755
Supplemental Table 1 Differences in stomatal conductance and transpiration rate between 756
the two B napus genotypes 757
Supplemental Table 2 Sequences of primera used for qRT-PCR 758
759
Acknowledgements 760
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 761
Shanghai Institutes for Biological Sciences) for providing nrt15-3 and nrt18-2 seeds 762
This study was supported by the National Natural Science Foundation of China (Grant 763
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 764
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 765
The Ministry of Education in China Open Novel Science Foundation of Hunan province 766
(13K062) the National Key Laboratory of Plant Molecular Genetics and the Twelfth 767
Five-Year National Science and technology support program (2012BAD15BO4) 768
769
Figure 1 B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 770
NO3- in roots at the seedling stage 771
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 772
genotype 814 Specific activities of the tonoplast proton pumps are expressed as μmol Pi 773
released mg-1 protein h-1 NO3- fluxes are expressed as pmol NO3
- cm-2 S-1 Mature vacuoles 774
were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 775
measuring solution between the two positions 1 μm and 11 μm from the vacuole surface 776
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles recording the 777
stable reading data The background was recorded by vibrating the electrode in measuring 778
solution without vacuoles 779
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 780
for NO3- content and NO3
- accumulation normalized against the specific activity of the 781
vacuole acid phosphatase (ACP) as described in Materials and Methods and plotted as μmol 782
NO3- per μmol p-nitrophenol the end product of ACP assay 783
Proton pump activities in root tissues between H and L genotypes are shown at the seedling 784
stage (A) Different letters at the top of the histogram bars denote significant differences of 785
V-ATPase in root tissues between H and L genotypes (Plt005) an asterisk () at the top of the 786
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
histogram bars indicates significant difference in V-PPase activity in root tissues between H 787
and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788
Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789
genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790
bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791
bars on the figures indicate SD (n=6) 792
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793
seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794
to the total NO3- in protoplasts 795
NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796
total NO3-in the cytosol and was calculated as total NO3
- in protoplasts ndash total NO3- in 797
vacuole Different letters at the top of histogram bars denote significant differences of total 798
NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799
(n=6) 800
801
Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802
roots to shoots at the seedling stage 803
H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804
Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805
the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806
as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807
the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808
the histogram bars denote significant differences at Plt005 809
Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810
in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811
genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812
of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813
SD (n=3) different letters at the top of the histogram bars denote significant differences at 814
Plt005 level 815
The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816
at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817
genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818
different letters at the top of the histogram bars denote significant differences at Plt005 819
820
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
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b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
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Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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Zhu ZJ Qian YR Pfeiffer W (2001) Effect of nitrogen form on the activity of tonoplast pyrophosphatase in tomato roots ActaBotanica Sinica 43(11) 1146-1149
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5
up-regulated (Zhang et al 2014) Based on these studies we argue that the expression and 132
functioning of NO3- long-distance transport genes NRT15 and NRT18 are regulated by 133
cytosolic NO3- concentration In addition the vacuolar and cytosolic NO3
- distribution is 134
likely regulated by proton-pumps located within the tonoplast (V-ATPase and V-PPase) 135
(Granstedt et al 1982 Glass et al 2002 Krebs et al 2010) Therefore NO3- use efficiency 136
must be affected by NO3- long-distant transport (between shoot and root) and short-distant 137
transport (between vacuole and cytosol) However the physiological mechanisms controlling 138
this regulation are still obscure 139
Previous studies showed that the chloride channel protein (CLCa) is mainly responsible 140
for vacuole NO3- short-distance transport as it is the main channel for NO3
-movement 141
between the vacuoles and cytosol (Angeli et al 2006 Wege et al 2014) The vacuole 142
proton-pumps (V-ATPase and V-PPase) located in the tonoplast supply energy for active 143
transport of NO3- and accumulation within the vacuole (Gaxiola et al 2001 Brux et al 2008 144
Krebs et al 2010) Despite that about 90 of the volume of mature plant cells is occupied by 145
vacuoles vacuolar NO3- cannot be efficiently assimilated because the enzyme nitrate 146
reductase (NR) is cytosolic (Shen et al 2003 Han et al 2015a) However re-translocation of 147
NO3- from the vacuole to the cytosol will permit its immediate assimilation and utilization 148
Generally NO3- concentrations in plant cell vacuoles and the cytoplasm are in the range 149
of 30-50 mol m-3 and 3-5 mol m-3 respectively (Martinoia et al 1981 Martinoia et al 2000) 150
Because vacuoles are obviously the organelle for high NO3- accumulation and storage in plant 151
tissues their function in NO3- use efficiency cannot be ignored (Martinoia et al 1981 Zhang 152
et al 2012 Han et al 2015b) NO3- assimilatory system in the cytoplasm is sufficient for its 153
assimilation when it is transported out of the vacuoles Therefore NO3- use efficiency could in 154
part be dependent on vacuolar-cytosolic NO3- short-distance transport in plant tissues 155
(Martinoia et al 1981 Shen et al 2003 Zhang et al 2012 Han et al 2015a) 156
Evidently NO3- use efficiency is regulated by both NO3
- long-distance transport from 157
root to shoot and short-distance transport and distribution between vacuoles and cytoplasm 158
within cells(Glass et al 2002 Dechorgnat et al 2011 Han et al 2015a) Although vacuoles 159
compartment excess NO3- that accumulates in plant cells (Granstedt et al 1982 Krebs et al 160
2010) neither NO3- inducible NR genes (NIA1and NIA2) (Fan et al 2007 Han et al 2015a) 161
nor the NO3- long-distance transport gene NRT15 (Lin et al 2008) are regulated by vacuolar 162
NO3- even though they are essential for NO3
- assimilation Only NO3- transported from the 163
vacuole to the cytosol can play a role in regulating NO3- inducible genes Consequently we 164
argue that both NO3- assimilation in cells and its long-distance transport from root to shoot are 165
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regulated by cytosolic NO3- concentration However this hypothesis needs to be substantiated 166
The mechanisms underlying both NO3- short-distance (Gaxiola et al 2001 Angeli et al 167
2006 Brux et al 2008 Krebs et al 2010) and long-distance transport (Lin et al 2008 Li et 168
al 2010) have been previously investigated yet the underlying mechanisms regulating the 169
flux of NO3- and the obvious relationship between the two transport pathways as well as their 170
relation to NUE are not well understood 171
The NRT family of genes play a partial role in vacuolar NO3- accumulation in petioles 172
(Chiu et al 2004) and seed tissues (Chopin et al 2007) whereas the proton pumps and 173
CLCa system in the tonoplast play a major role in accumulating NO3- in vacuoles (Gaxiola et 174
al 2001 Angeli et al 2006 Brux et al 2008 Krebs et al 2010) The vacuolar NO3- 175
short-distance transport system is spread throughout the plant tissues and is the principal 176
means by which vacuolar NO3- short-distance transport and distribution is controlled (Angeli 177
et al 2006Krebs et al 2010) 178
The NRT genes seem to work synergistically to control NO3- long-distance transport 179
between roots and shoots NRT19 is responsible for NO3- loading into the phloem (Wang et 180
al 2011) whereas NO3- loading and unloading into xylem are regulated by NRT15 and 181
NRT18 respectively (Lin et al 2008 Li et al 2010) Phloem transport mainly involves 182
organic N the inorganic-N (NO3-) concentrations in the phloem sap are typically very low 183
ranging from one-tenth to one-hundredth of that of the inorganic-N in xylem sap (Lin et al 184
2008Fan et al 2009) Therefore this study focused on NO3- short-distance transport 185
mediated through the tonoplast proton-pumps and the CLCa system and the long-distant 186
transport mechanisms responsible for xylem NO3- loading and unloading via NRT15 and 187
NRT18 respectively 188
Questions related to how long and short-distance transport of NO3- are coupled in plant 189
tissues and their role in determining NUE were addressed using a pair of high- and low-NUE 190
B napus genotypes and Arabidopsis thaliana Application of proton pump inhibitors and 191
ACC in the former and use of mutants with defective proton pumps in the latter allowed 192
experimental distinction of the physiological mechanisms regulating these processes Data 193
presented here provide strong evidence from both model plants supporting this linkage and 194
strongly suggest that cytosolic NO3- concentration in roots regulates NO3
- long-distance 195
transport from roots to shoots We also investigated how NO3- concentration in plant tissues 196
would be affected by NO3- long-distance transport vacuolar NO3
- sequestration and the 197
ensuing relationship with NO3- use efficiency We also proposed the physiological 198
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7
mechanisms likely to be important for enhancing NO3- use efficiency in plants These findings 199
will provide scientific rationales for improving NUE in important industrial and food crops 200
201
202
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Results 203
B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for NO3- 204
in root tissues 205
Our previous work identified high and low NUE B napus genotypes (Han et al 2015a 206
Han et al 2015b) NUE of B napus whether based on biomass or on grain yield (Table 1) 207
was significantly higher for the high-NUE genotype (H Xiangyou15) than for the low-NUE 208
genotype (L 814) That the activities of the tonoplast proton-pumps (V-ATPase and V-PPase) 209
of root tissues in the H genotype were lower than the L genotype at both seedling (Fig 1A) 210
and flowering stages (Fig S1A) Given that proton pumps in the tonoplast supply energy for 211
vacuolar NO3- accumulation (Krebs et al 2010) NO3
- influx into the vacuole was 212
consequently much slower in the H genotype compared with the L genotype at both seedling 213
(Fig 1B) and flowering stages (Fig S1B) Moreover the percentage of vacuolar NO3- relative 214
to the total NO3- in protoplasts of root tissues in the H genotype was lower than in the L 215
genotype at seedling (Fig 1C) and flowering stages (Fig S1C) and NO3- accumulation in the 216
cytosol increased significantly in the H genotype compared with the L genotype at seedling 217
(Fig 1D) and flowering stages (Fig S1D) 218
219
B napus with higher NUE showed enhanced long-distance transport of NO3- from roots 220
to shoots 221
The relative expression of BnNRT15 in roots of the H genotype was significantly higher 222
than that in the L genotype at both seedling and flowering stages while the relative expression 223
of BnNRT18 in roots of the H genotype was lower than that in the L genotype at both 224
seedling (Fig 2-AB) and flowering stages (Fig S2-AB) As a consequence total N 225
concentration (traced by 15N) in roots of the H genotype was significantly lower than that of 226
the L genotype at both seedling (Fig 2C) and flowering stages (Fig S2C) while shoot N 227
concentration in the H genotype was significantly higher than the L genotype at seedling stage 228
(Fig 2C) This resulted in significantly higher [15N] SR ratio in the H genotype compared 229
with the L genotype (Fig 2D Fig S2D) 230
No significant differences in total N per plant between the H and L genotypes were 231
observed at both seedling and flowering stages (Fig S3) However NO3- concentration in 232
roots of the H genotype was significantly lower than in the L genotype at both seedling and 233
flowering stages (Fig 2E Fig S2E) while NO3- concentration in shoot tissues of this 234
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genotype was significantly higher at seedling stage (Fig 2E) resulting in higher [NO3-] SR 235
ratios at both stages (Fig 2F Fig S2F) 236
NO3- concentration in the xylem sap xylem sap volume and total NO3
- in xylem sap 237
were significantly higher in the H genotype than in the L genotype at seedling stage (Fig 238
S4ACE) the amount of NO3- in xylem sap significantly increased in the H genotype relative 239
to the L genotype at flowering stage (Fig S4F) Together these data suggests greater 240
mobilization of NO3- from root to shoot in the H genotype 241
242
NO3- up-regulates NRT15 but down-regulates NRT18 243
244
A previous study showed that increased NRT18 expression in nrt15 mutants (Chen et al 245
2012) and the expression of NRT15 was induced by NO3- (Lin et al 2008) We tested the 246
relationship between NRT15 and NRT18 expression in A thaliana and B napus No 247
significant differences were observed in AtNRT15 expression in roots between wild type 248
(col-0) and mutant plants (nrt18-2) (Fig 3A) However AtNRT15 expression was 249
significantly up-regulated by NO3- in roots of nrt18-2 mutants (Fig 3B) Expression of 250
AtNRT18 significantly increased in roots of nrt15-3 mutants but not in col-0 plants and 251
there were no significant differences in AtNRT18 expression with or without NO3- treatment 252
in roots of nrt15-3 mutants (Fig 3CD) 253
In contrast BnNRT15 expression in roots of the H genotype increased significantly with 254
NO3- treatment (Fig 3E) while the reverse was observed in expression of BnNRT18 which 255
showed lower expression under NO3- treatment than control (Fig 3F) This suggests that NO3
- 256
induces the expression of NRT15 but down-regulates NRT18 in A thaliana and B napus 257
Further studies are needed to elucidate the mechanisms regulating this reverse regulation 258
259
Reduced VSC of NO3- in roots drives its long-distance transport from roots to shoots 260
Our previous study showed that Bafi (Bafilomycin A1) inhibits V-ATPase DCCD 261
(DCCD + Na2SO3) inhibits V-PPase and a 11 combination of Bafi and DCCD (B+D) 262
inhibits both V-ATPase and V-PPase (Han et al 2015a) These inhibitors were used to 263
control activities of the tonoplast proton pumps in the H B napus plants V-ATPase activity 264
significantly decreased under Bafil and B+D treatments relative to the control and DCCD 265
treatments whereas V-PPase activity declined significantly under DCCD and B+D treatments 266
relative to that in the control and Bafil treatments (Fig 4A) 267
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The V-ATPase activity in roots of A thaliana was significantly lower in the V-ATPase 268
mutants (vha-a2 and vha-a3) than the wild type (col-0) and V-PPase mutants (avp1) (Fig 5A) 269
In contrast the V-PPase activity in roots of V-PPase mutant (avp1) were significantly lower 270
than in the wild type (col-0) and V-ATPase mutants (vha-a2 and vha-a3) (Fig 5A) 271
NO3- influxes into vacuoles of the H B napus plants significantly decreased when treated 272
with inhibitors of proton pumps (Bafil DCCD B+D) (Fig 4B) Similarly NO3- influxes into 273
the vacuole of the Arabidopsis mutants (vha-a2 vha-a3 and avp1) was significantly lower 274
than those found in the wild type (col-0) (Fig 5B) Previous studies showed that VSC of NO3- 275
decreased when the activities of the proton pumps decline (Li et al 2005 Krebs et al 2010 276
Han et al 2015a) 277
We further investigated NO3- distribution between the vacuole and cytosol as affected by 278
proton pump inhibition in the H genotype of B napus and the mutants of A thaliana 279
defective in vacuolar proton pumps The percentage of vacuolar NO3- relative to total NO3
- in 280
root protoplasts of the H genotype treated with inhibitors (Bafil DCCD B+D) was lower than 281
that observed in the control (Fig 4C) Results were also similar when using A thaliana 282
mutants where the percentage of vacuolar NO3- relative to the total NO3
- in protoplasts was 283
lower in the mutants (vha-a2 vha-a3 and avp1) than in the wild type (col-0) (Fig 5C) 284
Consequently NO3- accumulation in the cytosol showed a significant increase when energy 285
pumps were suppressed in roots of the H genotype (Fig 4D) and NO3- accumulation in the 286
cytosol of root tissues of A thaliana mutants (vha-a2 vha-a3 and avp1) similarly increased 287
compared with col-0 (Fig 5D) 288
Based on previous observations (Lin et al 2008) expression of NRT15 is strongly 289
induced by NO3- but expression of NRT18 is down-regulated (Fig3 Chen et al 2012) We 290
then hypothesized that expression of these two genes is contrastingly regulated by 291
concentration of NO3- in the cytosol Our results were congruent with this hypothesis the 292
expression of BnNRT15 in root tissues of the H genotype of B napus was significantly higher 293
in plants treated with Bafil DCCD or B+D compared with the control (Fig 4E) whereas the 294
expression of BnNRT18 decreased substantially (Fig 4F) Similar results were also observed 295
in A thaliana where expression of AtNRT15 in roots of the mutants (vha-a2 vha-a3 and 296
avp1) were significantly higher than in the wild type (col-0) but expression of AtNRT18 in 297
the same mutants were considerably lower (Fig 5EF) 298
NO3- concentrations in the xylem sap the N-distribution between shoot and root (SR 299
ratios based on [15N]) and the [NO3-] in shoots relative to roots of the H genotype treated with 300
energy pumpsrsquo inhibitors were significantly higher than in the control (Fig 4GH Fig S5A) 301
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11
Similar trends were also observed when using mutants of A thaliana deficient in the energy 302
pumpsrsquo activities NO3- concentration in the xylem sap the [15N] SR ratios and [NO3
-] SR 303
ratios were all significantly higher in the mutants than in the wild type (Fig 5GH Fig S5B) 304
These data clearly showed that prevention of N sequestration in vacuoles would enhance its 305
translocation to shoot 306
Additional evidence linking NO3- long-distance transport and NO3
- short-distance 307
distribution within cells were provided through experiments comparing A thaliana wild type 308
(Ws) and mutant clca-2 (Fig S6) The chloride channel (CLCa) is the main channel for 309
vacuolar anion accumulation Vacuolar sequestration capacity of NO3-significantly declines in 310
clca mutants (Angeli et al 2006) NO3- influx into the vacuolar space of Ws roots was 311
significantly higher than that observed in the clca-2 mutants (Fig S6A) This resulted in a 312
smaller proportion of vacuolar NO3- in clca-2 plants relative to the total NO3
- in root tissue 313
protoplasts as compared with Ws (Fig S6B) Consequently the accumulation of NO3- in the 314
cytosol was significantly higher in clca-2 than in Ws plants (Fig S6C) The higher 315
NO3-concentration in the cytosol together with the higher expression of AtNRT15 coupled 316
with lower expression of AtNRT18 in clca-2 roots (Fig S6DE) resulted in significant 317
increase in xylem sap NO3- concentration [15N] SR ratios and [NO3
-] SR ratios compared 318
with Ws plants (Fig S6FGH) 319
320
Increased NO3- translocation to shoots enhanced NUE 321
NO3- assimilation efficiency is known to be higher in shoots than in roots (Smirnoff et al 322
1985 Andrews et al 1986 Tang et al 2012 Tang et al 2013) We therefore hypothesize 323
that increased NO3- translocation to shoot and the consequent higher shootroot ratio will 324
contribute to higher NUE Our data support this hypothesis The H B napus genotype showed 325
enhanced long-distance transport of NO3- from roots to shoots (Fig 2 Fig S2) This enhanced 326
NO3- transport requires higher carbon skeleton provided through higher photosynthetic rate 327
(Tang et al 2012 Tang et al 2013) Our results showed that chlorophyll content 328
intercellular CO2 concentration and photosynthetic rate were significantly higher in the H than 329
the L genotype at both seedling and flowering stages (Table 2) Moreover the NO3- 330
assimilating enzymes were strongly induced by NO3- providing sufficient capacity for N 331
assimilation (Smirnoff et al 1985 Andrews et al 1986) Nitrate reductase (NR) and 332
glutamine synthetase (GS) activities in roots of the H genotype were significantly lower than 333
in the L genotype both at seedling and flowering stages (Fig S7) In contrast NR activities in 334
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12
shoots of the H genotype were significantly higher than in the L genotype at both stages (Fig 335
S7AB) and GS activity in the H genotype was also higher than those of the L genotype at 336
seedling stage (Fig S7C) 337
Experiments comparing A thaliana wild type (col-0 Ws) with mutants defective in 338
vacuolar proton-pumps (vha-a2 vha-a3avp1) and transport channel (clca-2) yielded similar 339
results that is increases in NO3- shootroot ratio essentially contributed to enhanced NUE 340
(Fig 6) The NO3- shootroot ratios in A thaliana mutants were higher than that in the wild 341
type (Fig 5GH) Generally the efficiency of inorganic N (NO3-) assimilation into organic N 342
is higher in shoots than in roots (Smirnoff et al 1985 Andrews et al 1986) therefore the 343
NO3- concentration in A thaliana mutants decreased (Fig 6CD) leading to higher NUE 344
compared with the wild type (Fig 6AB) 345
346
BnNRT15BnNRT18 in B napus affects NO3- long-distance transport from roots to 347
shoots 348
Both B napus and A thaliana are members of Cruciferae Family The amino acid 349
sequence identity between BnNRT15 and AtNRT15 was 90 (Fig S8A) the amino acid 350
sequence similarity between BnNRT18 and AtNRT18 was 908 (Fig S8B) Comparisons of 351
nucleotide and amino acid sequences (Harper et al2012) of these genes 352
(httpbrassicanbiacukcgi-binmicroarray_databasecgi) showed that BnNRT15 353
(EV220114) and BnNRT18 (EV116423) of B napus are respectively highly homologous 354
with AtNRT15 and AtNRT18 and the two genes are mainly expressed in roots of both species 355
showing similar organ-specificity (Lin et al 2008 Li et al 2010) 356
NO3- long-distance transport from roots to shoots is therefore regulated by NRT15 and 357
NRT18 as reported before (Lin et al 2008 Li et al 2010) The [15N-traced] SR ratios and 358
the [NO3-] SR ratios were significantly lower in nrt15-3 mutants relative to the wild type 359
(col-0) while [15N] SR and [NO3-] SR ratios showed significant increase in nrt18-2mutant 360
relative to col-0 (Fig S9CD) A previous study showed that expression of NRT15 is 361
down-regulated by ACC and MeJA treatments while the expression of NRT18 is strongly 362
up-regulated in A thaliana (Zhang et al 2014) Consequently NO3- accumulated in plant 363
roots probably as an adaptive measure for abiotic stresses (Chen et al 2012) Both the H- 364
and L- B napus genotypes showed significantly lower expression of BnNRT15 in roots when 365
treated with ACC (Fig S10A) but the expression of BnNRT18 remained higher than in the 366
control plants (Fig S10B) This resulted in significantly lower [15N] SR ratios following 367
ACC treatment (Fig S10CDE) These data indicate that BnNRT15 and BnNRT18 play 368
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13
similar functions for NO3- long-distance transport between root and shoot in both B napus 369
and A thaliana (Lin et al 2008 Li et al 2010) 370
371
372
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14
Discussion 373
Nitrate long-distance transport from root to shoot is regulated by cytosolic NO3- in roots 374
NO3- long-distance transport from root to shoot is controlled by NRT gene family For 375
instance xylem NO3- loading and unloading are mainly controlled by NRT15 and NRT18 376
respectively (Lin et al 2008 Li et al 2010) Whereas the NO3- short-distance transport 377
between the vacuole and cytosol is controlled by tonoplast proton-pumps (V-ATPase and 378
V-PPase) (Krebs et al 2010) and by activity of the CLCa channel in the tonoplast membrane 379
(Angeli et al 2006 Wege et al 2014) tonoplast proton-pumps provide the energy required 380
for NO3- accumulation into the vacuole through CLCa (Gaxiola et al 2001 Brux et al 2008 381
Krebs et al 2010) The expression of NRT15 is up-regulated by NO3- in plant culture 382
solution (Lin et al 2008) whereas regulation of NRT18 expression is contrary to that of 383
NRT15 under both control and abiotic stress conditions (Li et al 2010 Chen et al 2012 384
Zhang et al 2014) 385
Despite the progress made in characterizing the genes involved in transport and 386
metabolism of NO3- in plants the relationship between long- and short-distance transport and 387
how these two pathways are regulated have not been thoroughly investigated We hypothesize 388
that NRT15 expression and its role in long-distance transport is regulated by NO3- in plant 389
tissue as was observed in culture solution (Lin et al 2008) and our data support this 390
hypothesis Both BnNRT15 and AtNRT15 expression were up-regulated by increasing 391
cytosolic NO3- in roots (Fig 4 Fig 5) The increase in cytosolic NO3
- was achieved through (i) 392
use of A thaliana mutants defective in proton-pumps (V-ATPase and V-PPase) or CLCa 393
activities in the tonoplast and (ii) inhibition of B napus tonoplast proton-pumps In both 394
model plants the influx of NO3- into vacuoles was substantially reduced resulting in higher 395
concentrations of NO3- in the cytosol (Fig 4A-D Fig 5A-D Fig S6A-C) Under those 396
conditions expression of BnNRT15 and AtNRT15 was up-regulated by the enhanced 397
cytosolic NO3- while expression of BnNRT18 and AtNRT18 was down-regulated (Fig 4EF 398
Fig 5EF FigS6DE) As a consequence more NO3- was loaded into the xylem sap and 399
transported from root to shoot (Fig S5AB FigS6F) This is also reflected as increased 400
shootroot ratios of N traced by [15N] and [NO3-] (Fig 4GH Fig 5GH Fig S6GH) 401
Interestingly the expression of both BnNRT15 and AtNRT15 is up-regulated by 402
increased cytosolic NO3- concentration in roots but were not affected by NO3
- sequestered 403
into vacuoles (Fig 4 Fig 5 Fig S6) This is because vacuolar NO3- is functionally separated 404
by the tonoplast and cannot be assimilated by enzymes that are localized in the cytosol 405
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15
Therefore NO3--inducible genes such as NR and NRT15 were not influenced by vacuolar 406
NO3- concentration (Martinoia et al 1981 Martinoia et al 2000 Zhang et al 2012) In 407
addition NO3- concentration in the cytosol is controlled by its short-distance transport through 408
the tonoplast which is mediated through the tonoplast proton-pumps providing the required 409
energy for its sequestration into vacuoles against concentration gradient (Gaxiola et al 2001 410
Brux et al 2008 Krebs et al 2010) The gradient of H+ between the inside of the vacuole 411
and the cytosol is maintained by an 2NO3-1H+ antiporter mechanism that facilitatesNO3
- 412
transport through CLCa (Angeli et al 2006 Wege et al 2014) Our data provide clear 413
evidence that NO3- long-distance transport from root to shoot is regulated by cytosolic NO3
- 414
concentration in roots 415
Regulatory mechanisms that control the expression of NRT18 and NRT15 as mediated 416
by NO3- are still unknown however previous studies showed that their expression is also 417
affected by abiotic stresses whereby NRT15 is down-regulated and NRT18 is up-regulated 418
(Li et al 2010 Zhang et al 2014) AtNRT18 expression in A thaliana nrt15 mutants is 419
highly up-regulated (Chen et al 2012) as was also observed here (Fig 3C) Moreover 420
AtNRT15 expression was enhanced by NO3- in culture solution and was not affected in 421
nrt18-2 mutant (Fig 3AB) On the other hand AtNRT18 expression was not affected by 422
cytosolic NO3- but down-regulated when AtNRT15 expression was up-regulated by NO3
-(Fig 423
S6DE) Analogous results were also obtained with B napus genotypes (Fig 3EF) Based on 424
these results we argue that NRT18 expression is not directly influenced by cytosolic NO3- but 425
rather by NRT15 expression (Fig 4 Fig 5 and Fig S6) suggesting NRT18 is probably 426
acting downstream of NRT15 Apparently the regulatory mechanisms of this NRT gene 427
family still awaits further studies to be elucidated 428
429
Response of NUE to NO3- long-distance transport 430
NO3- assimilation efficiency is higher in shoot than in root tissues and can be further 431
enhanced by carbon assimilation (Smirnoff et al 1985 Tang et al 2013) Therefore 432
translocation of higher proportion of NO3- from roots to shoots likely contributes to better 433
crop growth and higher NUE (Andrews et al 1986 Tang et al 2012) The present study 434
provides direct evidence for these results using contrasting B napus genotype identified 435
before (Zhang et al 2009 Han et al 2015a) Our data confirmed the genetic variation in 436
NUE between these two genotypes (Table 1) 437
We measured NO3- distribution in root and shoot of these genotypes to elucidate the 438
physiological mechanisms contributing to variation in NUE The data were consistent with 439
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our hypothesis that concentration of NO3- in the xylem sap and shoot would be significantly 440
higher in the H genotype than in the L genotype (Fig 2EFFig S2EF Fig S4) The NO3- 441
long-distance transport from root to shoot was affected by cytosolic NO3- in roots of both A 442
thaliana and B napus genotypes (Fig 4-5 Fig S6) The activities of the tonoplast 443
proton-pumps in roots were significantly lower in the H genotype than in the L genotype (Fig 444
1AB) Consequently less NO3- was sequestered into the vacuole and more NO3
- was retained 445
in cytosol (Fig 1CD) BnNRT15 expression was then up-regulated resulting in greater 446
loading into the xylem while BnNRT18 was down-regulated (Fig 2AB) probably to 447
suppress NO3- unloading from xylem sap Consequently a higher proportion of NO3
- was 448
transported from root to shoots in the H genotype(Fig 2C-D Fig S4) 449
Moreover photosynthetic carbon fixation (Table 2) and activities of both NR and GS 450
were higher in shoots of the H genotype (Fig S7) both of which promote higher N 451
assimilation These results agreed with previous reports (Smirnoff et al 1985 Andrews et al 452
1986 Tang et al 2012 Tang et al 2013) where higher NO3- transport from root to shoot 453
higher photosynthetic rate and N assimilation efficiency in shoots were suggested as essential 454
mechanisms for high-NUE in crop plants These qualities endowed the B napus H genotype 455
Xiangyou15 with higher NUE as compared with the L genotype 814 456
NO3- long-distance transport in B napus is not only regulated by BnNRT15 and 457
BnNRT18 in root tissues (Fig 4 Fig 5 Fig S6) but also by stomatal conductance and 458
transpiration by indirectly controlling xylem sap flow and long-distance transport 459
(Dechorgnat et al 2011 Krapp et al 2014) Stomatal conductance and transpiration rates of 460
the H genotype were significantly higher than those of the L genotype (Table S1) a result 461
consistent with other studies describing characteristics of high-NUE plants (Daniel-Vedele et 462
al 1998 Wilkinson et al 2007 Dechorgnat et al 2011 Krapp et al 2014) 463
464
Response of NUE to NO3- short-distance transport 465
NUE is also strongly controlled by NO3- short-distance distribution between the vacuole 466
and cytosol (Han et al 2015a) A lower proportion of NO3- accumulating in vacuoles and a 467
higher proportion retained in the cytosol will contribute to better NUE in crop plants (Han et 468
al 2015b) The energy required for vacuolar NO3- sequestration is provided by the tonoplast 469
proton-pumps (Gaxiola et al 2001 Brux et al 2008 Krebs et al 2010) while the 470
distribution channel CLCa enables transport of NO3- across the vacuolar membrane (Angeli et 471
al 2006 Wege et al 2014) The activities of the tonoplast proton-pumps (V-ATPase and 472
V-PPase) in leaves of the H genotype were significantly lower than those of the L genotype at 473
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17
flowering stages (Han et al 2015aHan et al 2015b) thus we expected similar differences in 474
root tissues Our results confirmed that the activities of tonoplast proton-pumps in root tissues 475
of B napus H genotype and A thaliana mutants were lower than those in the L genotype and 476
A thaliana wild type leading to less NO3- influx into vacuoles and more in the cytosol (Fig 1 477
Fig 5A-D) Moreover the enzymes catalyzing N assimilation are located in the cytoplasm 478
where NO3- could be assimilated (Martinoia et al 1981 Han et al 2015a) or immediately 479
loaded into xylem sap for long-distance transport from roots to shoots (Lin et al 2008) 480
Consequently the NO3- concentration in root tissues of the H genotype and A thaliana 481
mutants defective in tonoplast proton-pumps was significantly lower than that in the L 482
genotype (Fig 2E) and in A thaliana wild type (Fig 6C) Therefore the higher proportion of 483
NO3- transported to shoot and the lower concentrations retained in root vacuoles contributed 484
to the higher NUE in Xiangyou15 and A thaliana mutants (vha-a2 vha-a3 and avp1) (Table 485
1Fig 6A) Further studies are needed to assess the impacts of regulating vacuolar 486
sequestration of NO3- and rates of long-distance transport for enhancing NUE in other crop 487
species and genotypes 488
These data suggested that NO3- distribution between vacuoles and cytosol within cells 489
regulates NO3- long-distance transport from root to shoot and ultimately affects NUE NO3
- 490
loading into the xylem sap is an active transport process (Lin et al 2008) therefore not 491
regulated by absolute concentration of NO3- in the cytosol but rather by genes involved in 492
NO3- long-distance transport (NRT15 and NRT18) Apparently the expression of NRT15 the 493
gene responsible for xylem loading is regulated by NO3-concentration in the cytosol 494
Therefore both NO3- short-distance transport across the tonoplast and its long-distance 495
transport from root to shoot are important determinants of NUE in both B napus and A 496
thaliana (Table 1 and Fig 6) 497
498
Roles of NRT15 and NRT18 in NO3- long-distance transport in B napus and A 499
thaliana 500
Our results (Figs S9 S10) showed that the proportion of NO3- distributed from root to 501
shoot is strongly controlled by NRT15 in accord with previous studies (Lin et al 2008 Li et 502
al 2010) The data suggest that the high-NUE genotypes possess lower activities of tonoplast 503
proton-pumps (V-ATPase and V-PPase) resulting in less NO3- accumulation in vacuoles and 504
more NO3- retention in cytosol (Fig 7) The higher NO3
- in the cytosol then up-regulated 505
NRT15 which then down-regulates NRT18 As a result more NO3- is loaded into the xylem 506
system by NRT15 mechanism and less NO3- is unloaded via NRT18 (Fig 7) Differential 507
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18
regulation of these NRT genes in high-NUE plants increased transport of NO3- from root to 508
shoot through the xylem vascular tissues (Fig 7) This coordinated NO3- long- and 509
short-distance transport likely fine-tunes NO3- allocation and modulates the balance of NO3
- 510
distribution between roots and shoots (Fig 7) 511
Our research also revealed differences between A thaliana and B napus in the 512
expression of the wild-type NRT genes in response to NO3- in the protoplast Moreover using 513
A thaliananrt15-3 mutants we uncovered possible regulatory interactions between a 514
functioning (wild-type) NRT15 gene and NRT18 expression in response to NO3- 515
concentration Because mutants defective in nrt15 are not available for B napus we could 516
not confirm that the respective NRT15 and NRT18 systems behave in an analogous way in 517
these plant species Based on Harper et al (2012) research database our homology 518
comparisons and gene function annotations provided strong evidence for the roles of 519
BnNRT15 and BnNRT18 in B napus Finally our studies were conducted using two model 520
dicotyledonous plant species for which we had ample data on responses to specific inhibitors 521
(B napus) or specific mutants were available with results forming the bases for the model 522
presented in Figure 7 Further research using genetically diverse plant species (eg 523
monocotyledonous crops such as rice wheat and corn) will help validate this model and its 524
broader application across species The information generated in this study could have future 525
application for improving NUE in commercial crops with consequent reduction in nitrogen 526
use and benefits to the environment 527
528
Materials and Methods 529
Plant material 530
The two oilseed rape (Bnapus) cultivars used in this study have been characterized 531
before as high- (Xiangyou15 referred to as H genotype hereafter) and low- (814 referred to 532
as L genotype) nitrogen use efficiency (NUE) genotypes (Zhang et al 2009 Han et al 2015b) 533
Here we define NUE as the total biomass per unit of N uptake by the plant with total 534
biomass includes roots shoots and grains The two genotypes were provided by the Hunan 535
Sub-Center of Improvement Center of National Oil Crops Hunan China 536
The Arabidopsis thaliana wild-type Columbia-0 (col-0) was used as control for 537
V-ATPase (vha-a2 and vha-a3) V-PPase (avp1) nrt15-3 and nrt18-2 mutants whereas the A 538
thaliana wild-type Wassilewskija (Ws) was used as control for clca-2 mutants A thaliana 539
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19
mutants and transgenic lines (nrt15-3 and nrt18-2) have been described previously (Chen et 540
al 2012 Zhang et al 2014) The mutant lines vha-a2 (Salk_142642) and vha-a3 541
(Salk_122135) described by Krebs et al (2010) were obtained from the Arabidopsis 542
Biological Resources Center (ABRC) The mutant line avp1 (GK-005004) described in Li et 543
al (2005) was obtained from the European Arabidopsis Stock Centre while the mutant line 544
clca-2 (FST 171A06) described in Angeli et al (2006) was from the Institute National de la 545
Recherche Agronomique (INRA) collection in France together with the T-DNA mutants used 546
for genotyping to select pure mutant lines 547
548
Growth conditions 549
B napus plants were grown hydroponically in ceramic pots(20cmtimes15cm) filled with a 550
nutrient solution in the greenhouse under natural light as described in Han et al (2015b) The 551
pots were arranged in a completely randomized design with six biological replications The 552
nutrient solution was replaced every 3 days and its pH adjusted to 55 daily Experiments were 553
conducted at the field station of Hunan Agricultural University Southern China 554
A thaliana plants were grown in a nutrient solution in plastic pots as described in Arteca 555
et al (2000) and Gong et al (2003) The solution was changed every 3 days with pH adjusted 556
daily to 58 Pots were arranged in a completely randomized design with six biological 557
replications The nutrient solution used for both species consisted of 125mM KNO3 558
0625mM KH2PO4 05mM MgSO4 05mM Ca (NO3)2middot4H2O 0025mM Fe-EDTA 025 559
mlL-1 micronutrients (stock solution concentrations 70mM B 14mM Mn 1mM Zn 05mM 560
Cu and 02mM Mo) The experiments were conducted at Hunan Agricultural University in a 561
phytotron set at 70 relative humidity16h-light8h-dark cycle and constant temperature of 562
22degC 563
564
Experimental treatments and phenotyping 565
All measurements made on B napus plants were conducted either at the seedling stage 566
(2 months after transplanting) or at flowering (5 months after transplanting) The whole root 567
tissue was harvested and used for analyses at either stage For shoot measurements the fourth 568
leaf from the bottom was used for measurements made at seedling stage whereas the 12th leaf 569
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
from the bottom was harvested for measurements made at flowering stage For A thaliana 570
whole root and shoot of four-week-old plants were sampled and used for different assays 571
572
Vacuolar proton-pumps inhibitor treatments 573
Inhibition of vacuolar proton pumps of B napus (Xiangyou15) was conducted at the seedling 574
stage in hydroponic culture as described in Han et al (2015a) with minor modifications 575
Plants grown in hydroponic solution were used as control Inhibitor treatment was conducted 576
using several chemicals Bafi(25 nmol L-1 Bafilomycin A1) an inhibitor of V-ATPase 577
DCCD(10 μmol L-1DCCD + 50 mmolL-1 Na2SO3) specifically inhibits V-PPase and 578
Bafi+DCCD in a 11 ratio as inhibitor of both V-ATPase and V-PPase The inhibitors were 579
applied in the hydroponic solutions for 24 h Fresh plant tissue samples from all treatments 580
were collected for different assays 581
582
NO3- induced gene expression 583
Four-week old A thaliana mutants (nrt15-3nrt18-2) were grown in hydroponics with 584
225mM (NH4)2succinate for 3 days and shifted to hydroponics with 45mM NO3- for 12 h 585
then root tissues were collected to assess relative expression of AtNRT15 and AtNRT18 586
genes Seedlings of B napus (Xiangyou15) plants grown hydroponically were treated with 587
5mM (NH4)2succinate for 3 d and shifted to fresh hydroponic solution containing 15 mM 588
NO3- for 12 h Root tissue was then collected to assess the relative expression of BnNRT15 589
and BnNRT18 genes using quantitative RT-PCR 590
591
ACC treatment 592
Hydroponically grown seedlings of B napus were subjected to ACC treatment as described in 593
Zhang et al (2014) with minor modifications Seedlings of the two genotypes Xiangyou15 594
and 814 were transferred to a nutrient solution containing 002mM L-1 ACC for 6 h or grown 595
in normal hydroponic solution as control Root tissues were then collected to assay relative 596
expression of BnNRT15 and BnNRT18 using quantitative RT-PCR Another set of B napus 597
plants were grown in ACC-containing hydroponic solution but supplemented with 15NO3- (22 598
15N) replacing the unlabeled NO3- for 1 h Root and shoot tissues were collected separately 599
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21
for 15N measurement using a continuous-flow isotope ratio mass spectrometer coupled with a 600
C-N elemental analyzer (ANCA-MS PDZ Europa) 601
602
Biomass and N concentration 603
Grain yield dry biomass and N concentration were determined in plant samples at 604
harvest Plants were harvested (including senescing leaves) then dried in an oven for 30 min 605
at 105degC then at 70degC to a constant weight and weighed N concentration was determined 606
using the Kjeldahl method (Shi et al 2010 Han et al 2015b)02g of dried samples were 607
digested with H2SO4-H2O2 in a 100-ml Kjeldahl digestion flask after which N concentration 608
was determined using a Foss Auto Analyser Unit (Kjeldahl 8400) 609
610
V-ATPase and V-PPase activities 611
Roots (10 g) of B napus were collected at seedling and flowering stages and that of A 612
thaliana (05 g) sampled at seedling stage then used for determining the activities of 613
V-ATPase and V-PPase (Han et al 2015b) Activities of these two energy pumps within 614
microsomal membranes were followed colorimetrically based on released Pi as described by 615
Zhu et al (2001) and Krebs et al (2010) 616
617
Assay of NO3- flux in vacuoles 618
Roots of both species were collected similarly as that used for V-ATPase and V-PPase 619
activities then used for vacuole isolation and measurement of NO3- flux in vacuoles as 620
described by Robert et al (2007) with minor modification (Han et al 2015b) Net fluxes of 621
NO3- in vacuoles were measured non-invasively using SIET (scanning ion-selective electrode 622
technique SIET system BIO-003A Younger USA Science and Technology Corporation 623
USA) An NO3--selective microelectrode used for the assay of NO3
- flux was vibrated in the 624
measuring solution between two positions 1 μm and 11 μm from the vacuole surface 625
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles Background 626
signal was recorded by vibrating the electrode in the measuring solution in the absence of 627
vacuoles Ion flux was calculated by Fickrsquos law of diffusion J = -D(dcdx) where J represents 628
the ion flux (picomolescm-2s-1) dcdx is the ion concentration gradient and D is the ion 629
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
diffusion constant in a particular medium The direction of the flux was derived from Fickrsquos 630
law of diffusion that relates the concentration gradient 631
632
Isolation of intact protoplasts and vacuoles for determining NO3- concentration 633
Similar to the above assays root tissues (10 g) were collected from B napus at seedling 634
and flowering stages and from A thaliana (05 g) seedlings and used to isolate intact 635
protoplasts and vacuoles as described by Robert et al (2007) with minor modifications as 636
outlined in Huang et al (2012) and Han et al(2015b) The purified protoplasts were divided 637
into two equal aliquots with one of them used for isolation of vacuoles The purified 638
protoplasts and vacuoles were then sub-sampled and used to determine NO3- concentrations 639
(Vogeli-Lange and Wagner 1990) and for enzyme activity assays (Ma et al 2005) 640
NO3-concentrations in protoplasts and vacuoles were measured by a continuous-flow 641
auto-analyzer (Auto Analyser 3 Bran and Luebbe Germany) as described previously (Han et 642
al 2015b)The activities of acid phosphatase (ACP) and cytochrome oxidase (COX) were 643
determined using plant ACP colorimetry- and COX-assay kits (GenMedSci Inc) following 644
the manufacturerrsquos instructions ACP activity specific to vacuoles was determined and used to 645
normalize NO3- accumulation We measured NO3
- in the protoplast outside the vacuole which 646
includes the cytosol and organelles eg mitochondria and Golgi Apparatus (Robert et 647
al2007) Since most of the NO3- in the protoplast outside the vacuole is located in the cytosol 648
(Krebs et al 2010) we refer to NO3- distribution between vacuoles and cytosol rather than 649
vacuole versus protoplast 650
651
Quantitative RT-PCR 652
Total RNA extracted from B napus roots was prepared using Trizol reagent (Invitrogen) 653
cDNAs were synthesized using M-MLV reverse transcriptase (Promega) following 654
manufacturerrsquos protocol The relative expression of BnNRT15 (EV220114) BnNRT18 655
(EV116423) and Bnactin (AF1118121) genes in plant roots were determined by quantitative 656
RT-PCR and run on a LightCycler instrument (Roche) with the SYBR Green Real-Time PCR 657
Master Mix Kit (TOYOBO Japan) under the following conditions 95degC for 2min then 45 658
cycles of 95degC for 10s 60degC for 10s and 72degC for 20s The primer sequences of BnNRT15 659
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
BnNRT18 and Bnactin genes were obtained from httpbrassicanbiacuk as described in 660
Harper et al (2012) and expression levels were normalized to Bnactin as control 661
Total RNA extracted from the 4-week-old A thaliana roots under the treatments 662
conditions described above were prepared using Trizol reagent (Invitrogen) The cDNAs were 663
synthesized using M-MLV reverse transcriptase (Promega) based on the manufacturerrsquos 664
protocol and as described in Li et al (2010)The primer sequences of quantitative RT-PCR 665
used in these assays are listed in Supplemental Table 2 and were described previously (Chen 666
et al 2012 Zhang et al 2014) and their expression was normalized to Atactin2 as control 667
668
Determination of nitrate distribution using 15NO3- 669
Hydroponically grown B napus (seedling and flowering stages) and A thaliana 670
(seedling) were transferred to 01mM CaSO4 solution for 1min then to their respective 671
hydroponic nutrient solutions with15NO3- (22 excess) replacing unlabeled NO3
- for 1h 672
Plants were then transferred to 01mM CaSO4 solution for 1min after which roots were 673
washed with deionized water Root and shoot samples were separated and dried at 105degC for 674
30min followed by 70degC for 3 d then grounded and used for assaying 15N content using a 675
continuous-flow isotope ratio mass spectrometer coupled with a carbon-nitrogen elemental 676
analyzer (ANCA-MS PDZ Europa) 677
678
Xylem sap collection and assay of nitrate concentration 679
We used the method of Tang et al (2012) for collecting xylem sap Plants were cut 680
leaving1cm segments with intact roots at seedling and flowering stages for B napus and at 681
seedling stage for A thaliana Roots were immediately immersed in their respective nutrition 682
solutions Weighed cotton was put on the cut surface to absorb extruding xylem sap for 1 h 683
The cotton was then wrapped in plastic film and the volume of xylem sap was calculated as 684
the weight gain of the cotton Xylem sap was then squeezed from the cotton using a syringe 685
and used for subsequent assay of nitrate concentration using a continuous-flow auto-analyzer 686
(Auto Analyser 3 Bran and Luebbe Germany) 687
To determine nitrate concentration samples (10 g fresh root and shoot at seedling and 688
flowering of B napus and 05 g fresh root and shoot of 4-week-old A thaliana) were frozen 689
in liquid N2 and ground with a mortar and pestle the powder was then transferred to a beaker 690
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24
containing 70 ml deionized water and boiled for 30 min to extract nitrate then cooled and 691
made to 100 ml volume and 02 g activated carbon added to eliminate the effect of 692
chlorophyll The mixture was filtered and nitrate in the filtrate was determined using a 693
continuous-flow auto-analyser (Atuo-Analyser 3 Bran and Luebbe Germany) 694
695
Measurements of photosynthesis 696
All measurements of photosynthesis and related parameters were conducted at 1000 h 697
using intact leaves The 4th leaf from the bottom in B napus at seedling stage and the 12th leaf 698
from the bottom at flowering stage were used for determining chlorophyll concentration 699
photosynthetic rate intercellular CO2 concentration transpiration rate and stomatal 700
conductance Chlorophyll concentration was determined by SPAD-502 (Minolta Camera Co 701
Ltd Japan) (Luo et al 2006 Yandeau-Nelson et al 2011) Photosynthetic rate intercellular 702
CO2 concentration transpiration rate and stomatal conductance were measured using LI-6400 703
Portable Photosynthesis System (Li-Cor Biosciences Co Ltd USA) set at flow rate of 500 704
μmol s-1 photosynthetic photon flux density of 1000 μmol m-2 s-1 relative humidity of 65 705
CO2 concentration of 400ppm and ambient temperatures 706
707
NR and GS activities 708
Leaves with similar age as those used for photosynthesis in B napus were used for the 709
assay of nitrate reductase (NR) and glutamine synthetase (GS) activities NR activity was 710
measured by the modified method of Fan et al (2007) Fresh samples (05g) were frozen in 711
liquid N2 ground into powder in the presence of acid-washed sand and homogenized with 4 712
ml of extraction buffer (0025mol L-1phosphate buffer pH87 1211g L-1 cysteine 0372g L-1 713
EDTA) Homogenates were centrifuged at 30000timesg for 15 min at 4degC and the supernatants 714
were treated with sulfanilamide and α-naphthylamine reagents for colorimetric (540nm) 715
determination of nitrite as described by Fan et al (2007) 716
GS activity was determined using a modified reverseγ-glutamyltransferase method 717
(Wang et al2014) which measures the GS-catalyzed formation of glutamyl-γ-hydroxamate 718
from glutamine and hydroxylamine Fresh samples were frozen for 30 min at -20degC then 719
ground in 10ml Tris-HCl buffer and acid-washed sand The homogenates were filtered 720
through two layers of gauze and centrifuged at 8000timesg for 15 min at 4degC About 12ml of the 721
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
supernatant was added in a reaction mixture and treated as described (Wang et al2014) After 722
the GS-catalyzed reaction was stopped and the solution centrifuged glutamyl-γ723
-hydroxamate was quantified colorimetrically in the supernatant (at 485 nm) and the 724
concentration was determined using a standard curve 725
726
Statistical analyses 727
We used the SPSS software (Statistical Product and Service Solutions V130 USA) for 728
ANOVA and mean separation of main-effects and interactions using Duncanrsquos multiple range 729
test at Plt005Values are presented as means and SD of three or six replicates from three 730
independent experiments Different letters or an asterisk () associated with specific data (eg 731
at the top of histogram bars in figures or within tables) denote significant differences at 732
Plt005 733
734
Supplemental Data 735
Supplemental Figure 1 B napus with higher NUE showed lower vacuolar sequestration 736
capacity (VSC) for NO3-in roots at flowering stage 737
Supplemental Figure 2 B napus with higher NUE showed enhanced long-distance transport 738
of NO3- from roots to shoots at flowering stage 739
Supplemental Figure 3 The two B napus (H and L genotypes) showed the same total N per 740
plant at seedling stage (A) and flowering stage (B) 741
Supplemental Figure 4 B napus with higher NUE showed increased NO3- concentration in 742
the xylem sap at seedling and flowering stages 743
Supplemental Figure 5 NO3- concentration in the xylem sap as affected by inhibitor 744
treatments in B napus and in the energy pumpsrsquo mutants of A thaliana (col-0 vha-a2 vha-a3 745
avp1) 746
Supplemental Figure 6 Reduced VSC for NO3- in roots drives long-distance transport of 747
NO3- from roots to shoots in the A thaliana wild type (Ws) and mutant (clca-2) 748
Supplemental Figure 7 Differences of NR and GS activities between the two B napus (H 749
and L genotypes) at seedling and flowering stages 750
Supplemental Figure 8 Amino acid sequences of BnNRT15 and BnNRT18 751
Supplemental Figure 9 Functions of AtNRT15 and AtNRT18 genes in root tissues of A 752
thaliana in controlling NO3- long-distance transport from root to shoots 753
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
Supplemental Figure 10 B napus BnNRT15 and BnNRT18 genes in roots were 754
coordinately modulated to facilitate NO3- long distance transport from roots to shoots 755
Supplemental Table 1 Differences in stomatal conductance and transpiration rate between 756
the two B napus genotypes 757
Supplemental Table 2 Sequences of primera used for qRT-PCR 758
759
Acknowledgements 760
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 761
Shanghai Institutes for Biological Sciences) for providing nrt15-3 and nrt18-2 seeds 762
This study was supported by the National Natural Science Foundation of China (Grant 763
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 764
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 765
The Ministry of Education in China Open Novel Science Foundation of Hunan province 766
(13K062) the National Key Laboratory of Plant Molecular Genetics and the Twelfth 767
Five-Year National Science and technology support program (2012BAD15BO4) 768
769
Figure 1 B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 770
NO3- in roots at the seedling stage 771
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 772
genotype 814 Specific activities of the tonoplast proton pumps are expressed as μmol Pi 773
released mg-1 protein h-1 NO3- fluxes are expressed as pmol NO3
- cm-2 S-1 Mature vacuoles 774
were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 775
measuring solution between the two positions 1 μm and 11 μm from the vacuole surface 776
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles recording the 777
stable reading data The background was recorded by vibrating the electrode in measuring 778
solution without vacuoles 779
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 780
for NO3- content and NO3
- accumulation normalized against the specific activity of the 781
vacuole acid phosphatase (ACP) as described in Materials and Methods and plotted as μmol 782
NO3- per μmol p-nitrophenol the end product of ACP assay 783
Proton pump activities in root tissues between H and L genotypes are shown at the seedling 784
stage (A) Different letters at the top of the histogram bars denote significant differences of 785
V-ATPase in root tissues between H and L genotypes (Plt005) an asterisk () at the top of the 786
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
histogram bars indicates significant difference in V-PPase activity in root tissues between H 787
and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788
Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789
genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790
bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791
bars on the figures indicate SD (n=6) 792
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793
seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794
to the total NO3- in protoplasts 795
NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796
total NO3-in the cytosol and was calculated as total NO3
- in protoplasts ndash total NO3- in 797
vacuole Different letters at the top of histogram bars denote significant differences of total 798
NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799
(n=6) 800
801
Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802
roots to shoots at the seedling stage 803
H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804
Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805
the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806
as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807
the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808
the histogram bars denote significant differences at Plt005 809
Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810
in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811
genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812
of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813
SD (n=3) different letters at the top of the histogram bars denote significant differences at 814
Plt005 level 815
The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816
at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817
genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818
different letters at the top of the histogram bars denote significant differences at Plt005 819
820
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
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Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
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Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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- Parsed Citations
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6
regulated by cytosolic NO3- concentration However this hypothesis needs to be substantiated 166
The mechanisms underlying both NO3- short-distance (Gaxiola et al 2001 Angeli et al 167
2006 Brux et al 2008 Krebs et al 2010) and long-distance transport (Lin et al 2008 Li et 168
al 2010) have been previously investigated yet the underlying mechanisms regulating the 169
flux of NO3- and the obvious relationship between the two transport pathways as well as their 170
relation to NUE are not well understood 171
The NRT family of genes play a partial role in vacuolar NO3- accumulation in petioles 172
(Chiu et al 2004) and seed tissues (Chopin et al 2007) whereas the proton pumps and 173
CLCa system in the tonoplast play a major role in accumulating NO3- in vacuoles (Gaxiola et 174
al 2001 Angeli et al 2006 Brux et al 2008 Krebs et al 2010) The vacuolar NO3- 175
short-distance transport system is spread throughout the plant tissues and is the principal 176
means by which vacuolar NO3- short-distance transport and distribution is controlled (Angeli 177
et al 2006Krebs et al 2010) 178
The NRT genes seem to work synergistically to control NO3- long-distance transport 179
between roots and shoots NRT19 is responsible for NO3- loading into the phloem (Wang et 180
al 2011) whereas NO3- loading and unloading into xylem are regulated by NRT15 and 181
NRT18 respectively (Lin et al 2008 Li et al 2010) Phloem transport mainly involves 182
organic N the inorganic-N (NO3-) concentrations in the phloem sap are typically very low 183
ranging from one-tenth to one-hundredth of that of the inorganic-N in xylem sap (Lin et al 184
2008Fan et al 2009) Therefore this study focused on NO3- short-distance transport 185
mediated through the tonoplast proton-pumps and the CLCa system and the long-distant 186
transport mechanisms responsible for xylem NO3- loading and unloading via NRT15 and 187
NRT18 respectively 188
Questions related to how long and short-distance transport of NO3- are coupled in plant 189
tissues and their role in determining NUE were addressed using a pair of high- and low-NUE 190
B napus genotypes and Arabidopsis thaliana Application of proton pump inhibitors and 191
ACC in the former and use of mutants with defective proton pumps in the latter allowed 192
experimental distinction of the physiological mechanisms regulating these processes Data 193
presented here provide strong evidence from both model plants supporting this linkage and 194
strongly suggest that cytosolic NO3- concentration in roots regulates NO3
- long-distance 195
transport from roots to shoots We also investigated how NO3- concentration in plant tissues 196
would be affected by NO3- long-distance transport vacuolar NO3
- sequestration and the 197
ensuing relationship with NO3- use efficiency We also proposed the physiological 198
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7
mechanisms likely to be important for enhancing NO3- use efficiency in plants These findings 199
will provide scientific rationales for improving NUE in important industrial and food crops 200
201
202
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8
Results 203
B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for NO3- 204
in root tissues 205
Our previous work identified high and low NUE B napus genotypes (Han et al 2015a 206
Han et al 2015b) NUE of B napus whether based on biomass or on grain yield (Table 1) 207
was significantly higher for the high-NUE genotype (H Xiangyou15) than for the low-NUE 208
genotype (L 814) That the activities of the tonoplast proton-pumps (V-ATPase and V-PPase) 209
of root tissues in the H genotype were lower than the L genotype at both seedling (Fig 1A) 210
and flowering stages (Fig S1A) Given that proton pumps in the tonoplast supply energy for 211
vacuolar NO3- accumulation (Krebs et al 2010) NO3
- influx into the vacuole was 212
consequently much slower in the H genotype compared with the L genotype at both seedling 213
(Fig 1B) and flowering stages (Fig S1B) Moreover the percentage of vacuolar NO3- relative 214
to the total NO3- in protoplasts of root tissues in the H genotype was lower than in the L 215
genotype at seedling (Fig 1C) and flowering stages (Fig S1C) and NO3- accumulation in the 216
cytosol increased significantly in the H genotype compared with the L genotype at seedling 217
(Fig 1D) and flowering stages (Fig S1D) 218
219
B napus with higher NUE showed enhanced long-distance transport of NO3- from roots 220
to shoots 221
The relative expression of BnNRT15 in roots of the H genotype was significantly higher 222
than that in the L genotype at both seedling and flowering stages while the relative expression 223
of BnNRT18 in roots of the H genotype was lower than that in the L genotype at both 224
seedling (Fig 2-AB) and flowering stages (Fig S2-AB) As a consequence total N 225
concentration (traced by 15N) in roots of the H genotype was significantly lower than that of 226
the L genotype at both seedling (Fig 2C) and flowering stages (Fig S2C) while shoot N 227
concentration in the H genotype was significantly higher than the L genotype at seedling stage 228
(Fig 2C) This resulted in significantly higher [15N] SR ratio in the H genotype compared 229
with the L genotype (Fig 2D Fig S2D) 230
No significant differences in total N per plant between the H and L genotypes were 231
observed at both seedling and flowering stages (Fig S3) However NO3- concentration in 232
roots of the H genotype was significantly lower than in the L genotype at both seedling and 233
flowering stages (Fig 2E Fig S2E) while NO3- concentration in shoot tissues of this 234
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9
genotype was significantly higher at seedling stage (Fig 2E) resulting in higher [NO3-] SR 235
ratios at both stages (Fig 2F Fig S2F) 236
NO3- concentration in the xylem sap xylem sap volume and total NO3
- in xylem sap 237
were significantly higher in the H genotype than in the L genotype at seedling stage (Fig 238
S4ACE) the amount of NO3- in xylem sap significantly increased in the H genotype relative 239
to the L genotype at flowering stage (Fig S4F) Together these data suggests greater 240
mobilization of NO3- from root to shoot in the H genotype 241
242
NO3- up-regulates NRT15 but down-regulates NRT18 243
244
A previous study showed that increased NRT18 expression in nrt15 mutants (Chen et al 245
2012) and the expression of NRT15 was induced by NO3- (Lin et al 2008) We tested the 246
relationship between NRT15 and NRT18 expression in A thaliana and B napus No 247
significant differences were observed in AtNRT15 expression in roots between wild type 248
(col-0) and mutant plants (nrt18-2) (Fig 3A) However AtNRT15 expression was 249
significantly up-regulated by NO3- in roots of nrt18-2 mutants (Fig 3B) Expression of 250
AtNRT18 significantly increased in roots of nrt15-3 mutants but not in col-0 plants and 251
there were no significant differences in AtNRT18 expression with or without NO3- treatment 252
in roots of nrt15-3 mutants (Fig 3CD) 253
In contrast BnNRT15 expression in roots of the H genotype increased significantly with 254
NO3- treatment (Fig 3E) while the reverse was observed in expression of BnNRT18 which 255
showed lower expression under NO3- treatment than control (Fig 3F) This suggests that NO3
- 256
induces the expression of NRT15 but down-regulates NRT18 in A thaliana and B napus 257
Further studies are needed to elucidate the mechanisms regulating this reverse regulation 258
259
Reduced VSC of NO3- in roots drives its long-distance transport from roots to shoots 260
Our previous study showed that Bafi (Bafilomycin A1) inhibits V-ATPase DCCD 261
(DCCD + Na2SO3) inhibits V-PPase and a 11 combination of Bafi and DCCD (B+D) 262
inhibits both V-ATPase and V-PPase (Han et al 2015a) These inhibitors were used to 263
control activities of the tonoplast proton pumps in the H B napus plants V-ATPase activity 264
significantly decreased under Bafil and B+D treatments relative to the control and DCCD 265
treatments whereas V-PPase activity declined significantly under DCCD and B+D treatments 266
relative to that in the control and Bafil treatments (Fig 4A) 267
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10
The V-ATPase activity in roots of A thaliana was significantly lower in the V-ATPase 268
mutants (vha-a2 and vha-a3) than the wild type (col-0) and V-PPase mutants (avp1) (Fig 5A) 269
In contrast the V-PPase activity in roots of V-PPase mutant (avp1) were significantly lower 270
than in the wild type (col-0) and V-ATPase mutants (vha-a2 and vha-a3) (Fig 5A) 271
NO3- influxes into vacuoles of the H B napus plants significantly decreased when treated 272
with inhibitors of proton pumps (Bafil DCCD B+D) (Fig 4B) Similarly NO3- influxes into 273
the vacuole of the Arabidopsis mutants (vha-a2 vha-a3 and avp1) was significantly lower 274
than those found in the wild type (col-0) (Fig 5B) Previous studies showed that VSC of NO3- 275
decreased when the activities of the proton pumps decline (Li et al 2005 Krebs et al 2010 276
Han et al 2015a) 277
We further investigated NO3- distribution between the vacuole and cytosol as affected by 278
proton pump inhibition in the H genotype of B napus and the mutants of A thaliana 279
defective in vacuolar proton pumps The percentage of vacuolar NO3- relative to total NO3
- in 280
root protoplasts of the H genotype treated with inhibitors (Bafil DCCD B+D) was lower than 281
that observed in the control (Fig 4C) Results were also similar when using A thaliana 282
mutants where the percentage of vacuolar NO3- relative to the total NO3
- in protoplasts was 283
lower in the mutants (vha-a2 vha-a3 and avp1) than in the wild type (col-0) (Fig 5C) 284
Consequently NO3- accumulation in the cytosol showed a significant increase when energy 285
pumps were suppressed in roots of the H genotype (Fig 4D) and NO3- accumulation in the 286
cytosol of root tissues of A thaliana mutants (vha-a2 vha-a3 and avp1) similarly increased 287
compared with col-0 (Fig 5D) 288
Based on previous observations (Lin et al 2008) expression of NRT15 is strongly 289
induced by NO3- but expression of NRT18 is down-regulated (Fig3 Chen et al 2012) We 290
then hypothesized that expression of these two genes is contrastingly regulated by 291
concentration of NO3- in the cytosol Our results were congruent with this hypothesis the 292
expression of BnNRT15 in root tissues of the H genotype of B napus was significantly higher 293
in plants treated with Bafil DCCD or B+D compared with the control (Fig 4E) whereas the 294
expression of BnNRT18 decreased substantially (Fig 4F) Similar results were also observed 295
in A thaliana where expression of AtNRT15 in roots of the mutants (vha-a2 vha-a3 and 296
avp1) were significantly higher than in the wild type (col-0) but expression of AtNRT18 in 297
the same mutants were considerably lower (Fig 5EF) 298
NO3- concentrations in the xylem sap the N-distribution between shoot and root (SR 299
ratios based on [15N]) and the [NO3-] in shoots relative to roots of the H genotype treated with 300
energy pumpsrsquo inhibitors were significantly higher than in the control (Fig 4GH Fig S5A) 301
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11
Similar trends were also observed when using mutants of A thaliana deficient in the energy 302
pumpsrsquo activities NO3- concentration in the xylem sap the [15N] SR ratios and [NO3
-] SR 303
ratios were all significantly higher in the mutants than in the wild type (Fig 5GH Fig S5B) 304
These data clearly showed that prevention of N sequestration in vacuoles would enhance its 305
translocation to shoot 306
Additional evidence linking NO3- long-distance transport and NO3
- short-distance 307
distribution within cells were provided through experiments comparing A thaliana wild type 308
(Ws) and mutant clca-2 (Fig S6) The chloride channel (CLCa) is the main channel for 309
vacuolar anion accumulation Vacuolar sequestration capacity of NO3-significantly declines in 310
clca mutants (Angeli et al 2006) NO3- influx into the vacuolar space of Ws roots was 311
significantly higher than that observed in the clca-2 mutants (Fig S6A) This resulted in a 312
smaller proportion of vacuolar NO3- in clca-2 plants relative to the total NO3
- in root tissue 313
protoplasts as compared with Ws (Fig S6B) Consequently the accumulation of NO3- in the 314
cytosol was significantly higher in clca-2 than in Ws plants (Fig S6C) The higher 315
NO3-concentration in the cytosol together with the higher expression of AtNRT15 coupled 316
with lower expression of AtNRT18 in clca-2 roots (Fig S6DE) resulted in significant 317
increase in xylem sap NO3- concentration [15N] SR ratios and [NO3
-] SR ratios compared 318
with Ws plants (Fig S6FGH) 319
320
Increased NO3- translocation to shoots enhanced NUE 321
NO3- assimilation efficiency is known to be higher in shoots than in roots (Smirnoff et al 322
1985 Andrews et al 1986 Tang et al 2012 Tang et al 2013) We therefore hypothesize 323
that increased NO3- translocation to shoot and the consequent higher shootroot ratio will 324
contribute to higher NUE Our data support this hypothesis The H B napus genotype showed 325
enhanced long-distance transport of NO3- from roots to shoots (Fig 2 Fig S2) This enhanced 326
NO3- transport requires higher carbon skeleton provided through higher photosynthetic rate 327
(Tang et al 2012 Tang et al 2013) Our results showed that chlorophyll content 328
intercellular CO2 concentration and photosynthetic rate were significantly higher in the H than 329
the L genotype at both seedling and flowering stages (Table 2) Moreover the NO3- 330
assimilating enzymes were strongly induced by NO3- providing sufficient capacity for N 331
assimilation (Smirnoff et al 1985 Andrews et al 1986) Nitrate reductase (NR) and 332
glutamine synthetase (GS) activities in roots of the H genotype were significantly lower than 333
in the L genotype both at seedling and flowering stages (Fig S7) In contrast NR activities in 334
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12
shoots of the H genotype were significantly higher than in the L genotype at both stages (Fig 335
S7AB) and GS activity in the H genotype was also higher than those of the L genotype at 336
seedling stage (Fig S7C) 337
Experiments comparing A thaliana wild type (col-0 Ws) with mutants defective in 338
vacuolar proton-pumps (vha-a2 vha-a3avp1) and transport channel (clca-2) yielded similar 339
results that is increases in NO3- shootroot ratio essentially contributed to enhanced NUE 340
(Fig 6) The NO3- shootroot ratios in A thaliana mutants were higher than that in the wild 341
type (Fig 5GH) Generally the efficiency of inorganic N (NO3-) assimilation into organic N 342
is higher in shoots than in roots (Smirnoff et al 1985 Andrews et al 1986) therefore the 343
NO3- concentration in A thaliana mutants decreased (Fig 6CD) leading to higher NUE 344
compared with the wild type (Fig 6AB) 345
346
BnNRT15BnNRT18 in B napus affects NO3- long-distance transport from roots to 347
shoots 348
Both B napus and A thaliana are members of Cruciferae Family The amino acid 349
sequence identity between BnNRT15 and AtNRT15 was 90 (Fig S8A) the amino acid 350
sequence similarity between BnNRT18 and AtNRT18 was 908 (Fig S8B) Comparisons of 351
nucleotide and amino acid sequences (Harper et al2012) of these genes 352
(httpbrassicanbiacukcgi-binmicroarray_databasecgi) showed that BnNRT15 353
(EV220114) and BnNRT18 (EV116423) of B napus are respectively highly homologous 354
with AtNRT15 and AtNRT18 and the two genes are mainly expressed in roots of both species 355
showing similar organ-specificity (Lin et al 2008 Li et al 2010) 356
NO3- long-distance transport from roots to shoots is therefore regulated by NRT15 and 357
NRT18 as reported before (Lin et al 2008 Li et al 2010) The [15N-traced] SR ratios and 358
the [NO3-] SR ratios were significantly lower in nrt15-3 mutants relative to the wild type 359
(col-0) while [15N] SR and [NO3-] SR ratios showed significant increase in nrt18-2mutant 360
relative to col-0 (Fig S9CD) A previous study showed that expression of NRT15 is 361
down-regulated by ACC and MeJA treatments while the expression of NRT18 is strongly 362
up-regulated in A thaliana (Zhang et al 2014) Consequently NO3- accumulated in plant 363
roots probably as an adaptive measure for abiotic stresses (Chen et al 2012) Both the H- 364
and L- B napus genotypes showed significantly lower expression of BnNRT15 in roots when 365
treated with ACC (Fig S10A) but the expression of BnNRT18 remained higher than in the 366
control plants (Fig S10B) This resulted in significantly lower [15N] SR ratios following 367
ACC treatment (Fig S10CDE) These data indicate that BnNRT15 and BnNRT18 play 368
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13
similar functions for NO3- long-distance transport between root and shoot in both B napus 369
and A thaliana (Lin et al 2008 Li et al 2010) 370
371
372
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14
Discussion 373
Nitrate long-distance transport from root to shoot is regulated by cytosolic NO3- in roots 374
NO3- long-distance transport from root to shoot is controlled by NRT gene family For 375
instance xylem NO3- loading and unloading are mainly controlled by NRT15 and NRT18 376
respectively (Lin et al 2008 Li et al 2010) Whereas the NO3- short-distance transport 377
between the vacuole and cytosol is controlled by tonoplast proton-pumps (V-ATPase and 378
V-PPase) (Krebs et al 2010) and by activity of the CLCa channel in the tonoplast membrane 379
(Angeli et al 2006 Wege et al 2014) tonoplast proton-pumps provide the energy required 380
for NO3- accumulation into the vacuole through CLCa (Gaxiola et al 2001 Brux et al 2008 381
Krebs et al 2010) The expression of NRT15 is up-regulated by NO3- in plant culture 382
solution (Lin et al 2008) whereas regulation of NRT18 expression is contrary to that of 383
NRT15 under both control and abiotic stress conditions (Li et al 2010 Chen et al 2012 384
Zhang et al 2014) 385
Despite the progress made in characterizing the genes involved in transport and 386
metabolism of NO3- in plants the relationship between long- and short-distance transport and 387
how these two pathways are regulated have not been thoroughly investigated We hypothesize 388
that NRT15 expression and its role in long-distance transport is regulated by NO3- in plant 389
tissue as was observed in culture solution (Lin et al 2008) and our data support this 390
hypothesis Both BnNRT15 and AtNRT15 expression were up-regulated by increasing 391
cytosolic NO3- in roots (Fig 4 Fig 5) The increase in cytosolic NO3
- was achieved through (i) 392
use of A thaliana mutants defective in proton-pumps (V-ATPase and V-PPase) or CLCa 393
activities in the tonoplast and (ii) inhibition of B napus tonoplast proton-pumps In both 394
model plants the influx of NO3- into vacuoles was substantially reduced resulting in higher 395
concentrations of NO3- in the cytosol (Fig 4A-D Fig 5A-D Fig S6A-C) Under those 396
conditions expression of BnNRT15 and AtNRT15 was up-regulated by the enhanced 397
cytosolic NO3- while expression of BnNRT18 and AtNRT18 was down-regulated (Fig 4EF 398
Fig 5EF FigS6DE) As a consequence more NO3- was loaded into the xylem sap and 399
transported from root to shoot (Fig S5AB FigS6F) This is also reflected as increased 400
shootroot ratios of N traced by [15N] and [NO3-] (Fig 4GH Fig 5GH Fig S6GH) 401
Interestingly the expression of both BnNRT15 and AtNRT15 is up-regulated by 402
increased cytosolic NO3- concentration in roots but were not affected by NO3
- sequestered 403
into vacuoles (Fig 4 Fig 5 Fig S6) This is because vacuolar NO3- is functionally separated 404
by the tonoplast and cannot be assimilated by enzymes that are localized in the cytosol 405
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15
Therefore NO3--inducible genes such as NR and NRT15 were not influenced by vacuolar 406
NO3- concentration (Martinoia et al 1981 Martinoia et al 2000 Zhang et al 2012) In 407
addition NO3- concentration in the cytosol is controlled by its short-distance transport through 408
the tonoplast which is mediated through the tonoplast proton-pumps providing the required 409
energy for its sequestration into vacuoles against concentration gradient (Gaxiola et al 2001 410
Brux et al 2008 Krebs et al 2010) The gradient of H+ between the inside of the vacuole 411
and the cytosol is maintained by an 2NO3-1H+ antiporter mechanism that facilitatesNO3
- 412
transport through CLCa (Angeli et al 2006 Wege et al 2014) Our data provide clear 413
evidence that NO3- long-distance transport from root to shoot is regulated by cytosolic NO3
- 414
concentration in roots 415
Regulatory mechanisms that control the expression of NRT18 and NRT15 as mediated 416
by NO3- are still unknown however previous studies showed that their expression is also 417
affected by abiotic stresses whereby NRT15 is down-regulated and NRT18 is up-regulated 418
(Li et al 2010 Zhang et al 2014) AtNRT18 expression in A thaliana nrt15 mutants is 419
highly up-regulated (Chen et al 2012) as was also observed here (Fig 3C) Moreover 420
AtNRT15 expression was enhanced by NO3- in culture solution and was not affected in 421
nrt18-2 mutant (Fig 3AB) On the other hand AtNRT18 expression was not affected by 422
cytosolic NO3- but down-regulated when AtNRT15 expression was up-regulated by NO3
-(Fig 423
S6DE) Analogous results were also obtained with B napus genotypes (Fig 3EF) Based on 424
these results we argue that NRT18 expression is not directly influenced by cytosolic NO3- but 425
rather by NRT15 expression (Fig 4 Fig 5 and Fig S6) suggesting NRT18 is probably 426
acting downstream of NRT15 Apparently the regulatory mechanisms of this NRT gene 427
family still awaits further studies to be elucidated 428
429
Response of NUE to NO3- long-distance transport 430
NO3- assimilation efficiency is higher in shoot than in root tissues and can be further 431
enhanced by carbon assimilation (Smirnoff et al 1985 Tang et al 2013) Therefore 432
translocation of higher proportion of NO3- from roots to shoots likely contributes to better 433
crop growth and higher NUE (Andrews et al 1986 Tang et al 2012) The present study 434
provides direct evidence for these results using contrasting B napus genotype identified 435
before (Zhang et al 2009 Han et al 2015a) Our data confirmed the genetic variation in 436
NUE between these two genotypes (Table 1) 437
We measured NO3- distribution in root and shoot of these genotypes to elucidate the 438
physiological mechanisms contributing to variation in NUE The data were consistent with 439
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16
our hypothesis that concentration of NO3- in the xylem sap and shoot would be significantly 440
higher in the H genotype than in the L genotype (Fig 2EFFig S2EF Fig S4) The NO3- 441
long-distance transport from root to shoot was affected by cytosolic NO3- in roots of both A 442
thaliana and B napus genotypes (Fig 4-5 Fig S6) The activities of the tonoplast 443
proton-pumps in roots were significantly lower in the H genotype than in the L genotype (Fig 444
1AB) Consequently less NO3- was sequestered into the vacuole and more NO3
- was retained 445
in cytosol (Fig 1CD) BnNRT15 expression was then up-regulated resulting in greater 446
loading into the xylem while BnNRT18 was down-regulated (Fig 2AB) probably to 447
suppress NO3- unloading from xylem sap Consequently a higher proportion of NO3
- was 448
transported from root to shoots in the H genotype(Fig 2C-D Fig S4) 449
Moreover photosynthetic carbon fixation (Table 2) and activities of both NR and GS 450
were higher in shoots of the H genotype (Fig S7) both of which promote higher N 451
assimilation These results agreed with previous reports (Smirnoff et al 1985 Andrews et al 452
1986 Tang et al 2012 Tang et al 2013) where higher NO3- transport from root to shoot 453
higher photosynthetic rate and N assimilation efficiency in shoots were suggested as essential 454
mechanisms for high-NUE in crop plants These qualities endowed the B napus H genotype 455
Xiangyou15 with higher NUE as compared with the L genotype 814 456
NO3- long-distance transport in B napus is not only regulated by BnNRT15 and 457
BnNRT18 in root tissues (Fig 4 Fig 5 Fig S6) but also by stomatal conductance and 458
transpiration by indirectly controlling xylem sap flow and long-distance transport 459
(Dechorgnat et al 2011 Krapp et al 2014) Stomatal conductance and transpiration rates of 460
the H genotype were significantly higher than those of the L genotype (Table S1) a result 461
consistent with other studies describing characteristics of high-NUE plants (Daniel-Vedele et 462
al 1998 Wilkinson et al 2007 Dechorgnat et al 2011 Krapp et al 2014) 463
464
Response of NUE to NO3- short-distance transport 465
NUE is also strongly controlled by NO3- short-distance distribution between the vacuole 466
and cytosol (Han et al 2015a) A lower proportion of NO3- accumulating in vacuoles and a 467
higher proportion retained in the cytosol will contribute to better NUE in crop plants (Han et 468
al 2015b) The energy required for vacuolar NO3- sequestration is provided by the tonoplast 469
proton-pumps (Gaxiola et al 2001 Brux et al 2008 Krebs et al 2010) while the 470
distribution channel CLCa enables transport of NO3- across the vacuolar membrane (Angeli et 471
al 2006 Wege et al 2014) The activities of the tonoplast proton-pumps (V-ATPase and 472
V-PPase) in leaves of the H genotype were significantly lower than those of the L genotype at 473
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17
flowering stages (Han et al 2015aHan et al 2015b) thus we expected similar differences in 474
root tissues Our results confirmed that the activities of tonoplast proton-pumps in root tissues 475
of B napus H genotype and A thaliana mutants were lower than those in the L genotype and 476
A thaliana wild type leading to less NO3- influx into vacuoles and more in the cytosol (Fig 1 477
Fig 5A-D) Moreover the enzymes catalyzing N assimilation are located in the cytoplasm 478
where NO3- could be assimilated (Martinoia et al 1981 Han et al 2015a) or immediately 479
loaded into xylem sap for long-distance transport from roots to shoots (Lin et al 2008) 480
Consequently the NO3- concentration in root tissues of the H genotype and A thaliana 481
mutants defective in tonoplast proton-pumps was significantly lower than that in the L 482
genotype (Fig 2E) and in A thaliana wild type (Fig 6C) Therefore the higher proportion of 483
NO3- transported to shoot and the lower concentrations retained in root vacuoles contributed 484
to the higher NUE in Xiangyou15 and A thaliana mutants (vha-a2 vha-a3 and avp1) (Table 485
1Fig 6A) Further studies are needed to assess the impacts of regulating vacuolar 486
sequestration of NO3- and rates of long-distance transport for enhancing NUE in other crop 487
species and genotypes 488
These data suggested that NO3- distribution between vacuoles and cytosol within cells 489
regulates NO3- long-distance transport from root to shoot and ultimately affects NUE NO3
- 490
loading into the xylem sap is an active transport process (Lin et al 2008) therefore not 491
regulated by absolute concentration of NO3- in the cytosol but rather by genes involved in 492
NO3- long-distance transport (NRT15 and NRT18) Apparently the expression of NRT15 the 493
gene responsible for xylem loading is regulated by NO3-concentration in the cytosol 494
Therefore both NO3- short-distance transport across the tonoplast and its long-distance 495
transport from root to shoot are important determinants of NUE in both B napus and A 496
thaliana (Table 1 and Fig 6) 497
498
Roles of NRT15 and NRT18 in NO3- long-distance transport in B napus and A 499
thaliana 500
Our results (Figs S9 S10) showed that the proportion of NO3- distributed from root to 501
shoot is strongly controlled by NRT15 in accord with previous studies (Lin et al 2008 Li et 502
al 2010) The data suggest that the high-NUE genotypes possess lower activities of tonoplast 503
proton-pumps (V-ATPase and V-PPase) resulting in less NO3- accumulation in vacuoles and 504
more NO3- retention in cytosol (Fig 7) The higher NO3
- in the cytosol then up-regulated 505
NRT15 which then down-regulates NRT18 As a result more NO3- is loaded into the xylem 506
system by NRT15 mechanism and less NO3- is unloaded via NRT18 (Fig 7) Differential 507
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18
regulation of these NRT genes in high-NUE plants increased transport of NO3- from root to 508
shoot through the xylem vascular tissues (Fig 7) This coordinated NO3- long- and 509
short-distance transport likely fine-tunes NO3- allocation and modulates the balance of NO3
- 510
distribution between roots and shoots (Fig 7) 511
Our research also revealed differences between A thaliana and B napus in the 512
expression of the wild-type NRT genes in response to NO3- in the protoplast Moreover using 513
A thaliananrt15-3 mutants we uncovered possible regulatory interactions between a 514
functioning (wild-type) NRT15 gene and NRT18 expression in response to NO3- 515
concentration Because mutants defective in nrt15 are not available for B napus we could 516
not confirm that the respective NRT15 and NRT18 systems behave in an analogous way in 517
these plant species Based on Harper et al (2012) research database our homology 518
comparisons and gene function annotations provided strong evidence for the roles of 519
BnNRT15 and BnNRT18 in B napus Finally our studies were conducted using two model 520
dicotyledonous plant species for which we had ample data on responses to specific inhibitors 521
(B napus) or specific mutants were available with results forming the bases for the model 522
presented in Figure 7 Further research using genetically diverse plant species (eg 523
monocotyledonous crops such as rice wheat and corn) will help validate this model and its 524
broader application across species The information generated in this study could have future 525
application for improving NUE in commercial crops with consequent reduction in nitrogen 526
use and benefits to the environment 527
528
Materials and Methods 529
Plant material 530
The two oilseed rape (Bnapus) cultivars used in this study have been characterized 531
before as high- (Xiangyou15 referred to as H genotype hereafter) and low- (814 referred to 532
as L genotype) nitrogen use efficiency (NUE) genotypes (Zhang et al 2009 Han et al 2015b) 533
Here we define NUE as the total biomass per unit of N uptake by the plant with total 534
biomass includes roots shoots and grains The two genotypes were provided by the Hunan 535
Sub-Center of Improvement Center of National Oil Crops Hunan China 536
The Arabidopsis thaliana wild-type Columbia-0 (col-0) was used as control for 537
V-ATPase (vha-a2 and vha-a3) V-PPase (avp1) nrt15-3 and nrt18-2 mutants whereas the A 538
thaliana wild-type Wassilewskija (Ws) was used as control for clca-2 mutants A thaliana 539
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19
mutants and transgenic lines (nrt15-3 and nrt18-2) have been described previously (Chen et 540
al 2012 Zhang et al 2014) The mutant lines vha-a2 (Salk_142642) and vha-a3 541
(Salk_122135) described by Krebs et al (2010) were obtained from the Arabidopsis 542
Biological Resources Center (ABRC) The mutant line avp1 (GK-005004) described in Li et 543
al (2005) was obtained from the European Arabidopsis Stock Centre while the mutant line 544
clca-2 (FST 171A06) described in Angeli et al (2006) was from the Institute National de la 545
Recherche Agronomique (INRA) collection in France together with the T-DNA mutants used 546
for genotyping to select pure mutant lines 547
548
Growth conditions 549
B napus plants were grown hydroponically in ceramic pots(20cmtimes15cm) filled with a 550
nutrient solution in the greenhouse under natural light as described in Han et al (2015b) The 551
pots were arranged in a completely randomized design with six biological replications The 552
nutrient solution was replaced every 3 days and its pH adjusted to 55 daily Experiments were 553
conducted at the field station of Hunan Agricultural University Southern China 554
A thaliana plants were grown in a nutrient solution in plastic pots as described in Arteca 555
et al (2000) and Gong et al (2003) The solution was changed every 3 days with pH adjusted 556
daily to 58 Pots were arranged in a completely randomized design with six biological 557
replications The nutrient solution used for both species consisted of 125mM KNO3 558
0625mM KH2PO4 05mM MgSO4 05mM Ca (NO3)2middot4H2O 0025mM Fe-EDTA 025 559
mlL-1 micronutrients (stock solution concentrations 70mM B 14mM Mn 1mM Zn 05mM 560
Cu and 02mM Mo) The experiments were conducted at Hunan Agricultural University in a 561
phytotron set at 70 relative humidity16h-light8h-dark cycle and constant temperature of 562
22degC 563
564
Experimental treatments and phenotyping 565
All measurements made on B napus plants were conducted either at the seedling stage 566
(2 months after transplanting) or at flowering (5 months after transplanting) The whole root 567
tissue was harvested and used for analyses at either stage For shoot measurements the fourth 568
leaf from the bottom was used for measurements made at seedling stage whereas the 12th leaf 569
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20
from the bottom was harvested for measurements made at flowering stage For A thaliana 570
whole root and shoot of four-week-old plants were sampled and used for different assays 571
572
Vacuolar proton-pumps inhibitor treatments 573
Inhibition of vacuolar proton pumps of B napus (Xiangyou15) was conducted at the seedling 574
stage in hydroponic culture as described in Han et al (2015a) with minor modifications 575
Plants grown in hydroponic solution were used as control Inhibitor treatment was conducted 576
using several chemicals Bafi(25 nmol L-1 Bafilomycin A1) an inhibitor of V-ATPase 577
DCCD(10 μmol L-1DCCD + 50 mmolL-1 Na2SO3) specifically inhibits V-PPase and 578
Bafi+DCCD in a 11 ratio as inhibitor of both V-ATPase and V-PPase The inhibitors were 579
applied in the hydroponic solutions for 24 h Fresh plant tissue samples from all treatments 580
were collected for different assays 581
582
NO3- induced gene expression 583
Four-week old A thaliana mutants (nrt15-3nrt18-2) were grown in hydroponics with 584
225mM (NH4)2succinate for 3 days and shifted to hydroponics with 45mM NO3- for 12 h 585
then root tissues were collected to assess relative expression of AtNRT15 and AtNRT18 586
genes Seedlings of B napus (Xiangyou15) plants grown hydroponically were treated with 587
5mM (NH4)2succinate for 3 d and shifted to fresh hydroponic solution containing 15 mM 588
NO3- for 12 h Root tissue was then collected to assess the relative expression of BnNRT15 589
and BnNRT18 genes using quantitative RT-PCR 590
591
ACC treatment 592
Hydroponically grown seedlings of B napus were subjected to ACC treatment as described in 593
Zhang et al (2014) with minor modifications Seedlings of the two genotypes Xiangyou15 594
and 814 were transferred to a nutrient solution containing 002mM L-1 ACC for 6 h or grown 595
in normal hydroponic solution as control Root tissues were then collected to assay relative 596
expression of BnNRT15 and BnNRT18 using quantitative RT-PCR Another set of B napus 597
plants were grown in ACC-containing hydroponic solution but supplemented with 15NO3- (22 598
15N) replacing the unlabeled NO3- for 1 h Root and shoot tissues were collected separately 599
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21
for 15N measurement using a continuous-flow isotope ratio mass spectrometer coupled with a 600
C-N elemental analyzer (ANCA-MS PDZ Europa) 601
602
Biomass and N concentration 603
Grain yield dry biomass and N concentration were determined in plant samples at 604
harvest Plants were harvested (including senescing leaves) then dried in an oven for 30 min 605
at 105degC then at 70degC to a constant weight and weighed N concentration was determined 606
using the Kjeldahl method (Shi et al 2010 Han et al 2015b)02g of dried samples were 607
digested with H2SO4-H2O2 in a 100-ml Kjeldahl digestion flask after which N concentration 608
was determined using a Foss Auto Analyser Unit (Kjeldahl 8400) 609
610
V-ATPase and V-PPase activities 611
Roots (10 g) of B napus were collected at seedling and flowering stages and that of A 612
thaliana (05 g) sampled at seedling stage then used for determining the activities of 613
V-ATPase and V-PPase (Han et al 2015b) Activities of these two energy pumps within 614
microsomal membranes were followed colorimetrically based on released Pi as described by 615
Zhu et al (2001) and Krebs et al (2010) 616
617
Assay of NO3- flux in vacuoles 618
Roots of both species were collected similarly as that used for V-ATPase and V-PPase 619
activities then used for vacuole isolation and measurement of NO3- flux in vacuoles as 620
described by Robert et al (2007) with minor modification (Han et al 2015b) Net fluxes of 621
NO3- in vacuoles were measured non-invasively using SIET (scanning ion-selective electrode 622
technique SIET system BIO-003A Younger USA Science and Technology Corporation 623
USA) An NO3--selective microelectrode used for the assay of NO3
- flux was vibrated in the 624
measuring solution between two positions 1 μm and 11 μm from the vacuole surface 625
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles Background 626
signal was recorded by vibrating the electrode in the measuring solution in the absence of 627
vacuoles Ion flux was calculated by Fickrsquos law of diffusion J = -D(dcdx) where J represents 628
the ion flux (picomolescm-2s-1) dcdx is the ion concentration gradient and D is the ion 629
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22
diffusion constant in a particular medium The direction of the flux was derived from Fickrsquos 630
law of diffusion that relates the concentration gradient 631
632
Isolation of intact protoplasts and vacuoles for determining NO3- concentration 633
Similar to the above assays root tissues (10 g) were collected from B napus at seedling 634
and flowering stages and from A thaliana (05 g) seedlings and used to isolate intact 635
protoplasts and vacuoles as described by Robert et al (2007) with minor modifications as 636
outlined in Huang et al (2012) and Han et al(2015b) The purified protoplasts were divided 637
into two equal aliquots with one of them used for isolation of vacuoles The purified 638
protoplasts and vacuoles were then sub-sampled and used to determine NO3- concentrations 639
(Vogeli-Lange and Wagner 1990) and for enzyme activity assays (Ma et al 2005) 640
NO3-concentrations in protoplasts and vacuoles were measured by a continuous-flow 641
auto-analyzer (Auto Analyser 3 Bran and Luebbe Germany) as described previously (Han et 642
al 2015b)The activities of acid phosphatase (ACP) and cytochrome oxidase (COX) were 643
determined using plant ACP colorimetry- and COX-assay kits (GenMedSci Inc) following 644
the manufacturerrsquos instructions ACP activity specific to vacuoles was determined and used to 645
normalize NO3- accumulation We measured NO3
- in the protoplast outside the vacuole which 646
includes the cytosol and organelles eg mitochondria and Golgi Apparatus (Robert et 647
al2007) Since most of the NO3- in the protoplast outside the vacuole is located in the cytosol 648
(Krebs et al 2010) we refer to NO3- distribution between vacuoles and cytosol rather than 649
vacuole versus protoplast 650
651
Quantitative RT-PCR 652
Total RNA extracted from B napus roots was prepared using Trizol reagent (Invitrogen) 653
cDNAs were synthesized using M-MLV reverse transcriptase (Promega) following 654
manufacturerrsquos protocol The relative expression of BnNRT15 (EV220114) BnNRT18 655
(EV116423) and Bnactin (AF1118121) genes in plant roots were determined by quantitative 656
RT-PCR and run on a LightCycler instrument (Roche) with the SYBR Green Real-Time PCR 657
Master Mix Kit (TOYOBO Japan) under the following conditions 95degC for 2min then 45 658
cycles of 95degC for 10s 60degC for 10s and 72degC for 20s The primer sequences of BnNRT15 659
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23
BnNRT18 and Bnactin genes were obtained from httpbrassicanbiacuk as described in 660
Harper et al (2012) and expression levels were normalized to Bnactin as control 661
Total RNA extracted from the 4-week-old A thaliana roots under the treatments 662
conditions described above were prepared using Trizol reagent (Invitrogen) The cDNAs were 663
synthesized using M-MLV reverse transcriptase (Promega) based on the manufacturerrsquos 664
protocol and as described in Li et al (2010)The primer sequences of quantitative RT-PCR 665
used in these assays are listed in Supplemental Table 2 and were described previously (Chen 666
et al 2012 Zhang et al 2014) and their expression was normalized to Atactin2 as control 667
668
Determination of nitrate distribution using 15NO3- 669
Hydroponically grown B napus (seedling and flowering stages) and A thaliana 670
(seedling) were transferred to 01mM CaSO4 solution for 1min then to their respective 671
hydroponic nutrient solutions with15NO3- (22 excess) replacing unlabeled NO3
- for 1h 672
Plants were then transferred to 01mM CaSO4 solution for 1min after which roots were 673
washed with deionized water Root and shoot samples were separated and dried at 105degC for 674
30min followed by 70degC for 3 d then grounded and used for assaying 15N content using a 675
continuous-flow isotope ratio mass spectrometer coupled with a carbon-nitrogen elemental 676
analyzer (ANCA-MS PDZ Europa) 677
678
Xylem sap collection and assay of nitrate concentration 679
We used the method of Tang et al (2012) for collecting xylem sap Plants were cut 680
leaving1cm segments with intact roots at seedling and flowering stages for B napus and at 681
seedling stage for A thaliana Roots were immediately immersed in their respective nutrition 682
solutions Weighed cotton was put on the cut surface to absorb extruding xylem sap for 1 h 683
The cotton was then wrapped in plastic film and the volume of xylem sap was calculated as 684
the weight gain of the cotton Xylem sap was then squeezed from the cotton using a syringe 685
and used for subsequent assay of nitrate concentration using a continuous-flow auto-analyzer 686
(Auto Analyser 3 Bran and Luebbe Germany) 687
To determine nitrate concentration samples (10 g fresh root and shoot at seedling and 688
flowering of B napus and 05 g fresh root and shoot of 4-week-old A thaliana) were frozen 689
in liquid N2 and ground with a mortar and pestle the powder was then transferred to a beaker 690
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
containing 70 ml deionized water and boiled for 30 min to extract nitrate then cooled and 691
made to 100 ml volume and 02 g activated carbon added to eliminate the effect of 692
chlorophyll The mixture was filtered and nitrate in the filtrate was determined using a 693
continuous-flow auto-analyser (Atuo-Analyser 3 Bran and Luebbe Germany) 694
695
Measurements of photosynthesis 696
All measurements of photosynthesis and related parameters were conducted at 1000 h 697
using intact leaves The 4th leaf from the bottom in B napus at seedling stage and the 12th leaf 698
from the bottom at flowering stage were used for determining chlorophyll concentration 699
photosynthetic rate intercellular CO2 concentration transpiration rate and stomatal 700
conductance Chlorophyll concentration was determined by SPAD-502 (Minolta Camera Co 701
Ltd Japan) (Luo et al 2006 Yandeau-Nelson et al 2011) Photosynthetic rate intercellular 702
CO2 concentration transpiration rate and stomatal conductance were measured using LI-6400 703
Portable Photosynthesis System (Li-Cor Biosciences Co Ltd USA) set at flow rate of 500 704
μmol s-1 photosynthetic photon flux density of 1000 μmol m-2 s-1 relative humidity of 65 705
CO2 concentration of 400ppm and ambient temperatures 706
707
NR and GS activities 708
Leaves with similar age as those used for photosynthesis in B napus were used for the 709
assay of nitrate reductase (NR) and glutamine synthetase (GS) activities NR activity was 710
measured by the modified method of Fan et al (2007) Fresh samples (05g) were frozen in 711
liquid N2 ground into powder in the presence of acid-washed sand and homogenized with 4 712
ml of extraction buffer (0025mol L-1phosphate buffer pH87 1211g L-1 cysteine 0372g L-1 713
EDTA) Homogenates were centrifuged at 30000timesg for 15 min at 4degC and the supernatants 714
were treated with sulfanilamide and α-naphthylamine reagents for colorimetric (540nm) 715
determination of nitrite as described by Fan et al (2007) 716
GS activity was determined using a modified reverseγ-glutamyltransferase method 717
(Wang et al2014) which measures the GS-catalyzed formation of glutamyl-γ-hydroxamate 718
from glutamine and hydroxylamine Fresh samples were frozen for 30 min at -20degC then 719
ground in 10ml Tris-HCl buffer and acid-washed sand The homogenates were filtered 720
through two layers of gauze and centrifuged at 8000timesg for 15 min at 4degC About 12ml of the 721
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
supernatant was added in a reaction mixture and treated as described (Wang et al2014) After 722
the GS-catalyzed reaction was stopped and the solution centrifuged glutamyl-γ723
-hydroxamate was quantified colorimetrically in the supernatant (at 485 nm) and the 724
concentration was determined using a standard curve 725
726
Statistical analyses 727
We used the SPSS software (Statistical Product and Service Solutions V130 USA) for 728
ANOVA and mean separation of main-effects and interactions using Duncanrsquos multiple range 729
test at Plt005Values are presented as means and SD of three or six replicates from three 730
independent experiments Different letters or an asterisk () associated with specific data (eg 731
at the top of histogram bars in figures or within tables) denote significant differences at 732
Plt005 733
734
Supplemental Data 735
Supplemental Figure 1 B napus with higher NUE showed lower vacuolar sequestration 736
capacity (VSC) for NO3-in roots at flowering stage 737
Supplemental Figure 2 B napus with higher NUE showed enhanced long-distance transport 738
of NO3- from roots to shoots at flowering stage 739
Supplemental Figure 3 The two B napus (H and L genotypes) showed the same total N per 740
plant at seedling stage (A) and flowering stage (B) 741
Supplemental Figure 4 B napus with higher NUE showed increased NO3- concentration in 742
the xylem sap at seedling and flowering stages 743
Supplemental Figure 5 NO3- concentration in the xylem sap as affected by inhibitor 744
treatments in B napus and in the energy pumpsrsquo mutants of A thaliana (col-0 vha-a2 vha-a3 745
avp1) 746
Supplemental Figure 6 Reduced VSC for NO3- in roots drives long-distance transport of 747
NO3- from roots to shoots in the A thaliana wild type (Ws) and mutant (clca-2) 748
Supplemental Figure 7 Differences of NR and GS activities between the two B napus (H 749
and L genotypes) at seedling and flowering stages 750
Supplemental Figure 8 Amino acid sequences of BnNRT15 and BnNRT18 751
Supplemental Figure 9 Functions of AtNRT15 and AtNRT18 genes in root tissues of A 752
thaliana in controlling NO3- long-distance transport from root to shoots 753
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
Supplemental Figure 10 B napus BnNRT15 and BnNRT18 genes in roots were 754
coordinately modulated to facilitate NO3- long distance transport from roots to shoots 755
Supplemental Table 1 Differences in stomatal conductance and transpiration rate between 756
the two B napus genotypes 757
Supplemental Table 2 Sequences of primera used for qRT-PCR 758
759
Acknowledgements 760
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 761
Shanghai Institutes for Biological Sciences) for providing nrt15-3 and nrt18-2 seeds 762
This study was supported by the National Natural Science Foundation of China (Grant 763
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 764
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 765
The Ministry of Education in China Open Novel Science Foundation of Hunan province 766
(13K062) the National Key Laboratory of Plant Molecular Genetics and the Twelfth 767
Five-Year National Science and technology support program (2012BAD15BO4) 768
769
Figure 1 B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 770
NO3- in roots at the seedling stage 771
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 772
genotype 814 Specific activities of the tonoplast proton pumps are expressed as μmol Pi 773
released mg-1 protein h-1 NO3- fluxes are expressed as pmol NO3
- cm-2 S-1 Mature vacuoles 774
were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 775
measuring solution between the two positions 1 μm and 11 μm from the vacuole surface 776
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles recording the 777
stable reading data The background was recorded by vibrating the electrode in measuring 778
solution without vacuoles 779
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 780
for NO3- content and NO3
- accumulation normalized against the specific activity of the 781
vacuole acid phosphatase (ACP) as described in Materials and Methods and plotted as μmol 782
NO3- per μmol p-nitrophenol the end product of ACP assay 783
Proton pump activities in root tissues between H and L genotypes are shown at the seedling 784
stage (A) Different letters at the top of the histogram bars denote significant differences of 785
V-ATPase in root tissues between H and L genotypes (Plt005) an asterisk () at the top of the 786
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
histogram bars indicates significant difference in V-PPase activity in root tissues between H 787
and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788
Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789
genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790
bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791
bars on the figures indicate SD (n=6) 792
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793
seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794
to the total NO3- in protoplasts 795
NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796
total NO3-in the cytosol and was calculated as total NO3
- in protoplasts ndash total NO3- in 797
vacuole Different letters at the top of histogram bars denote significant differences of total 798
NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799
(n=6) 800
801
Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802
roots to shoots at the seedling stage 803
H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804
Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805
the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806
as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807
the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808
the histogram bars denote significant differences at Plt005 809
Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810
in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811
genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812
of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813
SD (n=3) different letters at the top of the histogram bars denote significant differences at 814
Plt005 level 815
The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816
at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817
genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818
different letters at the top of the histogram bars denote significant differences at Plt005 819
820
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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mechanisms likely to be important for enhancing NO3- use efficiency in plants These findings 199
will provide scientific rationales for improving NUE in important industrial and food crops 200
201
202
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Results 203
B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for NO3- 204
in root tissues 205
Our previous work identified high and low NUE B napus genotypes (Han et al 2015a 206
Han et al 2015b) NUE of B napus whether based on biomass or on grain yield (Table 1) 207
was significantly higher for the high-NUE genotype (H Xiangyou15) than for the low-NUE 208
genotype (L 814) That the activities of the tonoplast proton-pumps (V-ATPase and V-PPase) 209
of root tissues in the H genotype were lower than the L genotype at both seedling (Fig 1A) 210
and flowering stages (Fig S1A) Given that proton pumps in the tonoplast supply energy for 211
vacuolar NO3- accumulation (Krebs et al 2010) NO3
- influx into the vacuole was 212
consequently much slower in the H genotype compared with the L genotype at both seedling 213
(Fig 1B) and flowering stages (Fig S1B) Moreover the percentage of vacuolar NO3- relative 214
to the total NO3- in protoplasts of root tissues in the H genotype was lower than in the L 215
genotype at seedling (Fig 1C) and flowering stages (Fig S1C) and NO3- accumulation in the 216
cytosol increased significantly in the H genotype compared with the L genotype at seedling 217
(Fig 1D) and flowering stages (Fig S1D) 218
219
B napus with higher NUE showed enhanced long-distance transport of NO3- from roots 220
to shoots 221
The relative expression of BnNRT15 in roots of the H genotype was significantly higher 222
than that in the L genotype at both seedling and flowering stages while the relative expression 223
of BnNRT18 in roots of the H genotype was lower than that in the L genotype at both 224
seedling (Fig 2-AB) and flowering stages (Fig S2-AB) As a consequence total N 225
concentration (traced by 15N) in roots of the H genotype was significantly lower than that of 226
the L genotype at both seedling (Fig 2C) and flowering stages (Fig S2C) while shoot N 227
concentration in the H genotype was significantly higher than the L genotype at seedling stage 228
(Fig 2C) This resulted in significantly higher [15N] SR ratio in the H genotype compared 229
with the L genotype (Fig 2D Fig S2D) 230
No significant differences in total N per plant between the H and L genotypes were 231
observed at both seedling and flowering stages (Fig S3) However NO3- concentration in 232
roots of the H genotype was significantly lower than in the L genotype at both seedling and 233
flowering stages (Fig 2E Fig S2E) while NO3- concentration in shoot tissues of this 234
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genotype was significantly higher at seedling stage (Fig 2E) resulting in higher [NO3-] SR 235
ratios at both stages (Fig 2F Fig S2F) 236
NO3- concentration in the xylem sap xylem sap volume and total NO3
- in xylem sap 237
were significantly higher in the H genotype than in the L genotype at seedling stage (Fig 238
S4ACE) the amount of NO3- in xylem sap significantly increased in the H genotype relative 239
to the L genotype at flowering stage (Fig S4F) Together these data suggests greater 240
mobilization of NO3- from root to shoot in the H genotype 241
242
NO3- up-regulates NRT15 but down-regulates NRT18 243
244
A previous study showed that increased NRT18 expression in nrt15 mutants (Chen et al 245
2012) and the expression of NRT15 was induced by NO3- (Lin et al 2008) We tested the 246
relationship between NRT15 and NRT18 expression in A thaliana and B napus No 247
significant differences were observed in AtNRT15 expression in roots between wild type 248
(col-0) and mutant plants (nrt18-2) (Fig 3A) However AtNRT15 expression was 249
significantly up-regulated by NO3- in roots of nrt18-2 mutants (Fig 3B) Expression of 250
AtNRT18 significantly increased in roots of nrt15-3 mutants but not in col-0 plants and 251
there were no significant differences in AtNRT18 expression with or without NO3- treatment 252
in roots of nrt15-3 mutants (Fig 3CD) 253
In contrast BnNRT15 expression in roots of the H genotype increased significantly with 254
NO3- treatment (Fig 3E) while the reverse was observed in expression of BnNRT18 which 255
showed lower expression under NO3- treatment than control (Fig 3F) This suggests that NO3
- 256
induces the expression of NRT15 but down-regulates NRT18 in A thaliana and B napus 257
Further studies are needed to elucidate the mechanisms regulating this reverse regulation 258
259
Reduced VSC of NO3- in roots drives its long-distance transport from roots to shoots 260
Our previous study showed that Bafi (Bafilomycin A1) inhibits V-ATPase DCCD 261
(DCCD + Na2SO3) inhibits V-PPase and a 11 combination of Bafi and DCCD (B+D) 262
inhibits both V-ATPase and V-PPase (Han et al 2015a) These inhibitors were used to 263
control activities of the tonoplast proton pumps in the H B napus plants V-ATPase activity 264
significantly decreased under Bafil and B+D treatments relative to the control and DCCD 265
treatments whereas V-PPase activity declined significantly under DCCD and B+D treatments 266
relative to that in the control and Bafil treatments (Fig 4A) 267
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The V-ATPase activity in roots of A thaliana was significantly lower in the V-ATPase 268
mutants (vha-a2 and vha-a3) than the wild type (col-0) and V-PPase mutants (avp1) (Fig 5A) 269
In contrast the V-PPase activity in roots of V-PPase mutant (avp1) were significantly lower 270
than in the wild type (col-0) and V-ATPase mutants (vha-a2 and vha-a3) (Fig 5A) 271
NO3- influxes into vacuoles of the H B napus plants significantly decreased when treated 272
with inhibitors of proton pumps (Bafil DCCD B+D) (Fig 4B) Similarly NO3- influxes into 273
the vacuole of the Arabidopsis mutants (vha-a2 vha-a3 and avp1) was significantly lower 274
than those found in the wild type (col-0) (Fig 5B) Previous studies showed that VSC of NO3- 275
decreased when the activities of the proton pumps decline (Li et al 2005 Krebs et al 2010 276
Han et al 2015a) 277
We further investigated NO3- distribution between the vacuole and cytosol as affected by 278
proton pump inhibition in the H genotype of B napus and the mutants of A thaliana 279
defective in vacuolar proton pumps The percentage of vacuolar NO3- relative to total NO3
- in 280
root protoplasts of the H genotype treated with inhibitors (Bafil DCCD B+D) was lower than 281
that observed in the control (Fig 4C) Results were also similar when using A thaliana 282
mutants where the percentage of vacuolar NO3- relative to the total NO3
- in protoplasts was 283
lower in the mutants (vha-a2 vha-a3 and avp1) than in the wild type (col-0) (Fig 5C) 284
Consequently NO3- accumulation in the cytosol showed a significant increase when energy 285
pumps were suppressed in roots of the H genotype (Fig 4D) and NO3- accumulation in the 286
cytosol of root tissues of A thaliana mutants (vha-a2 vha-a3 and avp1) similarly increased 287
compared with col-0 (Fig 5D) 288
Based on previous observations (Lin et al 2008) expression of NRT15 is strongly 289
induced by NO3- but expression of NRT18 is down-regulated (Fig3 Chen et al 2012) We 290
then hypothesized that expression of these two genes is contrastingly regulated by 291
concentration of NO3- in the cytosol Our results were congruent with this hypothesis the 292
expression of BnNRT15 in root tissues of the H genotype of B napus was significantly higher 293
in plants treated with Bafil DCCD or B+D compared with the control (Fig 4E) whereas the 294
expression of BnNRT18 decreased substantially (Fig 4F) Similar results were also observed 295
in A thaliana where expression of AtNRT15 in roots of the mutants (vha-a2 vha-a3 and 296
avp1) were significantly higher than in the wild type (col-0) but expression of AtNRT18 in 297
the same mutants were considerably lower (Fig 5EF) 298
NO3- concentrations in the xylem sap the N-distribution between shoot and root (SR 299
ratios based on [15N]) and the [NO3-] in shoots relative to roots of the H genotype treated with 300
energy pumpsrsquo inhibitors were significantly higher than in the control (Fig 4GH Fig S5A) 301
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Similar trends were also observed when using mutants of A thaliana deficient in the energy 302
pumpsrsquo activities NO3- concentration in the xylem sap the [15N] SR ratios and [NO3
-] SR 303
ratios were all significantly higher in the mutants than in the wild type (Fig 5GH Fig S5B) 304
These data clearly showed that prevention of N sequestration in vacuoles would enhance its 305
translocation to shoot 306
Additional evidence linking NO3- long-distance transport and NO3
- short-distance 307
distribution within cells were provided through experiments comparing A thaliana wild type 308
(Ws) and mutant clca-2 (Fig S6) The chloride channel (CLCa) is the main channel for 309
vacuolar anion accumulation Vacuolar sequestration capacity of NO3-significantly declines in 310
clca mutants (Angeli et al 2006) NO3- influx into the vacuolar space of Ws roots was 311
significantly higher than that observed in the clca-2 mutants (Fig S6A) This resulted in a 312
smaller proportion of vacuolar NO3- in clca-2 plants relative to the total NO3
- in root tissue 313
protoplasts as compared with Ws (Fig S6B) Consequently the accumulation of NO3- in the 314
cytosol was significantly higher in clca-2 than in Ws plants (Fig S6C) The higher 315
NO3-concentration in the cytosol together with the higher expression of AtNRT15 coupled 316
with lower expression of AtNRT18 in clca-2 roots (Fig S6DE) resulted in significant 317
increase in xylem sap NO3- concentration [15N] SR ratios and [NO3
-] SR ratios compared 318
with Ws plants (Fig S6FGH) 319
320
Increased NO3- translocation to shoots enhanced NUE 321
NO3- assimilation efficiency is known to be higher in shoots than in roots (Smirnoff et al 322
1985 Andrews et al 1986 Tang et al 2012 Tang et al 2013) We therefore hypothesize 323
that increased NO3- translocation to shoot and the consequent higher shootroot ratio will 324
contribute to higher NUE Our data support this hypothesis The H B napus genotype showed 325
enhanced long-distance transport of NO3- from roots to shoots (Fig 2 Fig S2) This enhanced 326
NO3- transport requires higher carbon skeleton provided through higher photosynthetic rate 327
(Tang et al 2012 Tang et al 2013) Our results showed that chlorophyll content 328
intercellular CO2 concentration and photosynthetic rate were significantly higher in the H than 329
the L genotype at both seedling and flowering stages (Table 2) Moreover the NO3- 330
assimilating enzymes were strongly induced by NO3- providing sufficient capacity for N 331
assimilation (Smirnoff et al 1985 Andrews et al 1986) Nitrate reductase (NR) and 332
glutamine synthetase (GS) activities in roots of the H genotype were significantly lower than 333
in the L genotype both at seedling and flowering stages (Fig S7) In contrast NR activities in 334
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shoots of the H genotype were significantly higher than in the L genotype at both stages (Fig 335
S7AB) and GS activity in the H genotype was also higher than those of the L genotype at 336
seedling stage (Fig S7C) 337
Experiments comparing A thaliana wild type (col-0 Ws) with mutants defective in 338
vacuolar proton-pumps (vha-a2 vha-a3avp1) and transport channel (clca-2) yielded similar 339
results that is increases in NO3- shootroot ratio essentially contributed to enhanced NUE 340
(Fig 6) The NO3- shootroot ratios in A thaliana mutants were higher than that in the wild 341
type (Fig 5GH) Generally the efficiency of inorganic N (NO3-) assimilation into organic N 342
is higher in shoots than in roots (Smirnoff et al 1985 Andrews et al 1986) therefore the 343
NO3- concentration in A thaliana mutants decreased (Fig 6CD) leading to higher NUE 344
compared with the wild type (Fig 6AB) 345
346
BnNRT15BnNRT18 in B napus affects NO3- long-distance transport from roots to 347
shoots 348
Both B napus and A thaliana are members of Cruciferae Family The amino acid 349
sequence identity between BnNRT15 and AtNRT15 was 90 (Fig S8A) the amino acid 350
sequence similarity between BnNRT18 and AtNRT18 was 908 (Fig S8B) Comparisons of 351
nucleotide and amino acid sequences (Harper et al2012) of these genes 352
(httpbrassicanbiacukcgi-binmicroarray_databasecgi) showed that BnNRT15 353
(EV220114) and BnNRT18 (EV116423) of B napus are respectively highly homologous 354
with AtNRT15 and AtNRT18 and the two genes are mainly expressed in roots of both species 355
showing similar organ-specificity (Lin et al 2008 Li et al 2010) 356
NO3- long-distance transport from roots to shoots is therefore regulated by NRT15 and 357
NRT18 as reported before (Lin et al 2008 Li et al 2010) The [15N-traced] SR ratios and 358
the [NO3-] SR ratios were significantly lower in nrt15-3 mutants relative to the wild type 359
(col-0) while [15N] SR and [NO3-] SR ratios showed significant increase in nrt18-2mutant 360
relative to col-0 (Fig S9CD) A previous study showed that expression of NRT15 is 361
down-regulated by ACC and MeJA treatments while the expression of NRT18 is strongly 362
up-regulated in A thaliana (Zhang et al 2014) Consequently NO3- accumulated in plant 363
roots probably as an adaptive measure for abiotic stresses (Chen et al 2012) Both the H- 364
and L- B napus genotypes showed significantly lower expression of BnNRT15 in roots when 365
treated with ACC (Fig S10A) but the expression of BnNRT18 remained higher than in the 366
control plants (Fig S10B) This resulted in significantly lower [15N] SR ratios following 367
ACC treatment (Fig S10CDE) These data indicate that BnNRT15 and BnNRT18 play 368
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similar functions for NO3- long-distance transport between root and shoot in both B napus 369
and A thaliana (Lin et al 2008 Li et al 2010) 370
371
372
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Discussion 373
Nitrate long-distance transport from root to shoot is regulated by cytosolic NO3- in roots 374
NO3- long-distance transport from root to shoot is controlled by NRT gene family For 375
instance xylem NO3- loading and unloading are mainly controlled by NRT15 and NRT18 376
respectively (Lin et al 2008 Li et al 2010) Whereas the NO3- short-distance transport 377
between the vacuole and cytosol is controlled by tonoplast proton-pumps (V-ATPase and 378
V-PPase) (Krebs et al 2010) and by activity of the CLCa channel in the tonoplast membrane 379
(Angeli et al 2006 Wege et al 2014) tonoplast proton-pumps provide the energy required 380
for NO3- accumulation into the vacuole through CLCa (Gaxiola et al 2001 Brux et al 2008 381
Krebs et al 2010) The expression of NRT15 is up-regulated by NO3- in plant culture 382
solution (Lin et al 2008) whereas regulation of NRT18 expression is contrary to that of 383
NRT15 under both control and abiotic stress conditions (Li et al 2010 Chen et al 2012 384
Zhang et al 2014) 385
Despite the progress made in characterizing the genes involved in transport and 386
metabolism of NO3- in plants the relationship between long- and short-distance transport and 387
how these two pathways are regulated have not been thoroughly investigated We hypothesize 388
that NRT15 expression and its role in long-distance transport is regulated by NO3- in plant 389
tissue as was observed in culture solution (Lin et al 2008) and our data support this 390
hypothesis Both BnNRT15 and AtNRT15 expression were up-regulated by increasing 391
cytosolic NO3- in roots (Fig 4 Fig 5) The increase in cytosolic NO3
- was achieved through (i) 392
use of A thaliana mutants defective in proton-pumps (V-ATPase and V-PPase) or CLCa 393
activities in the tonoplast and (ii) inhibition of B napus tonoplast proton-pumps In both 394
model plants the influx of NO3- into vacuoles was substantially reduced resulting in higher 395
concentrations of NO3- in the cytosol (Fig 4A-D Fig 5A-D Fig S6A-C) Under those 396
conditions expression of BnNRT15 and AtNRT15 was up-regulated by the enhanced 397
cytosolic NO3- while expression of BnNRT18 and AtNRT18 was down-regulated (Fig 4EF 398
Fig 5EF FigS6DE) As a consequence more NO3- was loaded into the xylem sap and 399
transported from root to shoot (Fig S5AB FigS6F) This is also reflected as increased 400
shootroot ratios of N traced by [15N] and [NO3-] (Fig 4GH Fig 5GH Fig S6GH) 401
Interestingly the expression of both BnNRT15 and AtNRT15 is up-regulated by 402
increased cytosolic NO3- concentration in roots but were not affected by NO3
- sequestered 403
into vacuoles (Fig 4 Fig 5 Fig S6) This is because vacuolar NO3- is functionally separated 404
by the tonoplast and cannot be assimilated by enzymes that are localized in the cytosol 405
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15
Therefore NO3--inducible genes such as NR and NRT15 were not influenced by vacuolar 406
NO3- concentration (Martinoia et al 1981 Martinoia et al 2000 Zhang et al 2012) In 407
addition NO3- concentration in the cytosol is controlled by its short-distance transport through 408
the tonoplast which is mediated through the tonoplast proton-pumps providing the required 409
energy for its sequestration into vacuoles against concentration gradient (Gaxiola et al 2001 410
Brux et al 2008 Krebs et al 2010) The gradient of H+ between the inside of the vacuole 411
and the cytosol is maintained by an 2NO3-1H+ antiporter mechanism that facilitatesNO3
- 412
transport through CLCa (Angeli et al 2006 Wege et al 2014) Our data provide clear 413
evidence that NO3- long-distance transport from root to shoot is regulated by cytosolic NO3
- 414
concentration in roots 415
Regulatory mechanisms that control the expression of NRT18 and NRT15 as mediated 416
by NO3- are still unknown however previous studies showed that their expression is also 417
affected by abiotic stresses whereby NRT15 is down-regulated and NRT18 is up-regulated 418
(Li et al 2010 Zhang et al 2014) AtNRT18 expression in A thaliana nrt15 mutants is 419
highly up-regulated (Chen et al 2012) as was also observed here (Fig 3C) Moreover 420
AtNRT15 expression was enhanced by NO3- in culture solution and was not affected in 421
nrt18-2 mutant (Fig 3AB) On the other hand AtNRT18 expression was not affected by 422
cytosolic NO3- but down-regulated when AtNRT15 expression was up-regulated by NO3
-(Fig 423
S6DE) Analogous results were also obtained with B napus genotypes (Fig 3EF) Based on 424
these results we argue that NRT18 expression is not directly influenced by cytosolic NO3- but 425
rather by NRT15 expression (Fig 4 Fig 5 and Fig S6) suggesting NRT18 is probably 426
acting downstream of NRT15 Apparently the regulatory mechanisms of this NRT gene 427
family still awaits further studies to be elucidated 428
429
Response of NUE to NO3- long-distance transport 430
NO3- assimilation efficiency is higher in shoot than in root tissues and can be further 431
enhanced by carbon assimilation (Smirnoff et al 1985 Tang et al 2013) Therefore 432
translocation of higher proportion of NO3- from roots to shoots likely contributes to better 433
crop growth and higher NUE (Andrews et al 1986 Tang et al 2012) The present study 434
provides direct evidence for these results using contrasting B napus genotype identified 435
before (Zhang et al 2009 Han et al 2015a) Our data confirmed the genetic variation in 436
NUE between these two genotypes (Table 1) 437
We measured NO3- distribution in root and shoot of these genotypes to elucidate the 438
physiological mechanisms contributing to variation in NUE The data were consistent with 439
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16
our hypothesis that concentration of NO3- in the xylem sap and shoot would be significantly 440
higher in the H genotype than in the L genotype (Fig 2EFFig S2EF Fig S4) The NO3- 441
long-distance transport from root to shoot was affected by cytosolic NO3- in roots of both A 442
thaliana and B napus genotypes (Fig 4-5 Fig S6) The activities of the tonoplast 443
proton-pumps in roots were significantly lower in the H genotype than in the L genotype (Fig 444
1AB) Consequently less NO3- was sequestered into the vacuole and more NO3
- was retained 445
in cytosol (Fig 1CD) BnNRT15 expression was then up-regulated resulting in greater 446
loading into the xylem while BnNRT18 was down-regulated (Fig 2AB) probably to 447
suppress NO3- unloading from xylem sap Consequently a higher proportion of NO3
- was 448
transported from root to shoots in the H genotype(Fig 2C-D Fig S4) 449
Moreover photosynthetic carbon fixation (Table 2) and activities of both NR and GS 450
were higher in shoots of the H genotype (Fig S7) both of which promote higher N 451
assimilation These results agreed with previous reports (Smirnoff et al 1985 Andrews et al 452
1986 Tang et al 2012 Tang et al 2013) where higher NO3- transport from root to shoot 453
higher photosynthetic rate and N assimilation efficiency in shoots were suggested as essential 454
mechanisms for high-NUE in crop plants These qualities endowed the B napus H genotype 455
Xiangyou15 with higher NUE as compared with the L genotype 814 456
NO3- long-distance transport in B napus is not only regulated by BnNRT15 and 457
BnNRT18 in root tissues (Fig 4 Fig 5 Fig S6) but also by stomatal conductance and 458
transpiration by indirectly controlling xylem sap flow and long-distance transport 459
(Dechorgnat et al 2011 Krapp et al 2014) Stomatal conductance and transpiration rates of 460
the H genotype were significantly higher than those of the L genotype (Table S1) a result 461
consistent with other studies describing characteristics of high-NUE plants (Daniel-Vedele et 462
al 1998 Wilkinson et al 2007 Dechorgnat et al 2011 Krapp et al 2014) 463
464
Response of NUE to NO3- short-distance transport 465
NUE is also strongly controlled by NO3- short-distance distribution between the vacuole 466
and cytosol (Han et al 2015a) A lower proportion of NO3- accumulating in vacuoles and a 467
higher proportion retained in the cytosol will contribute to better NUE in crop plants (Han et 468
al 2015b) The energy required for vacuolar NO3- sequestration is provided by the tonoplast 469
proton-pumps (Gaxiola et al 2001 Brux et al 2008 Krebs et al 2010) while the 470
distribution channel CLCa enables transport of NO3- across the vacuolar membrane (Angeli et 471
al 2006 Wege et al 2014) The activities of the tonoplast proton-pumps (V-ATPase and 472
V-PPase) in leaves of the H genotype were significantly lower than those of the L genotype at 473
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17
flowering stages (Han et al 2015aHan et al 2015b) thus we expected similar differences in 474
root tissues Our results confirmed that the activities of tonoplast proton-pumps in root tissues 475
of B napus H genotype and A thaliana mutants were lower than those in the L genotype and 476
A thaliana wild type leading to less NO3- influx into vacuoles and more in the cytosol (Fig 1 477
Fig 5A-D) Moreover the enzymes catalyzing N assimilation are located in the cytoplasm 478
where NO3- could be assimilated (Martinoia et al 1981 Han et al 2015a) or immediately 479
loaded into xylem sap for long-distance transport from roots to shoots (Lin et al 2008) 480
Consequently the NO3- concentration in root tissues of the H genotype and A thaliana 481
mutants defective in tonoplast proton-pumps was significantly lower than that in the L 482
genotype (Fig 2E) and in A thaliana wild type (Fig 6C) Therefore the higher proportion of 483
NO3- transported to shoot and the lower concentrations retained in root vacuoles contributed 484
to the higher NUE in Xiangyou15 and A thaliana mutants (vha-a2 vha-a3 and avp1) (Table 485
1Fig 6A) Further studies are needed to assess the impacts of regulating vacuolar 486
sequestration of NO3- and rates of long-distance transport for enhancing NUE in other crop 487
species and genotypes 488
These data suggested that NO3- distribution between vacuoles and cytosol within cells 489
regulates NO3- long-distance transport from root to shoot and ultimately affects NUE NO3
- 490
loading into the xylem sap is an active transport process (Lin et al 2008) therefore not 491
regulated by absolute concentration of NO3- in the cytosol but rather by genes involved in 492
NO3- long-distance transport (NRT15 and NRT18) Apparently the expression of NRT15 the 493
gene responsible for xylem loading is regulated by NO3-concentration in the cytosol 494
Therefore both NO3- short-distance transport across the tonoplast and its long-distance 495
transport from root to shoot are important determinants of NUE in both B napus and A 496
thaliana (Table 1 and Fig 6) 497
498
Roles of NRT15 and NRT18 in NO3- long-distance transport in B napus and A 499
thaliana 500
Our results (Figs S9 S10) showed that the proportion of NO3- distributed from root to 501
shoot is strongly controlled by NRT15 in accord with previous studies (Lin et al 2008 Li et 502
al 2010) The data suggest that the high-NUE genotypes possess lower activities of tonoplast 503
proton-pumps (V-ATPase and V-PPase) resulting in less NO3- accumulation in vacuoles and 504
more NO3- retention in cytosol (Fig 7) The higher NO3
- in the cytosol then up-regulated 505
NRT15 which then down-regulates NRT18 As a result more NO3- is loaded into the xylem 506
system by NRT15 mechanism and less NO3- is unloaded via NRT18 (Fig 7) Differential 507
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regulation of these NRT genes in high-NUE plants increased transport of NO3- from root to 508
shoot through the xylem vascular tissues (Fig 7) This coordinated NO3- long- and 509
short-distance transport likely fine-tunes NO3- allocation and modulates the balance of NO3
- 510
distribution between roots and shoots (Fig 7) 511
Our research also revealed differences between A thaliana and B napus in the 512
expression of the wild-type NRT genes in response to NO3- in the protoplast Moreover using 513
A thaliananrt15-3 mutants we uncovered possible regulatory interactions between a 514
functioning (wild-type) NRT15 gene and NRT18 expression in response to NO3- 515
concentration Because mutants defective in nrt15 are not available for B napus we could 516
not confirm that the respective NRT15 and NRT18 systems behave in an analogous way in 517
these plant species Based on Harper et al (2012) research database our homology 518
comparisons and gene function annotations provided strong evidence for the roles of 519
BnNRT15 and BnNRT18 in B napus Finally our studies were conducted using two model 520
dicotyledonous plant species for which we had ample data on responses to specific inhibitors 521
(B napus) or specific mutants were available with results forming the bases for the model 522
presented in Figure 7 Further research using genetically diverse plant species (eg 523
monocotyledonous crops such as rice wheat and corn) will help validate this model and its 524
broader application across species The information generated in this study could have future 525
application for improving NUE in commercial crops with consequent reduction in nitrogen 526
use and benefits to the environment 527
528
Materials and Methods 529
Plant material 530
The two oilseed rape (Bnapus) cultivars used in this study have been characterized 531
before as high- (Xiangyou15 referred to as H genotype hereafter) and low- (814 referred to 532
as L genotype) nitrogen use efficiency (NUE) genotypes (Zhang et al 2009 Han et al 2015b) 533
Here we define NUE as the total biomass per unit of N uptake by the plant with total 534
biomass includes roots shoots and grains The two genotypes were provided by the Hunan 535
Sub-Center of Improvement Center of National Oil Crops Hunan China 536
The Arabidopsis thaliana wild-type Columbia-0 (col-0) was used as control for 537
V-ATPase (vha-a2 and vha-a3) V-PPase (avp1) nrt15-3 and nrt18-2 mutants whereas the A 538
thaliana wild-type Wassilewskija (Ws) was used as control for clca-2 mutants A thaliana 539
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
19
mutants and transgenic lines (nrt15-3 and nrt18-2) have been described previously (Chen et 540
al 2012 Zhang et al 2014) The mutant lines vha-a2 (Salk_142642) and vha-a3 541
(Salk_122135) described by Krebs et al (2010) were obtained from the Arabidopsis 542
Biological Resources Center (ABRC) The mutant line avp1 (GK-005004) described in Li et 543
al (2005) was obtained from the European Arabidopsis Stock Centre while the mutant line 544
clca-2 (FST 171A06) described in Angeli et al (2006) was from the Institute National de la 545
Recherche Agronomique (INRA) collection in France together with the T-DNA mutants used 546
for genotyping to select pure mutant lines 547
548
Growth conditions 549
B napus plants were grown hydroponically in ceramic pots(20cmtimes15cm) filled with a 550
nutrient solution in the greenhouse under natural light as described in Han et al (2015b) The 551
pots were arranged in a completely randomized design with six biological replications The 552
nutrient solution was replaced every 3 days and its pH adjusted to 55 daily Experiments were 553
conducted at the field station of Hunan Agricultural University Southern China 554
A thaliana plants were grown in a nutrient solution in plastic pots as described in Arteca 555
et al (2000) and Gong et al (2003) The solution was changed every 3 days with pH adjusted 556
daily to 58 Pots were arranged in a completely randomized design with six biological 557
replications The nutrient solution used for both species consisted of 125mM KNO3 558
0625mM KH2PO4 05mM MgSO4 05mM Ca (NO3)2middot4H2O 0025mM Fe-EDTA 025 559
mlL-1 micronutrients (stock solution concentrations 70mM B 14mM Mn 1mM Zn 05mM 560
Cu and 02mM Mo) The experiments were conducted at Hunan Agricultural University in a 561
phytotron set at 70 relative humidity16h-light8h-dark cycle and constant temperature of 562
22degC 563
564
Experimental treatments and phenotyping 565
All measurements made on B napus plants were conducted either at the seedling stage 566
(2 months after transplanting) or at flowering (5 months after transplanting) The whole root 567
tissue was harvested and used for analyses at either stage For shoot measurements the fourth 568
leaf from the bottom was used for measurements made at seedling stage whereas the 12th leaf 569
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
from the bottom was harvested for measurements made at flowering stage For A thaliana 570
whole root and shoot of four-week-old plants were sampled and used for different assays 571
572
Vacuolar proton-pumps inhibitor treatments 573
Inhibition of vacuolar proton pumps of B napus (Xiangyou15) was conducted at the seedling 574
stage in hydroponic culture as described in Han et al (2015a) with minor modifications 575
Plants grown in hydroponic solution were used as control Inhibitor treatment was conducted 576
using several chemicals Bafi(25 nmol L-1 Bafilomycin A1) an inhibitor of V-ATPase 577
DCCD(10 μmol L-1DCCD + 50 mmolL-1 Na2SO3) specifically inhibits V-PPase and 578
Bafi+DCCD in a 11 ratio as inhibitor of both V-ATPase and V-PPase The inhibitors were 579
applied in the hydroponic solutions for 24 h Fresh plant tissue samples from all treatments 580
were collected for different assays 581
582
NO3- induced gene expression 583
Four-week old A thaliana mutants (nrt15-3nrt18-2) were grown in hydroponics with 584
225mM (NH4)2succinate for 3 days and shifted to hydroponics with 45mM NO3- for 12 h 585
then root tissues were collected to assess relative expression of AtNRT15 and AtNRT18 586
genes Seedlings of B napus (Xiangyou15) plants grown hydroponically were treated with 587
5mM (NH4)2succinate for 3 d and shifted to fresh hydroponic solution containing 15 mM 588
NO3- for 12 h Root tissue was then collected to assess the relative expression of BnNRT15 589
and BnNRT18 genes using quantitative RT-PCR 590
591
ACC treatment 592
Hydroponically grown seedlings of B napus were subjected to ACC treatment as described in 593
Zhang et al (2014) with minor modifications Seedlings of the two genotypes Xiangyou15 594
and 814 were transferred to a nutrient solution containing 002mM L-1 ACC for 6 h or grown 595
in normal hydroponic solution as control Root tissues were then collected to assay relative 596
expression of BnNRT15 and BnNRT18 using quantitative RT-PCR Another set of B napus 597
plants were grown in ACC-containing hydroponic solution but supplemented with 15NO3- (22 598
15N) replacing the unlabeled NO3- for 1 h Root and shoot tissues were collected separately 599
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21
for 15N measurement using a continuous-flow isotope ratio mass spectrometer coupled with a 600
C-N elemental analyzer (ANCA-MS PDZ Europa) 601
602
Biomass and N concentration 603
Grain yield dry biomass and N concentration were determined in plant samples at 604
harvest Plants were harvested (including senescing leaves) then dried in an oven for 30 min 605
at 105degC then at 70degC to a constant weight and weighed N concentration was determined 606
using the Kjeldahl method (Shi et al 2010 Han et al 2015b)02g of dried samples were 607
digested with H2SO4-H2O2 in a 100-ml Kjeldahl digestion flask after which N concentration 608
was determined using a Foss Auto Analyser Unit (Kjeldahl 8400) 609
610
V-ATPase and V-PPase activities 611
Roots (10 g) of B napus were collected at seedling and flowering stages and that of A 612
thaliana (05 g) sampled at seedling stage then used for determining the activities of 613
V-ATPase and V-PPase (Han et al 2015b) Activities of these two energy pumps within 614
microsomal membranes were followed colorimetrically based on released Pi as described by 615
Zhu et al (2001) and Krebs et al (2010) 616
617
Assay of NO3- flux in vacuoles 618
Roots of both species were collected similarly as that used for V-ATPase and V-PPase 619
activities then used for vacuole isolation and measurement of NO3- flux in vacuoles as 620
described by Robert et al (2007) with minor modification (Han et al 2015b) Net fluxes of 621
NO3- in vacuoles were measured non-invasively using SIET (scanning ion-selective electrode 622
technique SIET system BIO-003A Younger USA Science and Technology Corporation 623
USA) An NO3--selective microelectrode used for the assay of NO3
- flux was vibrated in the 624
measuring solution between two positions 1 μm and 11 μm from the vacuole surface 625
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles Background 626
signal was recorded by vibrating the electrode in the measuring solution in the absence of 627
vacuoles Ion flux was calculated by Fickrsquos law of diffusion J = -D(dcdx) where J represents 628
the ion flux (picomolescm-2s-1) dcdx is the ion concentration gradient and D is the ion 629
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
diffusion constant in a particular medium The direction of the flux was derived from Fickrsquos 630
law of diffusion that relates the concentration gradient 631
632
Isolation of intact protoplasts and vacuoles for determining NO3- concentration 633
Similar to the above assays root tissues (10 g) were collected from B napus at seedling 634
and flowering stages and from A thaliana (05 g) seedlings and used to isolate intact 635
protoplasts and vacuoles as described by Robert et al (2007) with minor modifications as 636
outlined in Huang et al (2012) and Han et al(2015b) The purified protoplasts were divided 637
into two equal aliquots with one of them used for isolation of vacuoles The purified 638
protoplasts and vacuoles were then sub-sampled and used to determine NO3- concentrations 639
(Vogeli-Lange and Wagner 1990) and for enzyme activity assays (Ma et al 2005) 640
NO3-concentrations in protoplasts and vacuoles were measured by a continuous-flow 641
auto-analyzer (Auto Analyser 3 Bran and Luebbe Germany) as described previously (Han et 642
al 2015b)The activities of acid phosphatase (ACP) and cytochrome oxidase (COX) were 643
determined using plant ACP colorimetry- and COX-assay kits (GenMedSci Inc) following 644
the manufacturerrsquos instructions ACP activity specific to vacuoles was determined and used to 645
normalize NO3- accumulation We measured NO3
- in the protoplast outside the vacuole which 646
includes the cytosol and organelles eg mitochondria and Golgi Apparatus (Robert et 647
al2007) Since most of the NO3- in the protoplast outside the vacuole is located in the cytosol 648
(Krebs et al 2010) we refer to NO3- distribution between vacuoles and cytosol rather than 649
vacuole versus protoplast 650
651
Quantitative RT-PCR 652
Total RNA extracted from B napus roots was prepared using Trizol reagent (Invitrogen) 653
cDNAs were synthesized using M-MLV reverse transcriptase (Promega) following 654
manufacturerrsquos protocol The relative expression of BnNRT15 (EV220114) BnNRT18 655
(EV116423) and Bnactin (AF1118121) genes in plant roots were determined by quantitative 656
RT-PCR and run on a LightCycler instrument (Roche) with the SYBR Green Real-Time PCR 657
Master Mix Kit (TOYOBO Japan) under the following conditions 95degC for 2min then 45 658
cycles of 95degC for 10s 60degC for 10s and 72degC for 20s The primer sequences of BnNRT15 659
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
BnNRT18 and Bnactin genes were obtained from httpbrassicanbiacuk as described in 660
Harper et al (2012) and expression levels were normalized to Bnactin as control 661
Total RNA extracted from the 4-week-old A thaliana roots under the treatments 662
conditions described above were prepared using Trizol reagent (Invitrogen) The cDNAs were 663
synthesized using M-MLV reverse transcriptase (Promega) based on the manufacturerrsquos 664
protocol and as described in Li et al (2010)The primer sequences of quantitative RT-PCR 665
used in these assays are listed in Supplemental Table 2 and were described previously (Chen 666
et al 2012 Zhang et al 2014) and their expression was normalized to Atactin2 as control 667
668
Determination of nitrate distribution using 15NO3- 669
Hydroponically grown B napus (seedling and flowering stages) and A thaliana 670
(seedling) were transferred to 01mM CaSO4 solution for 1min then to their respective 671
hydroponic nutrient solutions with15NO3- (22 excess) replacing unlabeled NO3
- for 1h 672
Plants were then transferred to 01mM CaSO4 solution for 1min after which roots were 673
washed with deionized water Root and shoot samples were separated and dried at 105degC for 674
30min followed by 70degC for 3 d then grounded and used for assaying 15N content using a 675
continuous-flow isotope ratio mass spectrometer coupled with a carbon-nitrogen elemental 676
analyzer (ANCA-MS PDZ Europa) 677
678
Xylem sap collection and assay of nitrate concentration 679
We used the method of Tang et al (2012) for collecting xylem sap Plants were cut 680
leaving1cm segments with intact roots at seedling and flowering stages for B napus and at 681
seedling stage for A thaliana Roots were immediately immersed in their respective nutrition 682
solutions Weighed cotton was put on the cut surface to absorb extruding xylem sap for 1 h 683
The cotton was then wrapped in plastic film and the volume of xylem sap was calculated as 684
the weight gain of the cotton Xylem sap was then squeezed from the cotton using a syringe 685
and used for subsequent assay of nitrate concentration using a continuous-flow auto-analyzer 686
(Auto Analyser 3 Bran and Luebbe Germany) 687
To determine nitrate concentration samples (10 g fresh root and shoot at seedling and 688
flowering of B napus and 05 g fresh root and shoot of 4-week-old A thaliana) were frozen 689
in liquid N2 and ground with a mortar and pestle the powder was then transferred to a beaker 690
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
containing 70 ml deionized water and boiled for 30 min to extract nitrate then cooled and 691
made to 100 ml volume and 02 g activated carbon added to eliminate the effect of 692
chlorophyll The mixture was filtered and nitrate in the filtrate was determined using a 693
continuous-flow auto-analyser (Atuo-Analyser 3 Bran and Luebbe Germany) 694
695
Measurements of photosynthesis 696
All measurements of photosynthesis and related parameters were conducted at 1000 h 697
using intact leaves The 4th leaf from the bottom in B napus at seedling stage and the 12th leaf 698
from the bottom at flowering stage were used for determining chlorophyll concentration 699
photosynthetic rate intercellular CO2 concentration transpiration rate and stomatal 700
conductance Chlorophyll concentration was determined by SPAD-502 (Minolta Camera Co 701
Ltd Japan) (Luo et al 2006 Yandeau-Nelson et al 2011) Photosynthetic rate intercellular 702
CO2 concentration transpiration rate and stomatal conductance were measured using LI-6400 703
Portable Photosynthesis System (Li-Cor Biosciences Co Ltd USA) set at flow rate of 500 704
μmol s-1 photosynthetic photon flux density of 1000 μmol m-2 s-1 relative humidity of 65 705
CO2 concentration of 400ppm and ambient temperatures 706
707
NR and GS activities 708
Leaves with similar age as those used for photosynthesis in B napus were used for the 709
assay of nitrate reductase (NR) and glutamine synthetase (GS) activities NR activity was 710
measured by the modified method of Fan et al (2007) Fresh samples (05g) were frozen in 711
liquid N2 ground into powder in the presence of acid-washed sand and homogenized with 4 712
ml of extraction buffer (0025mol L-1phosphate buffer pH87 1211g L-1 cysteine 0372g L-1 713
EDTA) Homogenates were centrifuged at 30000timesg for 15 min at 4degC and the supernatants 714
were treated with sulfanilamide and α-naphthylamine reagents for colorimetric (540nm) 715
determination of nitrite as described by Fan et al (2007) 716
GS activity was determined using a modified reverseγ-glutamyltransferase method 717
(Wang et al2014) which measures the GS-catalyzed formation of glutamyl-γ-hydroxamate 718
from glutamine and hydroxylamine Fresh samples were frozen for 30 min at -20degC then 719
ground in 10ml Tris-HCl buffer and acid-washed sand The homogenates were filtered 720
through two layers of gauze and centrifuged at 8000timesg for 15 min at 4degC About 12ml of the 721
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
supernatant was added in a reaction mixture and treated as described (Wang et al2014) After 722
the GS-catalyzed reaction was stopped and the solution centrifuged glutamyl-γ723
-hydroxamate was quantified colorimetrically in the supernatant (at 485 nm) and the 724
concentration was determined using a standard curve 725
726
Statistical analyses 727
We used the SPSS software (Statistical Product and Service Solutions V130 USA) for 728
ANOVA and mean separation of main-effects and interactions using Duncanrsquos multiple range 729
test at Plt005Values are presented as means and SD of three or six replicates from three 730
independent experiments Different letters or an asterisk () associated with specific data (eg 731
at the top of histogram bars in figures or within tables) denote significant differences at 732
Plt005 733
734
Supplemental Data 735
Supplemental Figure 1 B napus with higher NUE showed lower vacuolar sequestration 736
capacity (VSC) for NO3-in roots at flowering stage 737
Supplemental Figure 2 B napus with higher NUE showed enhanced long-distance transport 738
of NO3- from roots to shoots at flowering stage 739
Supplemental Figure 3 The two B napus (H and L genotypes) showed the same total N per 740
plant at seedling stage (A) and flowering stage (B) 741
Supplemental Figure 4 B napus with higher NUE showed increased NO3- concentration in 742
the xylem sap at seedling and flowering stages 743
Supplemental Figure 5 NO3- concentration in the xylem sap as affected by inhibitor 744
treatments in B napus and in the energy pumpsrsquo mutants of A thaliana (col-0 vha-a2 vha-a3 745
avp1) 746
Supplemental Figure 6 Reduced VSC for NO3- in roots drives long-distance transport of 747
NO3- from roots to shoots in the A thaliana wild type (Ws) and mutant (clca-2) 748
Supplemental Figure 7 Differences of NR and GS activities between the two B napus (H 749
and L genotypes) at seedling and flowering stages 750
Supplemental Figure 8 Amino acid sequences of BnNRT15 and BnNRT18 751
Supplemental Figure 9 Functions of AtNRT15 and AtNRT18 genes in root tissues of A 752
thaliana in controlling NO3- long-distance transport from root to shoots 753
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
Supplemental Figure 10 B napus BnNRT15 and BnNRT18 genes in roots were 754
coordinately modulated to facilitate NO3- long distance transport from roots to shoots 755
Supplemental Table 1 Differences in stomatal conductance and transpiration rate between 756
the two B napus genotypes 757
Supplemental Table 2 Sequences of primera used for qRT-PCR 758
759
Acknowledgements 760
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 761
Shanghai Institutes for Biological Sciences) for providing nrt15-3 and nrt18-2 seeds 762
This study was supported by the National Natural Science Foundation of China (Grant 763
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 764
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 765
The Ministry of Education in China Open Novel Science Foundation of Hunan province 766
(13K062) the National Key Laboratory of Plant Molecular Genetics and the Twelfth 767
Five-Year National Science and technology support program (2012BAD15BO4) 768
769
Figure 1 B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 770
NO3- in roots at the seedling stage 771
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 772
genotype 814 Specific activities of the tonoplast proton pumps are expressed as μmol Pi 773
released mg-1 protein h-1 NO3- fluxes are expressed as pmol NO3
- cm-2 S-1 Mature vacuoles 774
were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 775
measuring solution between the two positions 1 μm and 11 μm from the vacuole surface 776
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles recording the 777
stable reading data The background was recorded by vibrating the electrode in measuring 778
solution without vacuoles 779
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 780
for NO3- content and NO3
- accumulation normalized against the specific activity of the 781
vacuole acid phosphatase (ACP) as described in Materials and Methods and plotted as μmol 782
NO3- per μmol p-nitrophenol the end product of ACP assay 783
Proton pump activities in root tissues between H and L genotypes are shown at the seedling 784
stage (A) Different letters at the top of the histogram bars denote significant differences of 785
V-ATPase in root tissues between H and L genotypes (Plt005) an asterisk () at the top of the 786
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
histogram bars indicates significant difference in V-PPase activity in root tissues between H 787
and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788
Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789
genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790
bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791
bars on the figures indicate SD (n=6) 792
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793
seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794
to the total NO3- in protoplasts 795
NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796
total NO3-in the cytosol and was calculated as total NO3
- in protoplasts ndash total NO3- in 797
vacuole Different letters at the top of histogram bars denote significant differences of total 798
NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799
(n=6) 800
801
Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802
roots to shoots at the seedling stage 803
H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804
Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805
the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806
as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807
the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808
the histogram bars denote significant differences at Plt005 809
Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810
in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811
genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812
of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813
SD (n=3) different letters at the top of the histogram bars denote significant differences at 814
Plt005 level 815
The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816
at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817
genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818
different letters at the top of the histogram bars denote significant differences at Plt005 819
820
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
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30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
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31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
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Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
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b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
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Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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8
Results 203
B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for NO3- 204
in root tissues 205
Our previous work identified high and low NUE B napus genotypes (Han et al 2015a 206
Han et al 2015b) NUE of B napus whether based on biomass or on grain yield (Table 1) 207
was significantly higher for the high-NUE genotype (H Xiangyou15) than for the low-NUE 208
genotype (L 814) That the activities of the tonoplast proton-pumps (V-ATPase and V-PPase) 209
of root tissues in the H genotype were lower than the L genotype at both seedling (Fig 1A) 210
and flowering stages (Fig S1A) Given that proton pumps in the tonoplast supply energy for 211
vacuolar NO3- accumulation (Krebs et al 2010) NO3
- influx into the vacuole was 212
consequently much slower in the H genotype compared with the L genotype at both seedling 213
(Fig 1B) and flowering stages (Fig S1B) Moreover the percentage of vacuolar NO3- relative 214
to the total NO3- in protoplasts of root tissues in the H genotype was lower than in the L 215
genotype at seedling (Fig 1C) and flowering stages (Fig S1C) and NO3- accumulation in the 216
cytosol increased significantly in the H genotype compared with the L genotype at seedling 217
(Fig 1D) and flowering stages (Fig S1D) 218
219
B napus with higher NUE showed enhanced long-distance transport of NO3- from roots 220
to shoots 221
The relative expression of BnNRT15 in roots of the H genotype was significantly higher 222
than that in the L genotype at both seedling and flowering stages while the relative expression 223
of BnNRT18 in roots of the H genotype was lower than that in the L genotype at both 224
seedling (Fig 2-AB) and flowering stages (Fig S2-AB) As a consequence total N 225
concentration (traced by 15N) in roots of the H genotype was significantly lower than that of 226
the L genotype at both seedling (Fig 2C) and flowering stages (Fig S2C) while shoot N 227
concentration in the H genotype was significantly higher than the L genotype at seedling stage 228
(Fig 2C) This resulted in significantly higher [15N] SR ratio in the H genotype compared 229
with the L genotype (Fig 2D Fig S2D) 230
No significant differences in total N per plant between the H and L genotypes were 231
observed at both seedling and flowering stages (Fig S3) However NO3- concentration in 232
roots of the H genotype was significantly lower than in the L genotype at both seedling and 233
flowering stages (Fig 2E Fig S2E) while NO3- concentration in shoot tissues of this 234
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9
genotype was significantly higher at seedling stage (Fig 2E) resulting in higher [NO3-] SR 235
ratios at both stages (Fig 2F Fig S2F) 236
NO3- concentration in the xylem sap xylem sap volume and total NO3
- in xylem sap 237
were significantly higher in the H genotype than in the L genotype at seedling stage (Fig 238
S4ACE) the amount of NO3- in xylem sap significantly increased in the H genotype relative 239
to the L genotype at flowering stage (Fig S4F) Together these data suggests greater 240
mobilization of NO3- from root to shoot in the H genotype 241
242
NO3- up-regulates NRT15 but down-regulates NRT18 243
244
A previous study showed that increased NRT18 expression in nrt15 mutants (Chen et al 245
2012) and the expression of NRT15 was induced by NO3- (Lin et al 2008) We tested the 246
relationship between NRT15 and NRT18 expression in A thaliana and B napus No 247
significant differences were observed in AtNRT15 expression in roots between wild type 248
(col-0) and mutant plants (nrt18-2) (Fig 3A) However AtNRT15 expression was 249
significantly up-regulated by NO3- in roots of nrt18-2 mutants (Fig 3B) Expression of 250
AtNRT18 significantly increased in roots of nrt15-3 mutants but not in col-0 plants and 251
there were no significant differences in AtNRT18 expression with or without NO3- treatment 252
in roots of nrt15-3 mutants (Fig 3CD) 253
In contrast BnNRT15 expression in roots of the H genotype increased significantly with 254
NO3- treatment (Fig 3E) while the reverse was observed in expression of BnNRT18 which 255
showed lower expression under NO3- treatment than control (Fig 3F) This suggests that NO3
- 256
induces the expression of NRT15 but down-regulates NRT18 in A thaliana and B napus 257
Further studies are needed to elucidate the mechanisms regulating this reverse regulation 258
259
Reduced VSC of NO3- in roots drives its long-distance transport from roots to shoots 260
Our previous study showed that Bafi (Bafilomycin A1) inhibits V-ATPase DCCD 261
(DCCD + Na2SO3) inhibits V-PPase and a 11 combination of Bafi and DCCD (B+D) 262
inhibits both V-ATPase and V-PPase (Han et al 2015a) These inhibitors were used to 263
control activities of the tonoplast proton pumps in the H B napus plants V-ATPase activity 264
significantly decreased under Bafil and B+D treatments relative to the control and DCCD 265
treatments whereas V-PPase activity declined significantly under DCCD and B+D treatments 266
relative to that in the control and Bafil treatments (Fig 4A) 267
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10
The V-ATPase activity in roots of A thaliana was significantly lower in the V-ATPase 268
mutants (vha-a2 and vha-a3) than the wild type (col-0) and V-PPase mutants (avp1) (Fig 5A) 269
In contrast the V-PPase activity in roots of V-PPase mutant (avp1) were significantly lower 270
than in the wild type (col-0) and V-ATPase mutants (vha-a2 and vha-a3) (Fig 5A) 271
NO3- influxes into vacuoles of the H B napus plants significantly decreased when treated 272
with inhibitors of proton pumps (Bafil DCCD B+D) (Fig 4B) Similarly NO3- influxes into 273
the vacuole of the Arabidopsis mutants (vha-a2 vha-a3 and avp1) was significantly lower 274
than those found in the wild type (col-0) (Fig 5B) Previous studies showed that VSC of NO3- 275
decreased when the activities of the proton pumps decline (Li et al 2005 Krebs et al 2010 276
Han et al 2015a) 277
We further investigated NO3- distribution between the vacuole and cytosol as affected by 278
proton pump inhibition in the H genotype of B napus and the mutants of A thaliana 279
defective in vacuolar proton pumps The percentage of vacuolar NO3- relative to total NO3
- in 280
root protoplasts of the H genotype treated with inhibitors (Bafil DCCD B+D) was lower than 281
that observed in the control (Fig 4C) Results were also similar when using A thaliana 282
mutants where the percentage of vacuolar NO3- relative to the total NO3
- in protoplasts was 283
lower in the mutants (vha-a2 vha-a3 and avp1) than in the wild type (col-0) (Fig 5C) 284
Consequently NO3- accumulation in the cytosol showed a significant increase when energy 285
pumps were suppressed in roots of the H genotype (Fig 4D) and NO3- accumulation in the 286
cytosol of root tissues of A thaliana mutants (vha-a2 vha-a3 and avp1) similarly increased 287
compared with col-0 (Fig 5D) 288
Based on previous observations (Lin et al 2008) expression of NRT15 is strongly 289
induced by NO3- but expression of NRT18 is down-regulated (Fig3 Chen et al 2012) We 290
then hypothesized that expression of these two genes is contrastingly regulated by 291
concentration of NO3- in the cytosol Our results were congruent with this hypothesis the 292
expression of BnNRT15 in root tissues of the H genotype of B napus was significantly higher 293
in plants treated with Bafil DCCD or B+D compared with the control (Fig 4E) whereas the 294
expression of BnNRT18 decreased substantially (Fig 4F) Similar results were also observed 295
in A thaliana where expression of AtNRT15 in roots of the mutants (vha-a2 vha-a3 and 296
avp1) were significantly higher than in the wild type (col-0) but expression of AtNRT18 in 297
the same mutants were considerably lower (Fig 5EF) 298
NO3- concentrations in the xylem sap the N-distribution between shoot and root (SR 299
ratios based on [15N]) and the [NO3-] in shoots relative to roots of the H genotype treated with 300
energy pumpsrsquo inhibitors were significantly higher than in the control (Fig 4GH Fig S5A) 301
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11
Similar trends were also observed when using mutants of A thaliana deficient in the energy 302
pumpsrsquo activities NO3- concentration in the xylem sap the [15N] SR ratios and [NO3
-] SR 303
ratios were all significantly higher in the mutants than in the wild type (Fig 5GH Fig S5B) 304
These data clearly showed that prevention of N sequestration in vacuoles would enhance its 305
translocation to shoot 306
Additional evidence linking NO3- long-distance transport and NO3
- short-distance 307
distribution within cells were provided through experiments comparing A thaliana wild type 308
(Ws) and mutant clca-2 (Fig S6) The chloride channel (CLCa) is the main channel for 309
vacuolar anion accumulation Vacuolar sequestration capacity of NO3-significantly declines in 310
clca mutants (Angeli et al 2006) NO3- influx into the vacuolar space of Ws roots was 311
significantly higher than that observed in the clca-2 mutants (Fig S6A) This resulted in a 312
smaller proportion of vacuolar NO3- in clca-2 plants relative to the total NO3
- in root tissue 313
protoplasts as compared with Ws (Fig S6B) Consequently the accumulation of NO3- in the 314
cytosol was significantly higher in clca-2 than in Ws plants (Fig S6C) The higher 315
NO3-concentration in the cytosol together with the higher expression of AtNRT15 coupled 316
with lower expression of AtNRT18 in clca-2 roots (Fig S6DE) resulted in significant 317
increase in xylem sap NO3- concentration [15N] SR ratios and [NO3
-] SR ratios compared 318
with Ws plants (Fig S6FGH) 319
320
Increased NO3- translocation to shoots enhanced NUE 321
NO3- assimilation efficiency is known to be higher in shoots than in roots (Smirnoff et al 322
1985 Andrews et al 1986 Tang et al 2012 Tang et al 2013) We therefore hypothesize 323
that increased NO3- translocation to shoot and the consequent higher shootroot ratio will 324
contribute to higher NUE Our data support this hypothesis The H B napus genotype showed 325
enhanced long-distance transport of NO3- from roots to shoots (Fig 2 Fig S2) This enhanced 326
NO3- transport requires higher carbon skeleton provided through higher photosynthetic rate 327
(Tang et al 2012 Tang et al 2013) Our results showed that chlorophyll content 328
intercellular CO2 concentration and photosynthetic rate were significantly higher in the H than 329
the L genotype at both seedling and flowering stages (Table 2) Moreover the NO3- 330
assimilating enzymes were strongly induced by NO3- providing sufficient capacity for N 331
assimilation (Smirnoff et al 1985 Andrews et al 1986) Nitrate reductase (NR) and 332
glutamine synthetase (GS) activities in roots of the H genotype were significantly lower than 333
in the L genotype both at seedling and flowering stages (Fig S7) In contrast NR activities in 334
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12
shoots of the H genotype were significantly higher than in the L genotype at both stages (Fig 335
S7AB) and GS activity in the H genotype was also higher than those of the L genotype at 336
seedling stage (Fig S7C) 337
Experiments comparing A thaliana wild type (col-0 Ws) with mutants defective in 338
vacuolar proton-pumps (vha-a2 vha-a3avp1) and transport channel (clca-2) yielded similar 339
results that is increases in NO3- shootroot ratio essentially contributed to enhanced NUE 340
(Fig 6) The NO3- shootroot ratios in A thaliana mutants were higher than that in the wild 341
type (Fig 5GH) Generally the efficiency of inorganic N (NO3-) assimilation into organic N 342
is higher in shoots than in roots (Smirnoff et al 1985 Andrews et al 1986) therefore the 343
NO3- concentration in A thaliana mutants decreased (Fig 6CD) leading to higher NUE 344
compared with the wild type (Fig 6AB) 345
346
BnNRT15BnNRT18 in B napus affects NO3- long-distance transport from roots to 347
shoots 348
Both B napus and A thaliana are members of Cruciferae Family The amino acid 349
sequence identity between BnNRT15 and AtNRT15 was 90 (Fig S8A) the amino acid 350
sequence similarity between BnNRT18 and AtNRT18 was 908 (Fig S8B) Comparisons of 351
nucleotide and amino acid sequences (Harper et al2012) of these genes 352
(httpbrassicanbiacukcgi-binmicroarray_databasecgi) showed that BnNRT15 353
(EV220114) and BnNRT18 (EV116423) of B napus are respectively highly homologous 354
with AtNRT15 and AtNRT18 and the two genes are mainly expressed in roots of both species 355
showing similar organ-specificity (Lin et al 2008 Li et al 2010) 356
NO3- long-distance transport from roots to shoots is therefore regulated by NRT15 and 357
NRT18 as reported before (Lin et al 2008 Li et al 2010) The [15N-traced] SR ratios and 358
the [NO3-] SR ratios were significantly lower in nrt15-3 mutants relative to the wild type 359
(col-0) while [15N] SR and [NO3-] SR ratios showed significant increase in nrt18-2mutant 360
relative to col-0 (Fig S9CD) A previous study showed that expression of NRT15 is 361
down-regulated by ACC and MeJA treatments while the expression of NRT18 is strongly 362
up-regulated in A thaliana (Zhang et al 2014) Consequently NO3- accumulated in plant 363
roots probably as an adaptive measure for abiotic stresses (Chen et al 2012) Both the H- 364
and L- B napus genotypes showed significantly lower expression of BnNRT15 in roots when 365
treated with ACC (Fig S10A) but the expression of BnNRT18 remained higher than in the 366
control plants (Fig S10B) This resulted in significantly lower [15N] SR ratios following 367
ACC treatment (Fig S10CDE) These data indicate that BnNRT15 and BnNRT18 play 368
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13
similar functions for NO3- long-distance transport between root and shoot in both B napus 369
and A thaliana (Lin et al 2008 Li et al 2010) 370
371
372
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14
Discussion 373
Nitrate long-distance transport from root to shoot is regulated by cytosolic NO3- in roots 374
NO3- long-distance transport from root to shoot is controlled by NRT gene family For 375
instance xylem NO3- loading and unloading are mainly controlled by NRT15 and NRT18 376
respectively (Lin et al 2008 Li et al 2010) Whereas the NO3- short-distance transport 377
between the vacuole and cytosol is controlled by tonoplast proton-pumps (V-ATPase and 378
V-PPase) (Krebs et al 2010) and by activity of the CLCa channel in the tonoplast membrane 379
(Angeli et al 2006 Wege et al 2014) tonoplast proton-pumps provide the energy required 380
for NO3- accumulation into the vacuole through CLCa (Gaxiola et al 2001 Brux et al 2008 381
Krebs et al 2010) The expression of NRT15 is up-regulated by NO3- in plant culture 382
solution (Lin et al 2008) whereas regulation of NRT18 expression is contrary to that of 383
NRT15 under both control and abiotic stress conditions (Li et al 2010 Chen et al 2012 384
Zhang et al 2014) 385
Despite the progress made in characterizing the genes involved in transport and 386
metabolism of NO3- in plants the relationship between long- and short-distance transport and 387
how these two pathways are regulated have not been thoroughly investigated We hypothesize 388
that NRT15 expression and its role in long-distance transport is regulated by NO3- in plant 389
tissue as was observed in culture solution (Lin et al 2008) and our data support this 390
hypothesis Both BnNRT15 and AtNRT15 expression were up-regulated by increasing 391
cytosolic NO3- in roots (Fig 4 Fig 5) The increase in cytosolic NO3
- was achieved through (i) 392
use of A thaliana mutants defective in proton-pumps (V-ATPase and V-PPase) or CLCa 393
activities in the tonoplast and (ii) inhibition of B napus tonoplast proton-pumps In both 394
model plants the influx of NO3- into vacuoles was substantially reduced resulting in higher 395
concentrations of NO3- in the cytosol (Fig 4A-D Fig 5A-D Fig S6A-C) Under those 396
conditions expression of BnNRT15 and AtNRT15 was up-regulated by the enhanced 397
cytosolic NO3- while expression of BnNRT18 and AtNRT18 was down-regulated (Fig 4EF 398
Fig 5EF FigS6DE) As a consequence more NO3- was loaded into the xylem sap and 399
transported from root to shoot (Fig S5AB FigS6F) This is also reflected as increased 400
shootroot ratios of N traced by [15N] and [NO3-] (Fig 4GH Fig 5GH Fig S6GH) 401
Interestingly the expression of both BnNRT15 and AtNRT15 is up-regulated by 402
increased cytosolic NO3- concentration in roots but were not affected by NO3
- sequestered 403
into vacuoles (Fig 4 Fig 5 Fig S6) This is because vacuolar NO3- is functionally separated 404
by the tonoplast and cannot be assimilated by enzymes that are localized in the cytosol 405
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15
Therefore NO3--inducible genes such as NR and NRT15 were not influenced by vacuolar 406
NO3- concentration (Martinoia et al 1981 Martinoia et al 2000 Zhang et al 2012) In 407
addition NO3- concentration in the cytosol is controlled by its short-distance transport through 408
the tonoplast which is mediated through the tonoplast proton-pumps providing the required 409
energy for its sequestration into vacuoles against concentration gradient (Gaxiola et al 2001 410
Brux et al 2008 Krebs et al 2010) The gradient of H+ between the inside of the vacuole 411
and the cytosol is maintained by an 2NO3-1H+ antiporter mechanism that facilitatesNO3
- 412
transport through CLCa (Angeli et al 2006 Wege et al 2014) Our data provide clear 413
evidence that NO3- long-distance transport from root to shoot is regulated by cytosolic NO3
- 414
concentration in roots 415
Regulatory mechanisms that control the expression of NRT18 and NRT15 as mediated 416
by NO3- are still unknown however previous studies showed that their expression is also 417
affected by abiotic stresses whereby NRT15 is down-regulated and NRT18 is up-regulated 418
(Li et al 2010 Zhang et al 2014) AtNRT18 expression in A thaliana nrt15 mutants is 419
highly up-regulated (Chen et al 2012) as was also observed here (Fig 3C) Moreover 420
AtNRT15 expression was enhanced by NO3- in culture solution and was not affected in 421
nrt18-2 mutant (Fig 3AB) On the other hand AtNRT18 expression was not affected by 422
cytosolic NO3- but down-regulated when AtNRT15 expression was up-regulated by NO3
-(Fig 423
S6DE) Analogous results were also obtained with B napus genotypes (Fig 3EF) Based on 424
these results we argue that NRT18 expression is not directly influenced by cytosolic NO3- but 425
rather by NRT15 expression (Fig 4 Fig 5 and Fig S6) suggesting NRT18 is probably 426
acting downstream of NRT15 Apparently the regulatory mechanisms of this NRT gene 427
family still awaits further studies to be elucidated 428
429
Response of NUE to NO3- long-distance transport 430
NO3- assimilation efficiency is higher in shoot than in root tissues and can be further 431
enhanced by carbon assimilation (Smirnoff et al 1985 Tang et al 2013) Therefore 432
translocation of higher proportion of NO3- from roots to shoots likely contributes to better 433
crop growth and higher NUE (Andrews et al 1986 Tang et al 2012) The present study 434
provides direct evidence for these results using contrasting B napus genotype identified 435
before (Zhang et al 2009 Han et al 2015a) Our data confirmed the genetic variation in 436
NUE between these two genotypes (Table 1) 437
We measured NO3- distribution in root and shoot of these genotypes to elucidate the 438
physiological mechanisms contributing to variation in NUE The data were consistent with 439
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16
our hypothesis that concentration of NO3- in the xylem sap and shoot would be significantly 440
higher in the H genotype than in the L genotype (Fig 2EFFig S2EF Fig S4) The NO3- 441
long-distance transport from root to shoot was affected by cytosolic NO3- in roots of both A 442
thaliana and B napus genotypes (Fig 4-5 Fig S6) The activities of the tonoplast 443
proton-pumps in roots were significantly lower in the H genotype than in the L genotype (Fig 444
1AB) Consequently less NO3- was sequestered into the vacuole and more NO3
- was retained 445
in cytosol (Fig 1CD) BnNRT15 expression was then up-regulated resulting in greater 446
loading into the xylem while BnNRT18 was down-regulated (Fig 2AB) probably to 447
suppress NO3- unloading from xylem sap Consequently a higher proportion of NO3
- was 448
transported from root to shoots in the H genotype(Fig 2C-D Fig S4) 449
Moreover photosynthetic carbon fixation (Table 2) and activities of both NR and GS 450
were higher in shoots of the H genotype (Fig S7) both of which promote higher N 451
assimilation These results agreed with previous reports (Smirnoff et al 1985 Andrews et al 452
1986 Tang et al 2012 Tang et al 2013) where higher NO3- transport from root to shoot 453
higher photosynthetic rate and N assimilation efficiency in shoots were suggested as essential 454
mechanisms for high-NUE in crop plants These qualities endowed the B napus H genotype 455
Xiangyou15 with higher NUE as compared with the L genotype 814 456
NO3- long-distance transport in B napus is not only regulated by BnNRT15 and 457
BnNRT18 in root tissues (Fig 4 Fig 5 Fig S6) but also by stomatal conductance and 458
transpiration by indirectly controlling xylem sap flow and long-distance transport 459
(Dechorgnat et al 2011 Krapp et al 2014) Stomatal conductance and transpiration rates of 460
the H genotype were significantly higher than those of the L genotype (Table S1) a result 461
consistent with other studies describing characteristics of high-NUE plants (Daniel-Vedele et 462
al 1998 Wilkinson et al 2007 Dechorgnat et al 2011 Krapp et al 2014) 463
464
Response of NUE to NO3- short-distance transport 465
NUE is also strongly controlled by NO3- short-distance distribution between the vacuole 466
and cytosol (Han et al 2015a) A lower proportion of NO3- accumulating in vacuoles and a 467
higher proportion retained in the cytosol will contribute to better NUE in crop plants (Han et 468
al 2015b) The energy required for vacuolar NO3- sequestration is provided by the tonoplast 469
proton-pumps (Gaxiola et al 2001 Brux et al 2008 Krebs et al 2010) while the 470
distribution channel CLCa enables transport of NO3- across the vacuolar membrane (Angeli et 471
al 2006 Wege et al 2014) The activities of the tonoplast proton-pumps (V-ATPase and 472
V-PPase) in leaves of the H genotype were significantly lower than those of the L genotype at 473
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17
flowering stages (Han et al 2015aHan et al 2015b) thus we expected similar differences in 474
root tissues Our results confirmed that the activities of tonoplast proton-pumps in root tissues 475
of B napus H genotype and A thaliana mutants were lower than those in the L genotype and 476
A thaliana wild type leading to less NO3- influx into vacuoles and more in the cytosol (Fig 1 477
Fig 5A-D) Moreover the enzymes catalyzing N assimilation are located in the cytoplasm 478
where NO3- could be assimilated (Martinoia et al 1981 Han et al 2015a) or immediately 479
loaded into xylem sap for long-distance transport from roots to shoots (Lin et al 2008) 480
Consequently the NO3- concentration in root tissues of the H genotype and A thaliana 481
mutants defective in tonoplast proton-pumps was significantly lower than that in the L 482
genotype (Fig 2E) and in A thaliana wild type (Fig 6C) Therefore the higher proportion of 483
NO3- transported to shoot and the lower concentrations retained in root vacuoles contributed 484
to the higher NUE in Xiangyou15 and A thaliana mutants (vha-a2 vha-a3 and avp1) (Table 485
1Fig 6A) Further studies are needed to assess the impacts of regulating vacuolar 486
sequestration of NO3- and rates of long-distance transport for enhancing NUE in other crop 487
species and genotypes 488
These data suggested that NO3- distribution between vacuoles and cytosol within cells 489
regulates NO3- long-distance transport from root to shoot and ultimately affects NUE NO3
- 490
loading into the xylem sap is an active transport process (Lin et al 2008) therefore not 491
regulated by absolute concentration of NO3- in the cytosol but rather by genes involved in 492
NO3- long-distance transport (NRT15 and NRT18) Apparently the expression of NRT15 the 493
gene responsible for xylem loading is regulated by NO3-concentration in the cytosol 494
Therefore both NO3- short-distance transport across the tonoplast and its long-distance 495
transport from root to shoot are important determinants of NUE in both B napus and A 496
thaliana (Table 1 and Fig 6) 497
498
Roles of NRT15 and NRT18 in NO3- long-distance transport in B napus and A 499
thaliana 500
Our results (Figs S9 S10) showed that the proportion of NO3- distributed from root to 501
shoot is strongly controlled by NRT15 in accord with previous studies (Lin et al 2008 Li et 502
al 2010) The data suggest that the high-NUE genotypes possess lower activities of tonoplast 503
proton-pumps (V-ATPase and V-PPase) resulting in less NO3- accumulation in vacuoles and 504
more NO3- retention in cytosol (Fig 7) The higher NO3
- in the cytosol then up-regulated 505
NRT15 which then down-regulates NRT18 As a result more NO3- is loaded into the xylem 506
system by NRT15 mechanism and less NO3- is unloaded via NRT18 (Fig 7) Differential 507
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18
regulation of these NRT genes in high-NUE plants increased transport of NO3- from root to 508
shoot through the xylem vascular tissues (Fig 7) This coordinated NO3- long- and 509
short-distance transport likely fine-tunes NO3- allocation and modulates the balance of NO3
- 510
distribution between roots and shoots (Fig 7) 511
Our research also revealed differences between A thaliana and B napus in the 512
expression of the wild-type NRT genes in response to NO3- in the protoplast Moreover using 513
A thaliananrt15-3 mutants we uncovered possible regulatory interactions between a 514
functioning (wild-type) NRT15 gene and NRT18 expression in response to NO3- 515
concentration Because mutants defective in nrt15 are not available for B napus we could 516
not confirm that the respective NRT15 and NRT18 systems behave in an analogous way in 517
these plant species Based on Harper et al (2012) research database our homology 518
comparisons and gene function annotations provided strong evidence for the roles of 519
BnNRT15 and BnNRT18 in B napus Finally our studies were conducted using two model 520
dicotyledonous plant species for which we had ample data on responses to specific inhibitors 521
(B napus) or specific mutants were available with results forming the bases for the model 522
presented in Figure 7 Further research using genetically diverse plant species (eg 523
monocotyledonous crops such as rice wheat and corn) will help validate this model and its 524
broader application across species The information generated in this study could have future 525
application for improving NUE in commercial crops with consequent reduction in nitrogen 526
use and benefits to the environment 527
528
Materials and Methods 529
Plant material 530
The two oilseed rape (Bnapus) cultivars used in this study have been characterized 531
before as high- (Xiangyou15 referred to as H genotype hereafter) and low- (814 referred to 532
as L genotype) nitrogen use efficiency (NUE) genotypes (Zhang et al 2009 Han et al 2015b) 533
Here we define NUE as the total biomass per unit of N uptake by the plant with total 534
biomass includes roots shoots and grains The two genotypes were provided by the Hunan 535
Sub-Center of Improvement Center of National Oil Crops Hunan China 536
The Arabidopsis thaliana wild-type Columbia-0 (col-0) was used as control for 537
V-ATPase (vha-a2 and vha-a3) V-PPase (avp1) nrt15-3 and nrt18-2 mutants whereas the A 538
thaliana wild-type Wassilewskija (Ws) was used as control for clca-2 mutants A thaliana 539
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19
mutants and transgenic lines (nrt15-3 and nrt18-2) have been described previously (Chen et 540
al 2012 Zhang et al 2014) The mutant lines vha-a2 (Salk_142642) and vha-a3 541
(Salk_122135) described by Krebs et al (2010) were obtained from the Arabidopsis 542
Biological Resources Center (ABRC) The mutant line avp1 (GK-005004) described in Li et 543
al (2005) was obtained from the European Arabidopsis Stock Centre while the mutant line 544
clca-2 (FST 171A06) described in Angeli et al (2006) was from the Institute National de la 545
Recherche Agronomique (INRA) collection in France together with the T-DNA mutants used 546
for genotyping to select pure mutant lines 547
548
Growth conditions 549
B napus plants were grown hydroponically in ceramic pots(20cmtimes15cm) filled with a 550
nutrient solution in the greenhouse under natural light as described in Han et al (2015b) The 551
pots were arranged in a completely randomized design with six biological replications The 552
nutrient solution was replaced every 3 days and its pH adjusted to 55 daily Experiments were 553
conducted at the field station of Hunan Agricultural University Southern China 554
A thaliana plants were grown in a nutrient solution in plastic pots as described in Arteca 555
et al (2000) and Gong et al (2003) The solution was changed every 3 days with pH adjusted 556
daily to 58 Pots were arranged in a completely randomized design with six biological 557
replications The nutrient solution used for both species consisted of 125mM KNO3 558
0625mM KH2PO4 05mM MgSO4 05mM Ca (NO3)2middot4H2O 0025mM Fe-EDTA 025 559
mlL-1 micronutrients (stock solution concentrations 70mM B 14mM Mn 1mM Zn 05mM 560
Cu and 02mM Mo) The experiments were conducted at Hunan Agricultural University in a 561
phytotron set at 70 relative humidity16h-light8h-dark cycle and constant temperature of 562
22degC 563
564
Experimental treatments and phenotyping 565
All measurements made on B napus plants were conducted either at the seedling stage 566
(2 months after transplanting) or at flowering (5 months after transplanting) The whole root 567
tissue was harvested and used for analyses at either stage For shoot measurements the fourth 568
leaf from the bottom was used for measurements made at seedling stage whereas the 12th leaf 569
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20
from the bottom was harvested for measurements made at flowering stage For A thaliana 570
whole root and shoot of four-week-old plants were sampled and used for different assays 571
572
Vacuolar proton-pumps inhibitor treatments 573
Inhibition of vacuolar proton pumps of B napus (Xiangyou15) was conducted at the seedling 574
stage in hydroponic culture as described in Han et al (2015a) with minor modifications 575
Plants grown in hydroponic solution were used as control Inhibitor treatment was conducted 576
using several chemicals Bafi(25 nmol L-1 Bafilomycin A1) an inhibitor of V-ATPase 577
DCCD(10 μmol L-1DCCD + 50 mmolL-1 Na2SO3) specifically inhibits V-PPase and 578
Bafi+DCCD in a 11 ratio as inhibitor of both V-ATPase and V-PPase The inhibitors were 579
applied in the hydroponic solutions for 24 h Fresh plant tissue samples from all treatments 580
were collected for different assays 581
582
NO3- induced gene expression 583
Four-week old A thaliana mutants (nrt15-3nrt18-2) were grown in hydroponics with 584
225mM (NH4)2succinate for 3 days and shifted to hydroponics with 45mM NO3- for 12 h 585
then root tissues were collected to assess relative expression of AtNRT15 and AtNRT18 586
genes Seedlings of B napus (Xiangyou15) plants grown hydroponically were treated with 587
5mM (NH4)2succinate for 3 d and shifted to fresh hydroponic solution containing 15 mM 588
NO3- for 12 h Root tissue was then collected to assess the relative expression of BnNRT15 589
and BnNRT18 genes using quantitative RT-PCR 590
591
ACC treatment 592
Hydroponically grown seedlings of B napus were subjected to ACC treatment as described in 593
Zhang et al (2014) with minor modifications Seedlings of the two genotypes Xiangyou15 594
and 814 were transferred to a nutrient solution containing 002mM L-1 ACC for 6 h or grown 595
in normal hydroponic solution as control Root tissues were then collected to assay relative 596
expression of BnNRT15 and BnNRT18 using quantitative RT-PCR Another set of B napus 597
plants were grown in ACC-containing hydroponic solution but supplemented with 15NO3- (22 598
15N) replacing the unlabeled NO3- for 1 h Root and shoot tissues were collected separately 599
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21
for 15N measurement using a continuous-flow isotope ratio mass spectrometer coupled with a 600
C-N elemental analyzer (ANCA-MS PDZ Europa) 601
602
Biomass and N concentration 603
Grain yield dry biomass and N concentration were determined in plant samples at 604
harvest Plants were harvested (including senescing leaves) then dried in an oven for 30 min 605
at 105degC then at 70degC to a constant weight and weighed N concentration was determined 606
using the Kjeldahl method (Shi et al 2010 Han et al 2015b)02g of dried samples were 607
digested with H2SO4-H2O2 in a 100-ml Kjeldahl digestion flask after which N concentration 608
was determined using a Foss Auto Analyser Unit (Kjeldahl 8400) 609
610
V-ATPase and V-PPase activities 611
Roots (10 g) of B napus were collected at seedling and flowering stages and that of A 612
thaliana (05 g) sampled at seedling stage then used for determining the activities of 613
V-ATPase and V-PPase (Han et al 2015b) Activities of these two energy pumps within 614
microsomal membranes were followed colorimetrically based on released Pi as described by 615
Zhu et al (2001) and Krebs et al (2010) 616
617
Assay of NO3- flux in vacuoles 618
Roots of both species were collected similarly as that used for V-ATPase and V-PPase 619
activities then used for vacuole isolation and measurement of NO3- flux in vacuoles as 620
described by Robert et al (2007) with minor modification (Han et al 2015b) Net fluxes of 621
NO3- in vacuoles were measured non-invasively using SIET (scanning ion-selective electrode 622
technique SIET system BIO-003A Younger USA Science and Technology Corporation 623
USA) An NO3--selective microelectrode used for the assay of NO3
- flux was vibrated in the 624
measuring solution between two positions 1 μm and 11 μm from the vacuole surface 625
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles Background 626
signal was recorded by vibrating the electrode in the measuring solution in the absence of 627
vacuoles Ion flux was calculated by Fickrsquos law of diffusion J = -D(dcdx) where J represents 628
the ion flux (picomolescm-2s-1) dcdx is the ion concentration gradient and D is the ion 629
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22
diffusion constant in a particular medium The direction of the flux was derived from Fickrsquos 630
law of diffusion that relates the concentration gradient 631
632
Isolation of intact protoplasts and vacuoles for determining NO3- concentration 633
Similar to the above assays root tissues (10 g) were collected from B napus at seedling 634
and flowering stages and from A thaliana (05 g) seedlings and used to isolate intact 635
protoplasts and vacuoles as described by Robert et al (2007) with minor modifications as 636
outlined in Huang et al (2012) and Han et al(2015b) The purified protoplasts were divided 637
into two equal aliquots with one of them used for isolation of vacuoles The purified 638
protoplasts and vacuoles were then sub-sampled and used to determine NO3- concentrations 639
(Vogeli-Lange and Wagner 1990) and for enzyme activity assays (Ma et al 2005) 640
NO3-concentrations in protoplasts and vacuoles were measured by a continuous-flow 641
auto-analyzer (Auto Analyser 3 Bran and Luebbe Germany) as described previously (Han et 642
al 2015b)The activities of acid phosphatase (ACP) and cytochrome oxidase (COX) were 643
determined using plant ACP colorimetry- and COX-assay kits (GenMedSci Inc) following 644
the manufacturerrsquos instructions ACP activity specific to vacuoles was determined and used to 645
normalize NO3- accumulation We measured NO3
- in the protoplast outside the vacuole which 646
includes the cytosol and organelles eg mitochondria and Golgi Apparatus (Robert et 647
al2007) Since most of the NO3- in the protoplast outside the vacuole is located in the cytosol 648
(Krebs et al 2010) we refer to NO3- distribution between vacuoles and cytosol rather than 649
vacuole versus protoplast 650
651
Quantitative RT-PCR 652
Total RNA extracted from B napus roots was prepared using Trizol reagent (Invitrogen) 653
cDNAs were synthesized using M-MLV reverse transcriptase (Promega) following 654
manufacturerrsquos protocol The relative expression of BnNRT15 (EV220114) BnNRT18 655
(EV116423) and Bnactin (AF1118121) genes in plant roots were determined by quantitative 656
RT-PCR and run on a LightCycler instrument (Roche) with the SYBR Green Real-Time PCR 657
Master Mix Kit (TOYOBO Japan) under the following conditions 95degC for 2min then 45 658
cycles of 95degC for 10s 60degC for 10s and 72degC for 20s The primer sequences of BnNRT15 659
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23
BnNRT18 and Bnactin genes were obtained from httpbrassicanbiacuk as described in 660
Harper et al (2012) and expression levels were normalized to Bnactin as control 661
Total RNA extracted from the 4-week-old A thaliana roots under the treatments 662
conditions described above were prepared using Trizol reagent (Invitrogen) The cDNAs were 663
synthesized using M-MLV reverse transcriptase (Promega) based on the manufacturerrsquos 664
protocol and as described in Li et al (2010)The primer sequences of quantitative RT-PCR 665
used in these assays are listed in Supplemental Table 2 and were described previously (Chen 666
et al 2012 Zhang et al 2014) and their expression was normalized to Atactin2 as control 667
668
Determination of nitrate distribution using 15NO3- 669
Hydroponically grown B napus (seedling and flowering stages) and A thaliana 670
(seedling) were transferred to 01mM CaSO4 solution for 1min then to their respective 671
hydroponic nutrient solutions with15NO3- (22 excess) replacing unlabeled NO3
- for 1h 672
Plants were then transferred to 01mM CaSO4 solution for 1min after which roots were 673
washed with deionized water Root and shoot samples were separated and dried at 105degC for 674
30min followed by 70degC for 3 d then grounded and used for assaying 15N content using a 675
continuous-flow isotope ratio mass spectrometer coupled with a carbon-nitrogen elemental 676
analyzer (ANCA-MS PDZ Europa) 677
678
Xylem sap collection and assay of nitrate concentration 679
We used the method of Tang et al (2012) for collecting xylem sap Plants were cut 680
leaving1cm segments with intact roots at seedling and flowering stages for B napus and at 681
seedling stage for A thaliana Roots were immediately immersed in their respective nutrition 682
solutions Weighed cotton was put on the cut surface to absorb extruding xylem sap for 1 h 683
The cotton was then wrapped in plastic film and the volume of xylem sap was calculated as 684
the weight gain of the cotton Xylem sap was then squeezed from the cotton using a syringe 685
and used for subsequent assay of nitrate concentration using a continuous-flow auto-analyzer 686
(Auto Analyser 3 Bran and Luebbe Germany) 687
To determine nitrate concentration samples (10 g fresh root and shoot at seedling and 688
flowering of B napus and 05 g fresh root and shoot of 4-week-old A thaliana) were frozen 689
in liquid N2 and ground with a mortar and pestle the powder was then transferred to a beaker 690
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24
containing 70 ml deionized water and boiled for 30 min to extract nitrate then cooled and 691
made to 100 ml volume and 02 g activated carbon added to eliminate the effect of 692
chlorophyll The mixture was filtered and nitrate in the filtrate was determined using a 693
continuous-flow auto-analyser (Atuo-Analyser 3 Bran and Luebbe Germany) 694
695
Measurements of photosynthesis 696
All measurements of photosynthesis and related parameters were conducted at 1000 h 697
using intact leaves The 4th leaf from the bottom in B napus at seedling stage and the 12th leaf 698
from the bottom at flowering stage were used for determining chlorophyll concentration 699
photosynthetic rate intercellular CO2 concentration transpiration rate and stomatal 700
conductance Chlorophyll concentration was determined by SPAD-502 (Minolta Camera Co 701
Ltd Japan) (Luo et al 2006 Yandeau-Nelson et al 2011) Photosynthetic rate intercellular 702
CO2 concentration transpiration rate and stomatal conductance were measured using LI-6400 703
Portable Photosynthesis System (Li-Cor Biosciences Co Ltd USA) set at flow rate of 500 704
μmol s-1 photosynthetic photon flux density of 1000 μmol m-2 s-1 relative humidity of 65 705
CO2 concentration of 400ppm and ambient temperatures 706
707
NR and GS activities 708
Leaves with similar age as those used for photosynthesis in B napus were used for the 709
assay of nitrate reductase (NR) and glutamine synthetase (GS) activities NR activity was 710
measured by the modified method of Fan et al (2007) Fresh samples (05g) were frozen in 711
liquid N2 ground into powder in the presence of acid-washed sand and homogenized with 4 712
ml of extraction buffer (0025mol L-1phosphate buffer pH87 1211g L-1 cysteine 0372g L-1 713
EDTA) Homogenates were centrifuged at 30000timesg for 15 min at 4degC and the supernatants 714
were treated with sulfanilamide and α-naphthylamine reagents for colorimetric (540nm) 715
determination of nitrite as described by Fan et al (2007) 716
GS activity was determined using a modified reverseγ-glutamyltransferase method 717
(Wang et al2014) which measures the GS-catalyzed formation of glutamyl-γ-hydroxamate 718
from glutamine and hydroxylamine Fresh samples were frozen for 30 min at -20degC then 719
ground in 10ml Tris-HCl buffer and acid-washed sand The homogenates were filtered 720
through two layers of gauze and centrifuged at 8000timesg for 15 min at 4degC About 12ml of the 721
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
supernatant was added in a reaction mixture and treated as described (Wang et al2014) After 722
the GS-catalyzed reaction was stopped and the solution centrifuged glutamyl-γ723
-hydroxamate was quantified colorimetrically in the supernatant (at 485 nm) and the 724
concentration was determined using a standard curve 725
726
Statistical analyses 727
We used the SPSS software (Statistical Product and Service Solutions V130 USA) for 728
ANOVA and mean separation of main-effects and interactions using Duncanrsquos multiple range 729
test at Plt005Values are presented as means and SD of three or six replicates from three 730
independent experiments Different letters or an asterisk () associated with specific data (eg 731
at the top of histogram bars in figures or within tables) denote significant differences at 732
Plt005 733
734
Supplemental Data 735
Supplemental Figure 1 B napus with higher NUE showed lower vacuolar sequestration 736
capacity (VSC) for NO3-in roots at flowering stage 737
Supplemental Figure 2 B napus with higher NUE showed enhanced long-distance transport 738
of NO3- from roots to shoots at flowering stage 739
Supplemental Figure 3 The two B napus (H and L genotypes) showed the same total N per 740
plant at seedling stage (A) and flowering stage (B) 741
Supplemental Figure 4 B napus with higher NUE showed increased NO3- concentration in 742
the xylem sap at seedling and flowering stages 743
Supplemental Figure 5 NO3- concentration in the xylem sap as affected by inhibitor 744
treatments in B napus and in the energy pumpsrsquo mutants of A thaliana (col-0 vha-a2 vha-a3 745
avp1) 746
Supplemental Figure 6 Reduced VSC for NO3- in roots drives long-distance transport of 747
NO3- from roots to shoots in the A thaliana wild type (Ws) and mutant (clca-2) 748
Supplemental Figure 7 Differences of NR and GS activities between the two B napus (H 749
and L genotypes) at seedling and flowering stages 750
Supplemental Figure 8 Amino acid sequences of BnNRT15 and BnNRT18 751
Supplemental Figure 9 Functions of AtNRT15 and AtNRT18 genes in root tissues of A 752
thaliana in controlling NO3- long-distance transport from root to shoots 753
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
Supplemental Figure 10 B napus BnNRT15 and BnNRT18 genes in roots were 754
coordinately modulated to facilitate NO3- long distance transport from roots to shoots 755
Supplemental Table 1 Differences in stomatal conductance and transpiration rate between 756
the two B napus genotypes 757
Supplemental Table 2 Sequences of primera used for qRT-PCR 758
759
Acknowledgements 760
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 761
Shanghai Institutes for Biological Sciences) for providing nrt15-3 and nrt18-2 seeds 762
This study was supported by the National Natural Science Foundation of China (Grant 763
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 764
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 765
The Ministry of Education in China Open Novel Science Foundation of Hunan province 766
(13K062) the National Key Laboratory of Plant Molecular Genetics and the Twelfth 767
Five-Year National Science and technology support program (2012BAD15BO4) 768
769
Figure 1 B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 770
NO3- in roots at the seedling stage 771
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 772
genotype 814 Specific activities of the tonoplast proton pumps are expressed as μmol Pi 773
released mg-1 protein h-1 NO3- fluxes are expressed as pmol NO3
- cm-2 S-1 Mature vacuoles 774
were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 775
measuring solution between the two positions 1 μm and 11 μm from the vacuole surface 776
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles recording the 777
stable reading data The background was recorded by vibrating the electrode in measuring 778
solution without vacuoles 779
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 780
for NO3- content and NO3
- accumulation normalized against the specific activity of the 781
vacuole acid phosphatase (ACP) as described in Materials and Methods and plotted as μmol 782
NO3- per μmol p-nitrophenol the end product of ACP assay 783
Proton pump activities in root tissues between H and L genotypes are shown at the seedling 784
stage (A) Different letters at the top of the histogram bars denote significant differences of 785
V-ATPase in root tissues between H and L genotypes (Plt005) an asterisk () at the top of the 786
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
histogram bars indicates significant difference in V-PPase activity in root tissues between H 787
and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788
Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789
genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790
bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791
bars on the figures indicate SD (n=6) 792
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793
seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794
to the total NO3- in protoplasts 795
NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796
total NO3-in the cytosol and was calculated as total NO3
- in protoplasts ndash total NO3- in 797
vacuole Different letters at the top of histogram bars denote significant differences of total 798
NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799
(n=6) 800
801
Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802
roots to shoots at the seedling stage 803
H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804
Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805
the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806
as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807
the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808
the histogram bars denote significant differences at Plt005 809
Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810
in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811
genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812
of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813
SD (n=3) different letters at the top of the histogram bars denote significant differences at 814
Plt005 level 815
The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816
at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817
genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818
different letters at the top of the histogram bars denote significant differences at Plt005 819
820
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
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b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
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Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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- Parsed Citations
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9
genotype was significantly higher at seedling stage (Fig 2E) resulting in higher [NO3-] SR 235
ratios at both stages (Fig 2F Fig S2F) 236
NO3- concentration in the xylem sap xylem sap volume and total NO3
- in xylem sap 237
were significantly higher in the H genotype than in the L genotype at seedling stage (Fig 238
S4ACE) the amount of NO3- in xylem sap significantly increased in the H genotype relative 239
to the L genotype at flowering stage (Fig S4F) Together these data suggests greater 240
mobilization of NO3- from root to shoot in the H genotype 241
242
NO3- up-regulates NRT15 but down-regulates NRT18 243
244
A previous study showed that increased NRT18 expression in nrt15 mutants (Chen et al 245
2012) and the expression of NRT15 was induced by NO3- (Lin et al 2008) We tested the 246
relationship between NRT15 and NRT18 expression in A thaliana and B napus No 247
significant differences were observed in AtNRT15 expression in roots between wild type 248
(col-0) and mutant plants (nrt18-2) (Fig 3A) However AtNRT15 expression was 249
significantly up-regulated by NO3- in roots of nrt18-2 mutants (Fig 3B) Expression of 250
AtNRT18 significantly increased in roots of nrt15-3 mutants but not in col-0 plants and 251
there were no significant differences in AtNRT18 expression with or without NO3- treatment 252
in roots of nrt15-3 mutants (Fig 3CD) 253
In contrast BnNRT15 expression in roots of the H genotype increased significantly with 254
NO3- treatment (Fig 3E) while the reverse was observed in expression of BnNRT18 which 255
showed lower expression under NO3- treatment than control (Fig 3F) This suggests that NO3
- 256
induces the expression of NRT15 but down-regulates NRT18 in A thaliana and B napus 257
Further studies are needed to elucidate the mechanisms regulating this reverse regulation 258
259
Reduced VSC of NO3- in roots drives its long-distance transport from roots to shoots 260
Our previous study showed that Bafi (Bafilomycin A1) inhibits V-ATPase DCCD 261
(DCCD + Na2SO3) inhibits V-PPase and a 11 combination of Bafi and DCCD (B+D) 262
inhibits both V-ATPase and V-PPase (Han et al 2015a) These inhibitors were used to 263
control activities of the tonoplast proton pumps in the H B napus plants V-ATPase activity 264
significantly decreased under Bafil and B+D treatments relative to the control and DCCD 265
treatments whereas V-PPase activity declined significantly under DCCD and B+D treatments 266
relative to that in the control and Bafil treatments (Fig 4A) 267
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10
The V-ATPase activity in roots of A thaliana was significantly lower in the V-ATPase 268
mutants (vha-a2 and vha-a3) than the wild type (col-0) and V-PPase mutants (avp1) (Fig 5A) 269
In contrast the V-PPase activity in roots of V-PPase mutant (avp1) were significantly lower 270
than in the wild type (col-0) and V-ATPase mutants (vha-a2 and vha-a3) (Fig 5A) 271
NO3- influxes into vacuoles of the H B napus plants significantly decreased when treated 272
with inhibitors of proton pumps (Bafil DCCD B+D) (Fig 4B) Similarly NO3- influxes into 273
the vacuole of the Arabidopsis mutants (vha-a2 vha-a3 and avp1) was significantly lower 274
than those found in the wild type (col-0) (Fig 5B) Previous studies showed that VSC of NO3- 275
decreased when the activities of the proton pumps decline (Li et al 2005 Krebs et al 2010 276
Han et al 2015a) 277
We further investigated NO3- distribution between the vacuole and cytosol as affected by 278
proton pump inhibition in the H genotype of B napus and the mutants of A thaliana 279
defective in vacuolar proton pumps The percentage of vacuolar NO3- relative to total NO3
- in 280
root protoplasts of the H genotype treated with inhibitors (Bafil DCCD B+D) was lower than 281
that observed in the control (Fig 4C) Results were also similar when using A thaliana 282
mutants where the percentage of vacuolar NO3- relative to the total NO3
- in protoplasts was 283
lower in the mutants (vha-a2 vha-a3 and avp1) than in the wild type (col-0) (Fig 5C) 284
Consequently NO3- accumulation in the cytosol showed a significant increase when energy 285
pumps were suppressed in roots of the H genotype (Fig 4D) and NO3- accumulation in the 286
cytosol of root tissues of A thaliana mutants (vha-a2 vha-a3 and avp1) similarly increased 287
compared with col-0 (Fig 5D) 288
Based on previous observations (Lin et al 2008) expression of NRT15 is strongly 289
induced by NO3- but expression of NRT18 is down-regulated (Fig3 Chen et al 2012) We 290
then hypothesized that expression of these two genes is contrastingly regulated by 291
concentration of NO3- in the cytosol Our results were congruent with this hypothesis the 292
expression of BnNRT15 in root tissues of the H genotype of B napus was significantly higher 293
in plants treated with Bafil DCCD or B+D compared with the control (Fig 4E) whereas the 294
expression of BnNRT18 decreased substantially (Fig 4F) Similar results were also observed 295
in A thaliana where expression of AtNRT15 in roots of the mutants (vha-a2 vha-a3 and 296
avp1) were significantly higher than in the wild type (col-0) but expression of AtNRT18 in 297
the same mutants were considerably lower (Fig 5EF) 298
NO3- concentrations in the xylem sap the N-distribution between shoot and root (SR 299
ratios based on [15N]) and the [NO3-] in shoots relative to roots of the H genotype treated with 300
energy pumpsrsquo inhibitors were significantly higher than in the control (Fig 4GH Fig S5A) 301
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11
Similar trends were also observed when using mutants of A thaliana deficient in the energy 302
pumpsrsquo activities NO3- concentration in the xylem sap the [15N] SR ratios and [NO3
-] SR 303
ratios were all significantly higher in the mutants than in the wild type (Fig 5GH Fig S5B) 304
These data clearly showed that prevention of N sequestration in vacuoles would enhance its 305
translocation to shoot 306
Additional evidence linking NO3- long-distance transport and NO3
- short-distance 307
distribution within cells were provided through experiments comparing A thaliana wild type 308
(Ws) and mutant clca-2 (Fig S6) The chloride channel (CLCa) is the main channel for 309
vacuolar anion accumulation Vacuolar sequestration capacity of NO3-significantly declines in 310
clca mutants (Angeli et al 2006) NO3- influx into the vacuolar space of Ws roots was 311
significantly higher than that observed in the clca-2 mutants (Fig S6A) This resulted in a 312
smaller proportion of vacuolar NO3- in clca-2 plants relative to the total NO3
- in root tissue 313
protoplasts as compared with Ws (Fig S6B) Consequently the accumulation of NO3- in the 314
cytosol was significantly higher in clca-2 than in Ws plants (Fig S6C) The higher 315
NO3-concentration in the cytosol together with the higher expression of AtNRT15 coupled 316
with lower expression of AtNRT18 in clca-2 roots (Fig S6DE) resulted in significant 317
increase in xylem sap NO3- concentration [15N] SR ratios and [NO3
-] SR ratios compared 318
with Ws plants (Fig S6FGH) 319
320
Increased NO3- translocation to shoots enhanced NUE 321
NO3- assimilation efficiency is known to be higher in shoots than in roots (Smirnoff et al 322
1985 Andrews et al 1986 Tang et al 2012 Tang et al 2013) We therefore hypothesize 323
that increased NO3- translocation to shoot and the consequent higher shootroot ratio will 324
contribute to higher NUE Our data support this hypothesis The H B napus genotype showed 325
enhanced long-distance transport of NO3- from roots to shoots (Fig 2 Fig S2) This enhanced 326
NO3- transport requires higher carbon skeleton provided through higher photosynthetic rate 327
(Tang et al 2012 Tang et al 2013) Our results showed that chlorophyll content 328
intercellular CO2 concentration and photosynthetic rate were significantly higher in the H than 329
the L genotype at both seedling and flowering stages (Table 2) Moreover the NO3- 330
assimilating enzymes were strongly induced by NO3- providing sufficient capacity for N 331
assimilation (Smirnoff et al 1985 Andrews et al 1986) Nitrate reductase (NR) and 332
glutamine synthetase (GS) activities in roots of the H genotype were significantly lower than 333
in the L genotype both at seedling and flowering stages (Fig S7) In contrast NR activities in 334
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12
shoots of the H genotype were significantly higher than in the L genotype at both stages (Fig 335
S7AB) and GS activity in the H genotype was also higher than those of the L genotype at 336
seedling stage (Fig S7C) 337
Experiments comparing A thaliana wild type (col-0 Ws) with mutants defective in 338
vacuolar proton-pumps (vha-a2 vha-a3avp1) and transport channel (clca-2) yielded similar 339
results that is increases in NO3- shootroot ratio essentially contributed to enhanced NUE 340
(Fig 6) The NO3- shootroot ratios in A thaliana mutants were higher than that in the wild 341
type (Fig 5GH) Generally the efficiency of inorganic N (NO3-) assimilation into organic N 342
is higher in shoots than in roots (Smirnoff et al 1985 Andrews et al 1986) therefore the 343
NO3- concentration in A thaliana mutants decreased (Fig 6CD) leading to higher NUE 344
compared with the wild type (Fig 6AB) 345
346
BnNRT15BnNRT18 in B napus affects NO3- long-distance transport from roots to 347
shoots 348
Both B napus and A thaliana are members of Cruciferae Family The amino acid 349
sequence identity between BnNRT15 and AtNRT15 was 90 (Fig S8A) the amino acid 350
sequence similarity between BnNRT18 and AtNRT18 was 908 (Fig S8B) Comparisons of 351
nucleotide and amino acid sequences (Harper et al2012) of these genes 352
(httpbrassicanbiacukcgi-binmicroarray_databasecgi) showed that BnNRT15 353
(EV220114) and BnNRT18 (EV116423) of B napus are respectively highly homologous 354
with AtNRT15 and AtNRT18 and the two genes are mainly expressed in roots of both species 355
showing similar organ-specificity (Lin et al 2008 Li et al 2010) 356
NO3- long-distance transport from roots to shoots is therefore regulated by NRT15 and 357
NRT18 as reported before (Lin et al 2008 Li et al 2010) The [15N-traced] SR ratios and 358
the [NO3-] SR ratios were significantly lower in nrt15-3 mutants relative to the wild type 359
(col-0) while [15N] SR and [NO3-] SR ratios showed significant increase in nrt18-2mutant 360
relative to col-0 (Fig S9CD) A previous study showed that expression of NRT15 is 361
down-regulated by ACC and MeJA treatments while the expression of NRT18 is strongly 362
up-regulated in A thaliana (Zhang et al 2014) Consequently NO3- accumulated in plant 363
roots probably as an adaptive measure for abiotic stresses (Chen et al 2012) Both the H- 364
and L- B napus genotypes showed significantly lower expression of BnNRT15 in roots when 365
treated with ACC (Fig S10A) but the expression of BnNRT18 remained higher than in the 366
control plants (Fig S10B) This resulted in significantly lower [15N] SR ratios following 367
ACC treatment (Fig S10CDE) These data indicate that BnNRT15 and BnNRT18 play 368
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13
similar functions for NO3- long-distance transport between root and shoot in both B napus 369
and A thaliana (Lin et al 2008 Li et al 2010) 370
371
372
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14
Discussion 373
Nitrate long-distance transport from root to shoot is regulated by cytosolic NO3- in roots 374
NO3- long-distance transport from root to shoot is controlled by NRT gene family For 375
instance xylem NO3- loading and unloading are mainly controlled by NRT15 and NRT18 376
respectively (Lin et al 2008 Li et al 2010) Whereas the NO3- short-distance transport 377
between the vacuole and cytosol is controlled by tonoplast proton-pumps (V-ATPase and 378
V-PPase) (Krebs et al 2010) and by activity of the CLCa channel in the tonoplast membrane 379
(Angeli et al 2006 Wege et al 2014) tonoplast proton-pumps provide the energy required 380
for NO3- accumulation into the vacuole through CLCa (Gaxiola et al 2001 Brux et al 2008 381
Krebs et al 2010) The expression of NRT15 is up-regulated by NO3- in plant culture 382
solution (Lin et al 2008) whereas regulation of NRT18 expression is contrary to that of 383
NRT15 under both control and abiotic stress conditions (Li et al 2010 Chen et al 2012 384
Zhang et al 2014) 385
Despite the progress made in characterizing the genes involved in transport and 386
metabolism of NO3- in plants the relationship between long- and short-distance transport and 387
how these two pathways are regulated have not been thoroughly investigated We hypothesize 388
that NRT15 expression and its role in long-distance transport is regulated by NO3- in plant 389
tissue as was observed in culture solution (Lin et al 2008) and our data support this 390
hypothesis Both BnNRT15 and AtNRT15 expression were up-regulated by increasing 391
cytosolic NO3- in roots (Fig 4 Fig 5) The increase in cytosolic NO3
- was achieved through (i) 392
use of A thaliana mutants defective in proton-pumps (V-ATPase and V-PPase) or CLCa 393
activities in the tonoplast and (ii) inhibition of B napus tonoplast proton-pumps In both 394
model plants the influx of NO3- into vacuoles was substantially reduced resulting in higher 395
concentrations of NO3- in the cytosol (Fig 4A-D Fig 5A-D Fig S6A-C) Under those 396
conditions expression of BnNRT15 and AtNRT15 was up-regulated by the enhanced 397
cytosolic NO3- while expression of BnNRT18 and AtNRT18 was down-regulated (Fig 4EF 398
Fig 5EF FigS6DE) As a consequence more NO3- was loaded into the xylem sap and 399
transported from root to shoot (Fig S5AB FigS6F) This is also reflected as increased 400
shootroot ratios of N traced by [15N] and [NO3-] (Fig 4GH Fig 5GH Fig S6GH) 401
Interestingly the expression of both BnNRT15 and AtNRT15 is up-regulated by 402
increased cytosolic NO3- concentration in roots but were not affected by NO3
- sequestered 403
into vacuoles (Fig 4 Fig 5 Fig S6) This is because vacuolar NO3- is functionally separated 404
by the tonoplast and cannot be assimilated by enzymes that are localized in the cytosol 405
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15
Therefore NO3--inducible genes such as NR and NRT15 were not influenced by vacuolar 406
NO3- concentration (Martinoia et al 1981 Martinoia et al 2000 Zhang et al 2012) In 407
addition NO3- concentration in the cytosol is controlled by its short-distance transport through 408
the tonoplast which is mediated through the tonoplast proton-pumps providing the required 409
energy for its sequestration into vacuoles against concentration gradient (Gaxiola et al 2001 410
Brux et al 2008 Krebs et al 2010) The gradient of H+ between the inside of the vacuole 411
and the cytosol is maintained by an 2NO3-1H+ antiporter mechanism that facilitatesNO3
- 412
transport through CLCa (Angeli et al 2006 Wege et al 2014) Our data provide clear 413
evidence that NO3- long-distance transport from root to shoot is regulated by cytosolic NO3
- 414
concentration in roots 415
Regulatory mechanisms that control the expression of NRT18 and NRT15 as mediated 416
by NO3- are still unknown however previous studies showed that their expression is also 417
affected by abiotic stresses whereby NRT15 is down-regulated and NRT18 is up-regulated 418
(Li et al 2010 Zhang et al 2014) AtNRT18 expression in A thaliana nrt15 mutants is 419
highly up-regulated (Chen et al 2012) as was also observed here (Fig 3C) Moreover 420
AtNRT15 expression was enhanced by NO3- in culture solution and was not affected in 421
nrt18-2 mutant (Fig 3AB) On the other hand AtNRT18 expression was not affected by 422
cytosolic NO3- but down-regulated when AtNRT15 expression was up-regulated by NO3
-(Fig 423
S6DE) Analogous results were also obtained with B napus genotypes (Fig 3EF) Based on 424
these results we argue that NRT18 expression is not directly influenced by cytosolic NO3- but 425
rather by NRT15 expression (Fig 4 Fig 5 and Fig S6) suggesting NRT18 is probably 426
acting downstream of NRT15 Apparently the regulatory mechanisms of this NRT gene 427
family still awaits further studies to be elucidated 428
429
Response of NUE to NO3- long-distance transport 430
NO3- assimilation efficiency is higher in shoot than in root tissues and can be further 431
enhanced by carbon assimilation (Smirnoff et al 1985 Tang et al 2013) Therefore 432
translocation of higher proportion of NO3- from roots to shoots likely contributes to better 433
crop growth and higher NUE (Andrews et al 1986 Tang et al 2012) The present study 434
provides direct evidence for these results using contrasting B napus genotype identified 435
before (Zhang et al 2009 Han et al 2015a) Our data confirmed the genetic variation in 436
NUE between these two genotypes (Table 1) 437
We measured NO3- distribution in root and shoot of these genotypes to elucidate the 438
physiological mechanisms contributing to variation in NUE The data were consistent with 439
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16
our hypothesis that concentration of NO3- in the xylem sap and shoot would be significantly 440
higher in the H genotype than in the L genotype (Fig 2EFFig S2EF Fig S4) The NO3- 441
long-distance transport from root to shoot was affected by cytosolic NO3- in roots of both A 442
thaliana and B napus genotypes (Fig 4-5 Fig S6) The activities of the tonoplast 443
proton-pumps in roots were significantly lower in the H genotype than in the L genotype (Fig 444
1AB) Consequently less NO3- was sequestered into the vacuole and more NO3
- was retained 445
in cytosol (Fig 1CD) BnNRT15 expression was then up-regulated resulting in greater 446
loading into the xylem while BnNRT18 was down-regulated (Fig 2AB) probably to 447
suppress NO3- unloading from xylem sap Consequently a higher proportion of NO3
- was 448
transported from root to shoots in the H genotype(Fig 2C-D Fig S4) 449
Moreover photosynthetic carbon fixation (Table 2) and activities of both NR and GS 450
were higher in shoots of the H genotype (Fig S7) both of which promote higher N 451
assimilation These results agreed with previous reports (Smirnoff et al 1985 Andrews et al 452
1986 Tang et al 2012 Tang et al 2013) where higher NO3- transport from root to shoot 453
higher photosynthetic rate and N assimilation efficiency in shoots were suggested as essential 454
mechanisms for high-NUE in crop plants These qualities endowed the B napus H genotype 455
Xiangyou15 with higher NUE as compared with the L genotype 814 456
NO3- long-distance transport in B napus is not only regulated by BnNRT15 and 457
BnNRT18 in root tissues (Fig 4 Fig 5 Fig S6) but also by stomatal conductance and 458
transpiration by indirectly controlling xylem sap flow and long-distance transport 459
(Dechorgnat et al 2011 Krapp et al 2014) Stomatal conductance and transpiration rates of 460
the H genotype were significantly higher than those of the L genotype (Table S1) a result 461
consistent with other studies describing characteristics of high-NUE plants (Daniel-Vedele et 462
al 1998 Wilkinson et al 2007 Dechorgnat et al 2011 Krapp et al 2014) 463
464
Response of NUE to NO3- short-distance transport 465
NUE is also strongly controlled by NO3- short-distance distribution between the vacuole 466
and cytosol (Han et al 2015a) A lower proportion of NO3- accumulating in vacuoles and a 467
higher proportion retained in the cytosol will contribute to better NUE in crop plants (Han et 468
al 2015b) The energy required for vacuolar NO3- sequestration is provided by the tonoplast 469
proton-pumps (Gaxiola et al 2001 Brux et al 2008 Krebs et al 2010) while the 470
distribution channel CLCa enables transport of NO3- across the vacuolar membrane (Angeli et 471
al 2006 Wege et al 2014) The activities of the tonoplast proton-pumps (V-ATPase and 472
V-PPase) in leaves of the H genotype were significantly lower than those of the L genotype at 473
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17
flowering stages (Han et al 2015aHan et al 2015b) thus we expected similar differences in 474
root tissues Our results confirmed that the activities of tonoplast proton-pumps in root tissues 475
of B napus H genotype and A thaliana mutants were lower than those in the L genotype and 476
A thaliana wild type leading to less NO3- influx into vacuoles and more in the cytosol (Fig 1 477
Fig 5A-D) Moreover the enzymes catalyzing N assimilation are located in the cytoplasm 478
where NO3- could be assimilated (Martinoia et al 1981 Han et al 2015a) or immediately 479
loaded into xylem sap for long-distance transport from roots to shoots (Lin et al 2008) 480
Consequently the NO3- concentration in root tissues of the H genotype and A thaliana 481
mutants defective in tonoplast proton-pumps was significantly lower than that in the L 482
genotype (Fig 2E) and in A thaliana wild type (Fig 6C) Therefore the higher proportion of 483
NO3- transported to shoot and the lower concentrations retained in root vacuoles contributed 484
to the higher NUE in Xiangyou15 and A thaliana mutants (vha-a2 vha-a3 and avp1) (Table 485
1Fig 6A) Further studies are needed to assess the impacts of regulating vacuolar 486
sequestration of NO3- and rates of long-distance transport for enhancing NUE in other crop 487
species and genotypes 488
These data suggested that NO3- distribution between vacuoles and cytosol within cells 489
regulates NO3- long-distance transport from root to shoot and ultimately affects NUE NO3
- 490
loading into the xylem sap is an active transport process (Lin et al 2008) therefore not 491
regulated by absolute concentration of NO3- in the cytosol but rather by genes involved in 492
NO3- long-distance transport (NRT15 and NRT18) Apparently the expression of NRT15 the 493
gene responsible for xylem loading is regulated by NO3-concentration in the cytosol 494
Therefore both NO3- short-distance transport across the tonoplast and its long-distance 495
transport from root to shoot are important determinants of NUE in both B napus and A 496
thaliana (Table 1 and Fig 6) 497
498
Roles of NRT15 and NRT18 in NO3- long-distance transport in B napus and A 499
thaliana 500
Our results (Figs S9 S10) showed that the proportion of NO3- distributed from root to 501
shoot is strongly controlled by NRT15 in accord with previous studies (Lin et al 2008 Li et 502
al 2010) The data suggest that the high-NUE genotypes possess lower activities of tonoplast 503
proton-pumps (V-ATPase and V-PPase) resulting in less NO3- accumulation in vacuoles and 504
more NO3- retention in cytosol (Fig 7) The higher NO3
- in the cytosol then up-regulated 505
NRT15 which then down-regulates NRT18 As a result more NO3- is loaded into the xylem 506
system by NRT15 mechanism and less NO3- is unloaded via NRT18 (Fig 7) Differential 507
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18
regulation of these NRT genes in high-NUE plants increased transport of NO3- from root to 508
shoot through the xylem vascular tissues (Fig 7) This coordinated NO3- long- and 509
short-distance transport likely fine-tunes NO3- allocation and modulates the balance of NO3
- 510
distribution between roots and shoots (Fig 7) 511
Our research also revealed differences between A thaliana and B napus in the 512
expression of the wild-type NRT genes in response to NO3- in the protoplast Moreover using 513
A thaliananrt15-3 mutants we uncovered possible regulatory interactions between a 514
functioning (wild-type) NRT15 gene and NRT18 expression in response to NO3- 515
concentration Because mutants defective in nrt15 are not available for B napus we could 516
not confirm that the respective NRT15 and NRT18 systems behave in an analogous way in 517
these plant species Based on Harper et al (2012) research database our homology 518
comparisons and gene function annotations provided strong evidence for the roles of 519
BnNRT15 and BnNRT18 in B napus Finally our studies were conducted using two model 520
dicotyledonous plant species for which we had ample data on responses to specific inhibitors 521
(B napus) or specific mutants were available with results forming the bases for the model 522
presented in Figure 7 Further research using genetically diverse plant species (eg 523
monocotyledonous crops such as rice wheat and corn) will help validate this model and its 524
broader application across species The information generated in this study could have future 525
application for improving NUE in commercial crops with consequent reduction in nitrogen 526
use and benefits to the environment 527
528
Materials and Methods 529
Plant material 530
The two oilseed rape (Bnapus) cultivars used in this study have been characterized 531
before as high- (Xiangyou15 referred to as H genotype hereafter) and low- (814 referred to 532
as L genotype) nitrogen use efficiency (NUE) genotypes (Zhang et al 2009 Han et al 2015b) 533
Here we define NUE as the total biomass per unit of N uptake by the plant with total 534
biomass includes roots shoots and grains The two genotypes were provided by the Hunan 535
Sub-Center of Improvement Center of National Oil Crops Hunan China 536
The Arabidopsis thaliana wild-type Columbia-0 (col-0) was used as control for 537
V-ATPase (vha-a2 and vha-a3) V-PPase (avp1) nrt15-3 and nrt18-2 mutants whereas the A 538
thaliana wild-type Wassilewskija (Ws) was used as control for clca-2 mutants A thaliana 539
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19
mutants and transgenic lines (nrt15-3 and nrt18-2) have been described previously (Chen et 540
al 2012 Zhang et al 2014) The mutant lines vha-a2 (Salk_142642) and vha-a3 541
(Salk_122135) described by Krebs et al (2010) were obtained from the Arabidopsis 542
Biological Resources Center (ABRC) The mutant line avp1 (GK-005004) described in Li et 543
al (2005) was obtained from the European Arabidopsis Stock Centre while the mutant line 544
clca-2 (FST 171A06) described in Angeli et al (2006) was from the Institute National de la 545
Recherche Agronomique (INRA) collection in France together with the T-DNA mutants used 546
for genotyping to select pure mutant lines 547
548
Growth conditions 549
B napus plants were grown hydroponically in ceramic pots(20cmtimes15cm) filled with a 550
nutrient solution in the greenhouse under natural light as described in Han et al (2015b) The 551
pots were arranged in a completely randomized design with six biological replications The 552
nutrient solution was replaced every 3 days and its pH adjusted to 55 daily Experiments were 553
conducted at the field station of Hunan Agricultural University Southern China 554
A thaliana plants were grown in a nutrient solution in plastic pots as described in Arteca 555
et al (2000) and Gong et al (2003) The solution was changed every 3 days with pH adjusted 556
daily to 58 Pots were arranged in a completely randomized design with six biological 557
replications The nutrient solution used for both species consisted of 125mM KNO3 558
0625mM KH2PO4 05mM MgSO4 05mM Ca (NO3)2middot4H2O 0025mM Fe-EDTA 025 559
mlL-1 micronutrients (stock solution concentrations 70mM B 14mM Mn 1mM Zn 05mM 560
Cu and 02mM Mo) The experiments were conducted at Hunan Agricultural University in a 561
phytotron set at 70 relative humidity16h-light8h-dark cycle and constant temperature of 562
22degC 563
564
Experimental treatments and phenotyping 565
All measurements made on B napus plants were conducted either at the seedling stage 566
(2 months after transplanting) or at flowering (5 months after transplanting) The whole root 567
tissue was harvested and used for analyses at either stage For shoot measurements the fourth 568
leaf from the bottom was used for measurements made at seedling stage whereas the 12th leaf 569
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20
from the bottom was harvested for measurements made at flowering stage For A thaliana 570
whole root and shoot of four-week-old plants were sampled and used for different assays 571
572
Vacuolar proton-pumps inhibitor treatments 573
Inhibition of vacuolar proton pumps of B napus (Xiangyou15) was conducted at the seedling 574
stage in hydroponic culture as described in Han et al (2015a) with minor modifications 575
Plants grown in hydroponic solution were used as control Inhibitor treatment was conducted 576
using several chemicals Bafi(25 nmol L-1 Bafilomycin A1) an inhibitor of V-ATPase 577
DCCD(10 μmol L-1DCCD + 50 mmolL-1 Na2SO3) specifically inhibits V-PPase and 578
Bafi+DCCD in a 11 ratio as inhibitor of both V-ATPase and V-PPase The inhibitors were 579
applied in the hydroponic solutions for 24 h Fresh plant tissue samples from all treatments 580
were collected for different assays 581
582
NO3- induced gene expression 583
Four-week old A thaliana mutants (nrt15-3nrt18-2) were grown in hydroponics with 584
225mM (NH4)2succinate for 3 days and shifted to hydroponics with 45mM NO3- for 12 h 585
then root tissues were collected to assess relative expression of AtNRT15 and AtNRT18 586
genes Seedlings of B napus (Xiangyou15) plants grown hydroponically were treated with 587
5mM (NH4)2succinate for 3 d and shifted to fresh hydroponic solution containing 15 mM 588
NO3- for 12 h Root tissue was then collected to assess the relative expression of BnNRT15 589
and BnNRT18 genes using quantitative RT-PCR 590
591
ACC treatment 592
Hydroponically grown seedlings of B napus were subjected to ACC treatment as described in 593
Zhang et al (2014) with minor modifications Seedlings of the two genotypes Xiangyou15 594
and 814 were transferred to a nutrient solution containing 002mM L-1 ACC for 6 h or grown 595
in normal hydroponic solution as control Root tissues were then collected to assay relative 596
expression of BnNRT15 and BnNRT18 using quantitative RT-PCR Another set of B napus 597
plants were grown in ACC-containing hydroponic solution but supplemented with 15NO3- (22 598
15N) replacing the unlabeled NO3- for 1 h Root and shoot tissues were collected separately 599
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21
for 15N measurement using a continuous-flow isotope ratio mass spectrometer coupled with a 600
C-N elemental analyzer (ANCA-MS PDZ Europa) 601
602
Biomass and N concentration 603
Grain yield dry biomass and N concentration were determined in plant samples at 604
harvest Plants were harvested (including senescing leaves) then dried in an oven for 30 min 605
at 105degC then at 70degC to a constant weight and weighed N concentration was determined 606
using the Kjeldahl method (Shi et al 2010 Han et al 2015b)02g of dried samples were 607
digested with H2SO4-H2O2 in a 100-ml Kjeldahl digestion flask after which N concentration 608
was determined using a Foss Auto Analyser Unit (Kjeldahl 8400) 609
610
V-ATPase and V-PPase activities 611
Roots (10 g) of B napus were collected at seedling and flowering stages and that of A 612
thaliana (05 g) sampled at seedling stage then used for determining the activities of 613
V-ATPase and V-PPase (Han et al 2015b) Activities of these two energy pumps within 614
microsomal membranes were followed colorimetrically based on released Pi as described by 615
Zhu et al (2001) and Krebs et al (2010) 616
617
Assay of NO3- flux in vacuoles 618
Roots of both species were collected similarly as that used for V-ATPase and V-PPase 619
activities then used for vacuole isolation and measurement of NO3- flux in vacuoles as 620
described by Robert et al (2007) with minor modification (Han et al 2015b) Net fluxes of 621
NO3- in vacuoles were measured non-invasively using SIET (scanning ion-selective electrode 622
technique SIET system BIO-003A Younger USA Science and Technology Corporation 623
USA) An NO3--selective microelectrode used for the assay of NO3
- flux was vibrated in the 624
measuring solution between two positions 1 μm and 11 μm from the vacuole surface 625
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles Background 626
signal was recorded by vibrating the electrode in the measuring solution in the absence of 627
vacuoles Ion flux was calculated by Fickrsquos law of diffusion J = -D(dcdx) where J represents 628
the ion flux (picomolescm-2s-1) dcdx is the ion concentration gradient and D is the ion 629
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22
diffusion constant in a particular medium The direction of the flux was derived from Fickrsquos 630
law of diffusion that relates the concentration gradient 631
632
Isolation of intact protoplasts and vacuoles for determining NO3- concentration 633
Similar to the above assays root tissues (10 g) were collected from B napus at seedling 634
and flowering stages and from A thaliana (05 g) seedlings and used to isolate intact 635
protoplasts and vacuoles as described by Robert et al (2007) with minor modifications as 636
outlined in Huang et al (2012) and Han et al(2015b) The purified protoplasts were divided 637
into two equal aliquots with one of them used for isolation of vacuoles The purified 638
protoplasts and vacuoles were then sub-sampled and used to determine NO3- concentrations 639
(Vogeli-Lange and Wagner 1990) and for enzyme activity assays (Ma et al 2005) 640
NO3-concentrations in protoplasts and vacuoles were measured by a continuous-flow 641
auto-analyzer (Auto Analyser 3 Bran and Luebbe Germany) as described previously (Han et 642
al 2015b)The activities of acid phosphatase (ACP) and cytochrome oxidase (COX) were 643
determined using plant ACP colorimetry- and COX-assay kits (GenMedSci Inc) following 644
the manufacturerrsquos instructions ACP activity specific to vacuoles was determined and used to 645
normalize NO3- accumulation We measured NO3
- in the protoplast outside the vacuole which 646
includes the cytosol and organelles eg mitochondria and Golgi Apparatus (Robert et 647
al2007) Since most of the NO3- in the protoplast outside the vacuole is located in the cytosol 648
(Krebs et al 2010) we refer to NO3- distribution between vacuoles and cytosol rather than 649
vacuole versus protoplast 650
651
Quantitative RT-PCR 652
Total RNA extracted from B napus roots was prepared using Trizol reagent (Invitrogen) 653
cDNAs were synthesized using M-MLV reverse transcriptase (Promega) following 654
manufacturerrsquos protocol The relative expression of BnNRT15 (EV220114) BnNRT18 655
(EV116423) and Bnactin (AF1118121) genes in plant roots were determined by quantitative 656
RT-PCR and run on a LightCycler instrument (Roche) with the SYBR Green Real-Time PCR 657
Master Mix Kit (TOYOBO Japan) under the following conditions 95degC for 2min then 45 658
cycles of 95degC for 10s 60degC for 10s and 72degC for 20s The primer sequences of BnNRT15 659
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
BnNRT18 and Bnactin genes were obtained from httpbrassicanbiacuk as described in 660
Harper et al (2012) and expression levels were normalized to Bnactin as control 661
Total RNA extracted from the 4-week-old A thaliana roots under the treatments 662
conditions described above were prepared using Trizol reagent (Invitrogen) The cDNAs were 663
synthesized using M-MLV reverse transcriptase (Promega) based on the manufacturerrsquos 664
protocol and as described in Li et al (2010)The primer sequences of quantitative RT-PCR 665
used in these assays are listed in Supplemental Table 2 and were described previously (Chen 666
et al 2012 Zhang et al 2014) and their expression was normalized to Atactin2 as control 667
668
Determination of nitrate distribution using 15NO3- 669
Hydroponically grown B napus (seedling and flowering stages) and A thaliana 670
(seedling) were transferred to 01mM CaSO4 solution for 1min then to their respective 671
hydroponic nutrient solutions with15NO3- (22 excess) replacing unlabeled NO3
- for 1h 672
Plants were then transferred to 01mM CaSO4 solution for 1min after which roots were 673
washed with deionized water Root and shoot samples were separated and dried at 105degC for 674
30min followed by 70degC for 3 d then grounded and used for assaying 15N content using a 675
continuous-flow isotope ratio mass spectrometer coupled with a carbon-nitrogen elemental 676
analyzer (ANCA-MS PDZ Europa) 677
678
Xylem sap collection and assay of nitrate concentration 679
We used the method of Tang et al (2012) for collecting xylem sap Plants were cut 680
leaving1cm segments with intact roots at seedling and flowering stages for B napus and at 681
seedling stage for A thaliana Roots were immediately immersed in their respective nutrition 682
solutions Weighed cotton was put on the cut surface to absorb extruding xylem sap for 1 h 683
The cotton was then wrapped in plastic film and the volume of xylem sap was calculated as 684
the weight gain of the cotton Xylem sap was then squeezed from the cotton using a syringe 685
and used for subsequent assay of nitrate concentration using a continuous-flow auto-analyzer 686
(Auto Analyser 3 Bran and Luebbe Germany) 687
To determine nitrate concentration samples (10 g fresh root and shoot at seedling and 688
flowering of B napus and 05 g fresh root and shoot of 4-week-old A thaliana) were frozen 689
in liquid N2 and ground with a mortar and pestle the powder was then transferred to a beaker 690
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
containing 70 ml deionized water and boiled for 30 min to extract nitrate then cooled and 691
made to 100 ml volume and 02 g activated carbon added to eliminate the effect of 692
chlorophyll The mixture was filtered and nitrate in the filtrate was determined using a 693
continuous-flow auto-analyser (Atuo-Analyser 3 Bran and Luebbe Germany) 694
695
Measurements of photosynthesis 696
All measurements of photosynthesis and related parameters were conducted at 1000 h 697
using intact leaves The 4th leaf from the bottom in B napus at seedling stage and the 12th leaf 698
from the bottom at flowering stage were used for determining chlorophyll concentration 699
photosynthetic rate intercellular CO2 concentration transpiration rate and stomatal 700
conductance Chlorophyll concentration was determined by SPAD-502 (Minolta Camera Co 701
Ltd Japan) (Luo et al 2006 Yandeau-Nelson et al 2011) Photosynthetic rate intercellular 702
CO2 concentration transpiration rate and stomatal conductance were measured using LI-6400 703
Portable Photosynthesis System (Li-Cor Biosciences Co Ltd USA) set at flow rate of 500 704
μmol s-1 photosynthetic photon flux density of 1000 μmol m-2 s-1 relative humidity of 65 705
CO2 concentration of 400ppm and ambient temperatures 706
707
NR and GS activities 708
Leaves with similar age as those used for photosynthesis in B napus were used for the 709
assay of nitrate reductase (NR) and glutamine synthetase (GS) activities NR activity was 710
measured by the modified method of Fan et al (2007) Fresh samples (05g) were frozen in 711
liquid N2 ground into powder in the presence of acid-washed sand and homogenized with 4 712
ml of extraction buffer (0025mol L-1phosphate buffer pH87 1211g L-1 cysteine 0372g L-1 713
EDTA) Homogenates were centrifuged at 30000timesg for 15 min at 4degC and the supernatants 714
were treated with sulfanilamide and α-naphthylamine reagents for colorimetric (540nm) 715
determination of nitrite as described by Fan et al (2007) 716
GS activity was determined using a modified reverseγ-glutamyltransferase method 717
(Wang et al2014) which measures the GS-catalyzed formation of glutamyl-γ-hydroxamate 718
from glutamine and hydroxylamine Fresh samples were frozen for 30 min at -20degC then 719
ground in 10ml Tris-HCl buffer and acid-washed sand The homogenates were filtered 720
through two layers of gauze and centrifuged at 8000timesg for 15 min at 4degC About 12ml of the 721
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
supernatant was added in a reaction mixture and treated as described (Wang et al2014) After 722
the GS-catalyzed reaction was stopped and the solution centrifuged glutamyl-γ723
-hydroxamate was quantified colorimetrically in the supernatant (at 485 nm) and the 724
concentration was determined using a standard curve 725
726
Statistical analyses 727
We used the SPSS software (Statistical Product and Service Solutions V130 USA) for 728
ANOVA and mean separation of main-effects and interactions using Duncanrsquos multiple range 729
test at Plt005Values are presented as means and SD of three or six replicates from three 730
independent experiments Different letters or an asterisk () associated with specific data (eg 731
at the top of histogram bars in figures or within tables) denote significant differences at 732
Plt005 733
734
Supplemental Data 735
Supplemental Figure 1 B napus with higher NUE showed lower vacuolar sequestration 736
capacity (VSC) for NO3-in roots at flowering stage 737
Supplemental Figure 2 B napus with higher NUE showed enhanced long-distance transport 738
of NO3- from roots to shoots at flowering stage 739
Supplemental Figure 3 The two B napus (H and L genotypes) showed the same total N per 740
plant at seedling stage (A) and flowering stage (B) 741
Supplemental Figure 4 B napus with higher NUE showed increased NO3- concentration in 742
the xylem sap at seedling and flowering stages 743
Supplemental Figure 5 NO3- concentration in the xylem sap as affected by inhibitor 744
treatments in B napus and in the energy pumpsrsquo mutants of A thaliana (col-0 vha-a2 vha-a3 745
avp1) 746
Supplemental Figure 6 Reduced VSC for NO3- in roots drives long-distance transport of 747
NO3- from roots to shoots in the A thaliana wild type (Ws) and mutant (clca-2) 748
Supplemental Figure 7 Differences of NR and GS activities between the two B napus (H 749
and L genotypes) at seedling and flowering stages 750
Supplemental Figure 8 Amino acid sequences of BnNRT15 and BnNRT18 751
Supplemental Figure 9 Functions of AtNRT15 and AtNRT18 genes in root tissues of A 752
thaliana in controlling NO3- long-distance transport from root to shoots 753
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
Supplemental Figure 10 B napus BnNRT15 and BnNRT18 genes in roots were 754
coordinately modulated to facilitate NO3- long distance transport from roots to shoots 755
Supplemental Table 1 Differences in stomatal conductance and transpiration rate between 756
the two B napus genotypes 757
Supplemental Table 2 Sequences of primera used for qRT-PCR 758
759
Acknowledgements 760
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 761
Shanghai Institutes for Biological Sciences) for providing nrt15-3 and nrt18-2 seeds 762
This study was supported by the National Natural Science Foundation of China (Grant 763
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 764
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 765
The Ministry of Education in China Open Novel Science Foundation of Hunan province 766
(13K062) the National Key Laboratory of Plant Molecular Genetics and the Twelfth 767
Five-Year National Science and technology support program (2012BAD15BO4) 768
769
Figure 1 B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 770
NO3- in roots at the seedling stage 771
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 772
genotype 814 Specific activities of the tonoplast proton pumps are expressed as μmol Pi 773
released mg-1 protein h-1 NO3- fluxes are expressed as pmol NO3
- cm-2 S-1 Mature vacuoles 774
were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 775
measuring solution between the two positions 1 μm and 11 μm from the vacuole surface 776
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles recording the 777
stable reading data The background was recorded by vibrating the electrode in measuring 778
solution without vacuoles 779
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 780
for NO3- content and NO3
- accumulation normalized against the specific activity of the 781
vacuole acid phosphatase (ACP) as described in Materials and Methods and plotted as μmol 782
NO3- per μmol p-nitrophenol the end product of ACP assay 783
Proton pump activities in root tissues between H and L genotypes are shown at the seedling 784
stage (A) Different letters at the top of the histogram bars denote significant differences of 785
V-ATPase in root tissues between H and L genotypes (Plt005) an asterisk () at the top of the 786
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
histogram bars indicates significant difference in V-PPase activity in root tissues between H 787
and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788
Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789
genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790
bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791
bars on the figures indicate SD (n=6) 792
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793
seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794
to the total NO3- in protoplasts 795
NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796
total NO3-in the cytosol and was calculated as total NO3
- in protoplasts ndash total NO3- in 797
vacuole Different letters at the top of histogram bars denote significant differences of total 798
NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799
(n=6) 800
801
Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802
roots to shoots at the seedling stage 803
H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804
Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805
the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806
as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807
the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808
the histogram bars denote significant differences at Plt005 809
Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810
in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811
genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812
of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813
SD (n=3) different letters at the top of the histogram bars denote significant differences at 814
Plt005 level 815
The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816
at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817
genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818
different letters at the top of the histogram bars denote significant differences at Plt005 819
820
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
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b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
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Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
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Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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10
The V-ATPase activity in roots of A thaliana was significantly lower in the V-ATPase 268
mutants (vha-a2 and vha-a3) than the wild type (col-0) and V-PPase mutants (avp1) (Fig 5A) 269
In contrast the V-PPase activity in roots of V-PPase mutant (avp1) were significantly lower 270
than in the wild type (col-0) and V-ATPase mutants (vha-a2 and vha-a3) (Fig 5A) 271
NO3- influxes into vacuoles of the H B napus plants significantly decreased when treated 272
with inhibitors of proton pumps (Bafil DCCD B+D) (Fig 4B) Similarly NO3- influxes into 273
the vacuole of the Arabidopsis mutants (vha-a2 vha-a3 and avp1) was significantly lower 274
than those found in the wild type (col-0) (Fig 5B) Previous studies showed that VSC of NO3- 275
decreased when the activities of the proton pumps decline (Li et al 2005 Krebs et al 2010 276
Han et al 2015a) 277
We further investigated NO3- distribution between the vacuole and cytosol as affected by 278
proton pump inhibition in the H genotype of B napus and the mutants of A thaliana 279
defective in vacuolar proton pumps The percentage of vacuolar NO3- relative to total NO3
- in 280
root protoplasts of the H genotype treated with inhibitors (Bafil DCCD B+D) was lower than 281
that observed in the control (Fig 4C) Results were also similar when using A thaliana 282
mutants where the percentage of vacuolar NO3- relative to the total NO3
- in protoplasts was 283
lower in the mutants (vha-a2 vha-a3 and avp1) than in the wild type (col-0) (Fig 5C) 284
Consequently NO3- accumulation in the cytosol showed a significant increase when energy 285
pumps were suppressed in roots of the H genotype (Fig 4D) and NO3- accumulation in the 286
cytosol of root tissues of A thaliana mutants (vha-a2 vha-a3 and avp1) similarly increased 287
compared with col-0 (Fig 5D) 288
Based on previous observations (Lin et al 2008) expression of NRT15 is strongly 289
induced by NO3- but expression of NRT18 is down-regulated (Fig3 Chen et al 2012) We 290
then hypothesized that expression of these two genes is contrastingly regulated by 291
concentration of NO3- in the cytosol Our results were congruent with this hypothesis the 292
expression of BnNRT15 in root tissues of the H genotype of B napus was significantly higher 293
in plants treated with Bafil DCCD or B+D compared with the control (Fig 4E) whereas the 294
expression of BnNRT18 decreased substantially (Fig 4F) Similar results were also observed 295
in A thaliana where expression of AtNRT15 in roots of the mutants (vha-a2 vha-a3 and 296
avp1) were significantly higher than in the wild type (col-0) but expression of AtNRT18 in 297
the same mutants were considerably lower (Fig 5EF) 298
NO3- concentrations in the xylem sap the N-distribution between shoot and root (SR 299
ratios based on [15N]) and the [NO3-] in shoots relative to roots of the H genotype treated with 300
energy pumpsrsquo inhibitors were significantly higher than in the control (Fig 4GH Fig S5A) 301
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Similar trends were also observed when using mutants of A thaliana deficient in the energy 302
pumpsrsquo activities NO3- concentration in the xylem sap the [15N] SR ratios and [NO3
-] SR 303
ratios were all significantly higher in the mutants than in the wild type (Fig 5GH Fig S5B) 304
These data clearly showed that prevention of N sequestration in vacuoles would enhance its 305
translocation to shoot 306
Additional evidence linking NO3- long-distance transport and NO3
- short-distance 307
distribution within cells were provided through experiments comparing A thaliana wild type 308
(Ws) and mutant clca-2 (Fig S6) The chloride channel (CLCa) is the main channel for 309
vacuolar anion accumulation Vacuolar sequestration capacity of NO3-significantly declines in 310
clca mutants (Angeli et al 2006) NO3- influx into the vacuolar space of Ws roots was 311
significantly higher than that observed in the clca-2 mutants (Fig S6A) This resulted in a 312
smaller proportion of vacuolar NO3- in clca-2 plants relative to the total NO3
- in root tissue 313
protoplasts as compared with Ws (Fig S6B) Consequently the accumulation of NO3- in the 314
cytosol was significantly higher in clca-2 than in Ws plants (Fig S6C) The higher 315
NO3-concentration in the cytosol together with the higher expression of AtNRT15 coupled 316
with lower expression of AtNRT18 in clca-2 roots (Fig S6DE) resulted in significant 317
increase in xylem sap NO3- concentration [15N] SR ratios and [NO3
-] SR ratios compared 318
with Ws plants (Fig S6FGH) 319
320
Increased NO3- translocation to shoots enhanced NUE 321
NO3- assimilation efficiency is known to be higher in shoots than in roots (Smirnoff et al 322
1985 Andrews et al 1986 Tang et al 2012 Tang et al 2013) We therefore hypothesize 323
that increased NO3- translocation to shoot and the consequent higher shootroot ratio will 324
contribute to higher NUE Our data support this hypothesis The H B napus genotype showed 325
enhanced long-distance transport of NO3- from roots to shoots (Fig 2 Fig S2) This enhanced 326
NO3- transport requires higher carbon skeleton provided through higher photosynthetic rate 327
(Tang et al 2012 Tang et al 2013) Our results showed that chlorophyll content 328
intercellular CO2 concentration and photosynthetic rate were significantly higher in the H than 329
the L genotype at both seedling and flowering stages (Table 2) Moreover the NO3- 330
assimilating enzymes were strongly induced by NO3- providing sufficient capacity for N 331
assimilation (Smirnoff et al 1985 Andrews et al 1986) Nitrate reductase (NR) and 332
glutamine synthetase (GS) activities in roots of the H genotype were significantly lower than 333
in the L genotype both at seedling and flowering stages (Fig S7) In contrast NR activities in 334
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12
shoots of the H genotype were significantly higher than in the L genotype at both stages (Fig 335
S7AB) and GS activity in the H genotype was also higher than those of the L genotype at 336
seedling stage (Fig S7C) 337
Experiments comparing A thaliana wild type (col-0 Ws) with mutants defective in 338
vacuolar proton-pumps (vha-a2 vha-a3avp1) and transport channel (clca-2) yielded similar 339
results that is increases in NO3- shootroot ratio essentially contributed to enhanced NUE 340
(Fig 6) The NO3- shootroot ratios in A thaliana mutants were higher than that in the wild 341
type (Fig 5GH) Generally the efficiency of inorganic N (NO3-) assimilation into organic N 342
is higher in shoots than in roots (Smirnoff et al 1985 Andrews et al 1986) therefore the 343
NO3- concentration in A thaliana mutants decreased (Fig 6CD) leading to higher NUE 344
compared with the wild type (Fig 6AB) 345
346
BnNRT15BnNRT18 in B napus affects NO3- long-distance transport from roots to 347
shoots 348
Both B napus and A thaliana are members of Cruciferae Family The amino acid 349
sequence identity between BnNRT15 and AtNRT15 was 90 (Fig S8A) the amino acid 350
sequence similarity between BnNRT18 and AtNRT18 was 908 (Fig S8B) Comparisons of 351
nucleotide and amino acid sequences (Harper et al2012) of these genes 352
(httpbrassicanbiacukcgi-binmicroarray_databasecgi) showed that BnNRT15 353
(EV220114) and BnNRT18 (EV116423) of B napus are respectively highly homologous 354
with AtNRT15 and AtNRT18 and the two genes are mainly expressed in roots of both species 355
showing similar organ-specificity (Lin et al 2008 Li et al 2010) 356
NO3- long-distance transport from roots to shoots is therefore regulated by NRT15 and 357
NRT18 as reported before (Lin et al 2008 Li et al 2010) The [15N-traced] SR ratios and 358
the [NO3-] SR ratios were significantly lower in nrt15-3 mutants relative to the wild type 359
(col-0) while [15N] SR and [NO3-] SR ratios showed significant increase in nrt18-2mutant 360
relative to col-0 (Fig S9CD) A previous study showed that expression of NRT15 is 361
down-regulated by ACC and MeJA treatments while the expression of NRT18 is strongly 362
up-regulated in A thaliana (Zhang et al 2014) Consequently NO3- accumulated in plant 363
roots probably as an adaptive measure for abiotic stresses (Chen et al 2012) Both the H- 364
and L- B napus genotypes showed significantly lower expression of BnNRT15 in roots when 365
treated with ACC (Fig S10A) but the expression of BnNRT18 remained higher than in the 366
control plants (Fig S10B) This resulted in significantly lower [15N] SR ratios following 367
ACC treatment (Fig S10CDE) These data indicate that BnNRT15 and BnNRT18 play 368
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similar functions for NO3- long-distance transport between root and shoot in both B napus 369
and A thaliana (Lin et al 2008 Li et al 2010) 370
371
372
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14
Discussion 373
Nitrate long-distance transport from root to shoot is regulated by cytosolic NO3- in roots 374
NO3- long-distance transport from root to shoot is controlled by NRT gene family For 375
instance xylem NO3- loading and unloading are mainly controlled by NRT15 and NRT18 376
respectively (Lin et al 2008 Li et al 2010) Whereas the NO3- short-distance transport 377
between the vacuole and cytosol is controlled by tonoplast proton-pumps (V-ATPase and 378
V-PPase) (Krebs et al 2010) and by activity of the CLCa channel in the tonoplast membrane 379
(Angeli et al 2006 Wege et al 2014) tonoplast proton-pumps provide the energy required 380
for NO3- accumulation into the vacuole through CLCa (Gaxiola et al 2001 Brux et al 2008 381
Krebs et al 2010) The expression of NRT15 is up-regulated by NO3- in plant culture 382
solution (Lin et al 2008) whereas regulation of NRT18 expression is contrary to that of 383
NRT15 under both control and abiotic stress conditions (Li et al 2010 Chen et al 2012 384
Zhang et al 2014) 385
Despite the progress made in characterizing the genes involved in transport and 386
metabolism of NO3- in plants the relationship between long- and short-distance transport and 387
how these two pathways are regulated have not been thoroughly investigated We hypothesize 388
that NRT15 expression and its role in long-distance transport is regulated by NO3- in plant 389
tissue as was observed in culture solution (Lin et al 2008) and our data support this 390
hypothesis Both BnNRT15 and AtNRT15 expression were up-regulated by increasing 391
cytosolic NO3- in roots (Fig 4 Fig 5) The increase in cytosolic NO3
- was achieved through (i) 392
use of A thaliana mutants defective in proton-pumps (V-ATPase and V-PPase) or CLCa 393
activities in the tonoplast and (ii) inhibition of B napus tonoplast proton-pumps In both 394
model plants the influx of NO3- into vacuoles was substantially reduced resulting in higher 395
concentrations of NO3- in the cytosol (Fig 4A-D Fig 5A-D Fig S6A-C) Under those 396
conditions expression of BnNRT15 and AtNRT15 was up-regulated by the enhanced 397
cytosolic NO3- while expression of BnNRT18 and AtNRT18 was down-regulated (Fig 4EF 398
Fig 5EF FigS6DE) As a consequence more NO3- was loaded into the xylem sap and 399
transported from root to shoot (Fig S5AB FigS6F) This is also reflected as increased 400
shootroot ratios of N traced by [15N] and [NO3-] (Fig 4GH Fig 5GH Fig S6GH) 401
Interestingly the expression of both BnNRT15 and AtNRT15 is up-regulated by 402
increased cytosolic NO3- concentration in roots but were not affected by NO3
- sequestered 403
into vacuoles (Fig 4 Fig 5 Fig S6) This is because vacuolar NO3- is functionally separated 404
by the tonoplast and cannot be assimilated by enzymes that are localized in the cytosol 405
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15
Therefore NO3--inducible genes such as NR and NRT15 were not influenced by vacuolar 406
NO3- concentration (Martinoia et al 1981 Martinoia et al 2000 Zhang et al 2012) In 407
addition NO3- concentration in the cytosol is controlled by its short-distance transport through 408
the tonoplast which is mediated through the tonoplast proton-pumps providing the required 409
energy for its sequestration into vacuoles against concentration gradient (Gaxiola et al 2001 410
Brux et al 2008 Krebs et al 2010) The gradient of H+ between the inside of the vacuole 411
and the cytosol is maintained by an 2NO3-1H+ antiporter mechanism that facilitatesNO3
- 412
transport through CLCa (Angeli et al 2006 Wege et al 2014) Our data provide clear 413
evidence that NO3- long-distance transport from root to shoot is regulated by cytosolic NO3
- 414
concentration in roots 415
Regulatory mechanisms that control the expression of NRT18 and NRT15 as mediated 416
by NO3- are still unknown however previous studies showed that their expression is also 417
affected by abiotic stresses whereby NRT15 is down-regulated and NRT18 is up-regulated 418
(Li et al 2010 Zhang et al 2014) AtNRT18 expression in A thaliana nrt15 mutants is 419
highly up-regulated (Chen et al 2012) as was also observed here (Fig 3C) Moreover 420
AtNRT15 expression was enhanced by NO3- in culture solution and was not affected in 421
nrt18-2 mutant (Fig 3AB) On the other hand AtNRT18 expression was not affected by 422
cytosolic NO3- but down-regulated when AtNRT15 expression was up-regulated by NO3
-(Fig 423
S6DE) Analogous results were also obtained with B napus genotypes (Fig 3EF) Based on 424
these results we argue that NRT18 expression is not directly influenced by cytosolic NO3- but 425
rather by NRT15 expression (Fig 4 Fig 5 and Fig S6) suggesting NRT18 is probably 426
acting downstream of NRT15 Apparently the regulatory mechanisms of this NRT gene 427
family still awaits further studies to be elucidated 428
429
Response of NUE to NO3- long-distance transport 430
NO3- assimilation efficiency is higher in shoot than in root tissues and can be further 431
enhanced by carbon assimilation (Smirnoff et al 1985 Tang et al 2013) Therefore 432
translocation of higher proportion of NO3- from roots to shoots likely contributes to better 433
crop growth and higher NUE (Andrews et al 1986 Tang et al 2012) The present study 434
provides direct evidence for these results using contrasting B napus genotype identified 435
before (Zhang et al 2009 Han et al 2015a) Our data confirmed the genetic variation in 436
NUE between these two genotypes (Table 1) 437
We measured NO3- distribution in root and shoot of these genotypes to elucidate the 438
physiological mechanisms contributing to variation in NUE The data were consistent with 439
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16
our hypothesis that concentration of NO3- in the xylem sap and shoot would be significantly 440
higher in the H genotype than in the L genotype (Fig 2EFFig S2EF Fig S4) The NO3- 441
long-distance transport from root to shoot was affected by cytosolic NO3- in roots of both A 442
thaliana and B napus genotypes (Fig 4-5 Fig S6) The activities of the tonoplast 443
proton-pumps in roots were significantly lower in the H genotype than in the L genotype (Fig 444
1AB) Consequently less NO3- was sequestered into the vacuole and more NO3
- was retained 445
in cytosol (Fig 1CD) BnNRT15 expression was then up-regulated resulting in greater 446
loading into the xylem while BnNRT18 was down-regulated (Fig 2AB) probably to 447
suppress NO3- unloading from xylem sap Consequently a higher proportion of NO3
- was 448
transported from root to shoots in the H genotype(Fig 2C-D Fig S4) 449
Moreover photosynthetic carbon fixation (Table 2) and activities of both NR and GS 450
were higher in shoots of the H genotype (Fig S7) both of which promote higher N 451
assimilation These results agreed with previous reports (Smirnoff et al 1985 Andrews et al 452
1986 Tang et al 2012 Tang et al 2013) where higher NO3- transport from root to shoot 453
higher photosynthetic rate and N assimilation efficiency in shoots were suggested as essential 454
mechanisms for high-NUE in crop plants These qualities endowed the B napus H genotype 455
Xiangyou15 with higher NUE as compared with the L genotype 814 456
NO3- long-distance transport in B napus is not only regulated by BnNRT15 and 457
BnNRT18 in root tissues (Fig 4 Fig 5 Fig S6) but also by stomatal conductance and 458
transpiration by indirectly controlling xylem sap flow and long-distance transport 459
(Dechorgnat et al 2011 Krapp et al 2014) Stomatal conductance and transpiration rates of 460
the H genotype were significantly higher than those of the L genotype (Table S1) a result 461
consistent with other studies describing characteristics of high-NUE plants (Daniel-Vedele et 462
al 1998 Wilkinson et al 2007 Dechorgnat et al 2011 Krapp et al 2014) 463
464
Response of NUE to NO3- short-distance transport 465
NUE is also strongly controlled by NO3- short-distance distribution between the vacuole 466
and cytosol (Han et al 2015a) A lower proportion of NO3- accumulating in vacuoles and a 467
higher proportion retained in the cytosol will contribute to better NUE in crop plants (Han et 468
al 2015b) The energy required for vacuolar NO3- sequestration is provided by the tonoplast 469
proton-pumps (Gaxiola et al 2001 Brux et al 2008 Krebs et al 2010) while the 470
distribution channel CLCa enables transport of NO3- across the vacuolar membrane (Angeli et 471
al 2006 Wege et al 2014) The activities of the tonoplast proton-pumps (V-ATPase and 472
V-PPase) in leaves of the H genotype were significantly lower than those of the L genotype at 473
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17
flowering stages (Han et al 2015aHan et al 2015b) thus we expected similar differences in 474
root tissues Our results confirmed that the activities of tonoplast proton-pumps in root tissues 475
of B napus H genotype and A thaliana mutants were lower than those in the L genotype and 476
A thaliana wild type leading to less NO3- influx into vacuoles and more in the cytosol (Fig 1 477
Fig 5A-D) Moreover the enzymes catalyzing N assimilation are located in the cytoplasm 478
where NO3- could be assimilated (Martinoia et al 1981 Han et al 2015a) or immediately 479
loaded into xylem sap for long-distance transport from roots to shoots (Lin et al 2008) 480
Consequently the NO3- concentration in root tissues of the H genotype and A thaliana 481
mutants defective in tonoplast proton-pumps was significantly lower than that in the L 482
genotype (Fig 2E) and in A thaliana wild type (Fig 6C) Therefore the higher proportion of 483
NO3- transported to shoot and the lower concentrations retained in root vacuoles contributed 484
to the higher NUE in Xiangyou15 and A thaliana mutants (vha-a2 vha-a3 and avp1) (Table 485
1Fig 6A) Further studies are needed to assess the impacts of regulating vacuolar 486
sequestration of NO3- and rates of long-distance transport for enhancing NUE in other crop 487
species and genotypes 488
These data suggested that NO3- distribution between vacuoles and cytosol within cells 489
regulates NO3- long-distance transport from root to shoot and ultimately affects NUE NO3
- 490
loading into the xylem sap is an active transport process (Lin et al 2008) therefore not 491
regulated by absolute concentration of NO3- in the cytosol but rather by genes involved in 492
NO3- long-distance transport (NRT15 and NRT18) Apparently the expression of NRT15 the 493
gene responsible for xylem loading is regulated by NO3-concentration in the cytosol 494
Therefore both NO3- short-distance transport across the tonoplast and its long-distance 495
transport from root to shoot are important determinants of NUE in both B napus and A 496
thaliana (Table 1 and Fig 6) 497
498
Roles of NRT15 and NRT18 in NO3- long-distance transport in B napus and A 499
thaliana 500
Our results (Figs S9 S10) showed that the proportion of NO3- distributed from root to 501
shoot is strongly controlled by NRT15 in accord with previous studies (Lin et al 2008 Li et 502
al 2010) The data suggest that the high-NUE genotypes possess lower activities of tonoplast 503
proton-pumps (V-ATPase and V-PPase) resulting in less NO3- accumulation in vacuoles and 504
more NO3- retention in cytosol (Fig 7) The higher NO3
- in the cytosol then up-regulated 505
NRT15 which then down-regulates NRT18 As a result more NO3- is loaded into the xylem 506
system by NRT15 mechanism and less NO3- is unloaded via NRT18 (Fig 7) Differential 507
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18
regulation of these NRT genes in high-NUE plants increased transport of NO3- from root to 508
shoot through the xylem vascular tissues (Fig 7) This coordinated NO3- long- and 509
short-distance transport likely fine-tunes NO3- allocation and modulates the balance of NO3
- 510
distribution between roots and shoots (Fig 7) 511
Our research also revealed differences between A thaliana and B napus in the 512
expression of the wild-type NRT genes in response to NO3- in the protoplast Moreover using 513
A thaliananrt15-3 mutants we uncovered possible regulatory interactions between a 514
functioning (wild-type) NRT15 gene and NRT18 expression in response to NO3- 515
concentration Because mutants defective in nrt15 are not available for B napus we could 516
not confirm that the respective NRT15 and NRT18 systems behave in an analogous way in 517
these plant species Based on Harper et al (2012) research database our homology 518
comparisons and gene function annotations provided strong evidence for the roles of 519
BnNRT15 and BnNRT18 in B napus Finally our studies were conducted using two model 520
dicotyledonous plant species for which we had ample data on responses to specific inhibitors 521
(B napus) or specific mutants were available with results forming the bases for the model 522
presented in Figure 7 Further research using genetically diverse plant species (eg 523
monocotyledonous crops such as rice wheat and corn) will help validate this model and its 524
broader application across species The information generated in this study could have future 525
application for improving NUE in commercial crops with consequent reduction in nitrogen 526
use and benefits to the environment 527
528
Materials and Methods 529
Plant material 530
The two oilseed rape (Bnapus) cultivars used in this study have been characterized 531
before as high- (Xiangyou15 referred to as H genotype hereafter) and low- (814 referred to 532
as L genotype) nitrogen use efficiency (NUE) genotypes (Zhang et al 2009 Han et al 2015b) 533
Here we define NUE as the total biomass per unit of N uptake by the plant with total 534
biomass includes roots shoots and grains The two genotypes were provided by the Hunan 535
Sub-Center of Improvement Center of National Oil Crops Hunan China 536
The Arabidopsis thaliana wild-type Columbia-0 (col-0) was used as control for 537
V-ATPase (vha-a2 and vha-a3) V-PPase (avp1) nrt15-3 and nrt18-2 mutants whereas the A 538
thaliana wild-type Wassilewskija (Ws) was used as control for clca-2 mutants A thaliana 539
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19
mutants and transgenic lines (nrt15-3 and nrt18-2) have been described previously (Chen et 540
al 2012 Zhang et al 2014) The mutant lines vha-a2 (Salk_142642) and vha-a3 541
(Salk_122135) described by Krebs et al (2010) were obtained from the Arabidopsis 542
Biological Resources Center (ABRC) The mutant line avp1 (GK-005004) described in Li et 543
al (2005) was obtained from the European Arabidopsis Stock Centre while the mutant line 544
clca-2 (FST 171A06) described in Angeli et al (2006) was from the Institute National de la 545
Recherche Agronomique (INRA) collection in France together with the T-DNA mutants used 546
for genotyping to select pure mutant lines 547
548
Growth conditions 549
B napus plants were grown hydroponically in ceramic pots(20cmtimes15cm) filled with a 550
nutrient solution in the greenhouse under natural light as described in Han et al (2015b) The 551
pots were arranged in a completely randomized design with six biological replications The 552
nutrient solution was replaced every 3 days and its pH adjusted to 55 daily Experiments were 553
conducted at the field station of Hunan Agricultural University Southern China 554
A thaliana plants were grown in a nutrient solution in plastic pots as described in Arteca 555
et al (2000) and Gong et al (2003) The solution was changed every 3 days with pH adjusted 556
daily to 58 Pots were arranged in a completely randomized design with six biological 557
replications The nutrient solution used for both species consisted of 125mM KNO3 558
0625mM KH2PO4 05mM MgSO4 05mM Ca (NO3)2middot4H2O 0025mM Fe-EDTA 025 559
mlL-1 micronutrients (stock solution concentrations 70mM B 14mM Mn 1mM Zn 05mM 560
Cu and 02mM Mo) The experiments were conducted at Hunan Agricultural University in a 561
phytotron set at 70 relative humidity16h-light8h-dark cycle and constant temperature of 562
22degC 563
564
Experimental treatments and phenotyping 565
All measurements made on B napus plants were conducted either at the seedling stage 566
(2 months after transplanting) or at flowering (5 months after transplanting) The whole root 567
tissue was harvested and used for analyses at either stage For shoot measurements the fourth 568
leaf from the bottom was used for measurements made at seedling stage whereas the 12th leaf 569
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
from the bottom was harvested for measurements made at flowering stage For A thaliana 570
whole root and shoot of four-week-old plants were sampled and used for different assays 571
572
Vacuolar proton-pumps inhibitor treatments 573
Inhibition of vacuolar proton pumps of B napus (Xiangyou15) was conducted at the seedling 574
stage in hydroponic culture as described in Han et al (2015a) with minor modifications 575
Plants grown in hydroponic solution were used as control Inhibitor treatment was conducted 576
using several chemicals Bafi(25 nmol L-1 Bafilomycin A1) an inhibitor of V-ATPase 577
DCCD(10 μmol L-1DCCD + 50 mmolL-1 Na2SO3) specifically inhibits V-PPase and 578
Bafi+DCCD in a 11 ratio as inhibitor of both V-ATPase and V-PPase The inhibitors were 579
applied in the hydroponic solutions for 24 h Fresh plant tissue samples from all treatments 580
were collected for different assays 581
582
NO3- induced gene expression 583
Four-week old A thaliana mutants (nrt15-3nrt18-2) were grown in hydroponics with 584
225mM (NH4)2succinate for 3 days and shifted to hydroponics with 45mM NO3- for 12 h 585
then root tissues were collected to assess relative expression of AtNRT15 and AtNRT18 586
genes Seedlings of B napus (Xiangyou15) plants grown hydroponically were treated with 587
5mM (NH4)2succinate for 3 d and shifted to fresh hydroponic solution containing 15 mM 588
NO3- for 12 h Root tissue was then collected to assess the relative expression of BnNRT15 589
and BnNRT18 genes using quantitative RT-PCR 590
591
ACC treatment 592
Hydroponically grown seedlings of B napus were subjected to ACC treatment as described in 593
Zhang et al (2014) with minor modifications Seedlings of the two genotypes Xiangyou15 594
and 814 were transferred to a nutrient solution containing 002mM L-1 ACC for 6 h or grown 595
in normal hydroponic solution as control Root tissues were then collected to assay relative 596
expression of BnNRT15 and BnNRT18 using quantitative RT-PCR Another set of B napus 597
plants were grown in ACC-containing hydroponic solution but supplemented with 15NO3- (22 598
15N) replacing the unlabeled NO3- for 1 h Root and shoot tissues were collected separately 599
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21
for 15N measurement using a continuous-flow isotope ratio mass spectrometer coupled with a 600
C-N elemental analyzer (ANCA-MS PDZ Europa) 601
602
Biomass and N concentration 603
Grain yield dry biomass and N concentration were determined in plant samples at 604
harvest Plants were harvested (including senescing leaves) then dried in an oven for 30 min 605
at 105degC then at 70degC to a constant weight and weighed N concentration was determined 606
using the Kjeldahl method (Shi et al 2010 Han et al 2015b)02g of dried samples were 607
digested with H2SO4-H2O2 in a 100-ml Kjeldahl digestion flask after which N concentration 608
was determined using a Foss Auto Analyser Unit (Kjeldahl 8400) 609
610
V-ATPase and V-PPase activities 611
Roots (10 g) of B napus were collected at seedling and flowering stages and that of A 612
thaliana (05 g) sampled at seedling stage then used for determining the activities of 613
V-ATPase and V-PPase (Han et al 2015b) Activities of these two energy pumps within 614
microsomal membranes were followed colorimetrically based on released Pi as described by 615
Zhu et al (2001) and Krebs et al (2010) 616
617
Assay of NO3- flux in vacuoles 618
Roots of both species were collected similarly as that used for V-ATPase and V-PPase 619
activities then used for vacuole isolation and measurement of NO3- flux in vacuoles as 620
described by Robert et al (2007) with minor modification (Han et al 2015b) Net fluxes of 621
NO3- in vacuoles were measured non-invasively using SIET (scanning ion-selective electrode 622
technique SIET system BIO-003A Younger USA Science and Technology Corporation 623
USA) An NO3--selective microelectrode used for the assay of NO3
- flux was vibrated in the 624
measuring solution between two positions 1 μm and 11 μm from the vacuole surface 625
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles Background 626
signal was recorded by vibrating the electrode in the measuring solution in the absence of 627
vacuoles Ion flux was calculated by Fickrsquos law of diffusion J = -D(dcdx) where J represents 628
the ion flux (picomolescm-2s-1) dcdx is the ion concentration gradient and D is the ion 629
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
diffusion constant in a particular medium The direction of the flux was derived from Fickrsquos 630
law of diffusion that relates the concentration gradient 631
632
Isolation of intact protoplasts and vacuoles for determining NO3- concentration 633
Similar to the above assays root tissues (10 g) were collected from B napus at seedling 634
and flowering stages and from A thaliana (05 g) seedlings and used to isolate intact 635
protoplasts and vacuoles as described by Robert et al (2007) with minor modifications as 636
outlined in Huang et al (2012) and Han et al(2015b) The purified protoplasts were divided 637
into two equal aliquots with one of them used for isolation of vacuoles The purified 638
protoplasts and vacuoles were then sub-sampled and used to determine NO3- concentrations 639
(Vogeli-Lange and Wagner 1990) and for enzyme activity assays (Ma et al 2005) 640
NO3-concentrations in protoplasts and vacuoles were measured by a continuous-flow 641
auto-analyzer (Auto Analyser 3 Bran and Luebbe Germany) as described previously (Han et 642
al 2015b)The activities of acid phosphatase (ACP) and cytochrome oxidase (COX) were 643
determined using plant ACP colorimetry- and COX-assay kits (GenMedSci Inc) following 644
the manufacturerrsquos instructions ACP activity specific to vacuoles was determined and used to 645
normalize NO3- accumulation We measured NO3
- in the protoplast outside the vacuole which 646
includes the cytosol and organelles eg mitochondria and Golgi Apparatus (Robert et 647
al2007) Since most of the NO3- in the protoplast outside the vacuole is located in the cytosol 648
(Krebs et al 2010) we refer to NO3- distribution between vacuoles and cytosol rather than 649
vacuole versus protoplast 650
651
Quantitative RT-PCR 652
Total RNA extracted from B napus roots was prepared using Trizol reagent (Invitrogen) 653
cDNAs were synthesized using M-MLV reverse transcriptase (Promega) following 654
manufacturerrsquos protocol The relative expression of BnNRT15 (EV220114) BnNRT18 655
(EV116423) and Bnactin (AF1118121) genes in plant roots were determined by quantitative 656
RT-PCR and run on a LightCycler instrument (Roche) with the SYBR Green Real-Time PCR 657
Master Mix Kit (TOYOBO Japan) under the following conditions 95degC for 2min then 45 658
cycles of 95degC for 10s 60degC for 10s and 72degC for 20s The primer sequences of BnNRT15 659
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
BnNRT18 and Bnactin genes were obtained from httpbrassicanbiacuk as described in 660
Harper et al (2012) and expression levels were normalized to Bnactin as control 661
Total RNA extracted from the 4-week-old A thaliana roots under the treatments 662
conditions described above were prepared using Trizol reagent (Invitrogen) The cDNAs were 663
synthesized using M-MLV reverse transcriptase (Promega) based on the manufacturerrsquos 664
protocol and as described in Li et al (2010)The primer sequences of quantitative RT-PCR 665
used in these assays are listed in Supplemental Table 2 and were described previously (Chen 666
et al 2012 Zhang et al 2014) and their expression was normalized to Atactin2 as control 667
668
Determination of nitrate distribution using 15NO3- 669
Hydroponically grown B napus (seedling and flowering stages) and A thaliana 670
(seedling) were transferred to 01mM CaSO4 solution for 1min then to their respective 671
hydroponic nutrient solutions with15NO3- (22 excess) replacing unlabeled NO3
- for 1h 672
Plants were then transferred to 01mM CaSO4 solution for 1min after which roots were 673
washed with deionized water Root and shoot samples were separated and dried at 105degC for 674
30min followed by 70degC for 3 d then grounded and used for assaying 15N content using a 675
continuous-flow isotope ratio mass spectrometer coupled with a carbon-nitrogen elemental 676
analyzer (ANCA-MS PDZ Europa) 677
678
Xylem sap collection and assay of nitrate concentration 679
We used the method of Tang et al (2012) for collecting xylem sap Plants were cut 680
leaving1cm segments with intact roots at seedling and flowering stages for B napus and at 681
seedling stage for A thaliana Roots were immediately immersed in their respective nutrition 682
solutions Weighed cotton was put on the cut surface to absorb extruding xylem sap for 1 h 683
The cotton was then wrapped in plastic film and the volume of xylem sap was calculated as 684
the weight gain of the cotton Xylem sap was then squeezed from the cotton using a syringe 685
and used for subsequent assay of nitrate concentration using a continuous-flow auto-analyzer 686
(Auto Analyser 3 Bran and Luebbe Germany) 687
To determine nitrate concentration samples (10 g fresh root and shoot at seedling and 688
flowering of B napus and 05 g fresh root and shoot of 4-week-old A thaliana) were frozen 689
in liquid N2 and ground with a mortar and pestle the powder was then transferred to a beaker 690
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
containing 70 ml deionized water and boiled for 30 min to extract nitrate then cooled and 691
made to 100 ml volume and 02 g activated carbon added to eliminate the effect of 692
chlorophyll The mixture was filtered and nitrate in the filtrate was determined using a 693
continuous-flow auto-analyser (Atuo-Analyser 3 Bran and Luebbe Germany) 694
695
Measurements of photosynthesis 696
All measurements of photosynthesis and related parameters were conducted at 1000 h 697
using intact leaves The 4th leaf from the bottom in B napus at seedling stage and the 12th leaf 698
from the bottom at flowering stage were used for determining chlorophyll concentration 699
photosynthetic rate intercellular CO2 concentration transpiration rate and stomatal 700
conductance Chlorophyll concentration was determined by SPAD-502 (Minolta Camera Co 701
Ltd Japan) (Luo et al 2006 Yandeau-Nelson et al 2011) Photosynthetic rate intercellular 702
CO2 concentration transpiration rate and stomatal conductance were measured using LI-6400 703
Portable Photosynthesis System (Li-Cor Biosciences Co Ltd USA) set at flow rate of 500 704
μmol s-1 photosynthetic photon flux density of 1000 μmol m-2 s-1 relative humidity of 65 705
CO2 concentration of 400ppm and ambient temperatures 706
707
NR and GS activities 708
Leaves with similar age as those used for photosynthesis in B napus were used for the 709
assay of nitrate reductase (NR) and glutamine synthetase (GS) activities NR activity was 710
measured by the modified method of Fan et al (2007) Fresh samples (05g) were frozen in 711
liquid N2 ground into powder in the presence of acid-washed sand and homogenized with 4 712
ml of extraction buffer (0025mol L-1phosphate buffer pH87 1211g L-1 cysteine 0372g L-1 713
EDTA) Homogenates were centrifuged at 30000timesg for 15 min at 4degC and the supernatants 714
were treated with sulfanilamide and α-naphthylamine reagents for colorimetric (540nm) 715
determination of nitrite as described by Fan et al (2007) 716
GS activity was determined using a modified reverseγ-glutamyltransferase method 717
(Wang et al2014) which measures the GS-catalyzed formation of glutamyl-γ-hydroxamate 718
from glutamine and hydroxylamine Fresh samples were frozen for 30 min at -20degC then 719
ground in 10ml Tris-HCl buffer and acid-washed sand The homogenates were filtered 720
through two layers of gauze and centrifuged at 8000timesg for 15 min at 4degC About 12ml of the 721
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
supernatant was added in a reaction mixture and treated as described (Wang et al2014) After 722
the GS-catalyzed reaction was stopped and the solution centrifuged glutamyl-γ723
-hydroxamate was quantified colorimetrically in the supernatant (at 485 nm) and the 724
concentration was determined using a standard curve 725
726
Statistical analyses 727
We used the SPSS software (Statistical Product and Service Solutions V130 USA) for 728
ANOVA and mean separation of main-effects and interactions using Duncanrsquos multiple range 729
test at Plt005Values are presented as means and SD of three or six replicates from three 730
independent experiments Different letters or an asterisk () associated with specific data (eg 731
at the top of histogram bars in figures or within tables) denote significant differences at 732
Plt005 733
734
Supplemental Data 735
Supplemental Figure 1 B napus with higher NUE showed lower vacuolar sequestration 736
capacity (VSC) for NO3-in roots at flowering stage 737
Supplemental Figure 2 B napus with higher NUE showed enhanced long-distance transport 738
of NO3- from roots to shoots at flowering stage 739
Supplemental Figure 3 The two B napus (H and L genotypes) showed the same total N per 740
plant at seedling stage (A) and flowering stage (B) 741
Supplemental Figure 4 B napus with higher NUE showed increased NO3- concentration in 742
the xylem sap at seedling and flowering stages 743
Supplemental Figure 5 NO3- concentration in the xylem sap as affected by inhibitor 744
treatments in B napus and in the energy pumpsrsquo mutants of A thaliana (col-0 vha-a2 vha-a3 745
avp1) 746
Supplemental Figure 6 Reduced VSC for NO3- in roots drives long-distance transport of 747
NO3- from roots to shoots in the A thaliana wild type (Ws) and mutant (clca-2) 748
Supplemental Figure 7 Differences of NR and GS activities between the two B napus (H 749
and L genotypes) at seedling and flowering stages 750
Supplemental Figure 8 Amino acid sequences of BnNRT15 and BnNRT18 751
Supplemental Figure 9 Functions of AtNRT15 and AtNRT18 genes in root tissues of A 752
thaliana in controlling NO3- long-distance transport from root to shoots 753
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
Supplemental Figure 10 B napus BnNRT15 and BnNRT18 genes in roots were 754
coordinately modulated to facilitate NO3- long distance transport from roots to shoots 755
Supplemental Table 1 Differences in stomatal conductance and transpiration rate between 756
the two B napus genotypes 757
Supplemental Table 2 Sequences of primera used for qRT-PCR 758
759
Acknowledgements 760
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 761
Shanghai Institutes for Biological Sciences) for providing nrt15-3 and nrt18-2 seeds 762
This study was supported by the National Natural Science Foundation of China (Grant 763
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 764
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 765
The Ministry of Education in China Open Novel Science Foundation of Hunan province 766
(13K062) the National Key Laboratory of Plant Molecular Genetics and the Twelfth 767
Five-Year National Science and technology support program (2012BAD15BO4) 768
769
Figure 1 B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 770
NO3- in roots at the seedling stage 771
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 772
genotype 814 Specific activities of the tonoplast proton pumps are expressed as μmol Pi 773
released mg-1 protein h-1 NO3- fluxes are expressed as pmol NO3
- cm-2 S-1 Mature vacuoles 774
were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 775
measuring solution between the two positions 1 μm and 11 μm from the vacuole surface 776
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles recording the 777
stable reading data The background was recorded by vibrating the electrode in measuring 778
solution without vacuoles 779
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 780
for NO3- content and NO3
- accumulation normalized against the specific activity of the 781
vacuole acid phosphatase (ACP) as described in Materials and Methods and plotted as μmol 782
NO3- per μmol p-nitrophenol the end product of ACP assay 783
Proton pump activities in root tissues between H and L genotypes are shown at the seedling 784
stage (A) Different letters at the top of the histogram bars denote significant differences of 785
V-ATPase in root tissues between H and L genotypes (Plt005) an asterisk () at the top of the 786
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
histogram bars indicates significant difference in V-PPase activity in root tissues between H 787
and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788
Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789
genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790
bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791
bars on the figures indicate SD (n=6) 792
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793
seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794
to the total NO3- in protoplasts 795
NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796
total NO3-in the cytosol and was calculated as total NO3
- in protoplasts ndash total NO3- in 797
vacuole Different letters at the top of histogram bars denote significant differences of total 798
NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799
(n=6) 800
801
Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802
roots to shoots at the seedling stage 803
H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804
Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805
the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806
as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807
the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808
the histogram bars denote significant differences at Plt005 809
Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810
in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811
genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812
of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813
SD (n=3) different letters at the top of the histogram bars denote significant differences at 814
Plt005 level 815
The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816
at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817
genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818
different letters at the top of the histogram bars denote significant differences at Plt005 819
820
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
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Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
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b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
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Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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11
Similar trends were also observed when using mutants of A thaliana deficient in the energy 302
pumpsrsquo activities NO3- concentration in the xylem sap the [15N] SR ratios and [NO3
-] SR 303
ratios were all significantly higher in the mutants than in the wild type (Fig 5GH Fig S5B) 304
These data clearly showed that prevention of N sequestration in vacuoles would enhance its 305
translocation to shoot 306
Additional evidence linking NO3- long-distance transport and NO3
- short-distance 307
distribution within cells were provided through experiments comparing A thaliana wild type 308
(Ws) and mutant clca-2 (Fig S6) The chloride channel (CLCa) is the main channel for 309
vacuolar anion accumulation Vacuolar sequestration capacity of NO3-significantly declines in 310
clca mutants (Angeli et al 2006) NO3- influx into the vacuolar space of Ws roots was 311
significantly higher than that observed in the clca-2 mutants (Fig S6A) This resulted in a 312
smaller proportion of vacuolar NO3- in clca-2 plants relative to the total NO3
- in root tissue 313
protoplasts as compared with Ws (Fig S6B) Consequently the accumulation of NO3- in the 314
cytosol was significantly higher in clca-2 than in Ws plants (Fig S6C) The higher 315
NO3-concentration in the cytosol together with the higher expression of AtNRT15 coupled 316
with lower expression of AtNRT18 in clca-2 roots (Fig S6DE) resulted in significant 317
increase in xylem sap NO3- concentration [15N] SR ratios and [NO3
-] SR ratios compared 318
with Ws plants (Fig S6FGH) 319
320
Increased NO3- translocation to shoots enhanced NUE 321
NO3- assimilation efficiency is known to be higher in shoots than in roots (Smirnoff et al 322
1985 Andrews et al 1986 Tang et al 2012 Tang et al 2013) We therefore hypothesize 323
that increased NO3- translocation to shoot and the consequent higher shootroot ratio will 324
contribute to higher NUE Our data support this hypothesis The H B napus genotype showed 325
enhanced long-distance transport of NO3- from roots to shoots (Fig 2 Fig S2) This enhanced 326
NO3- transport requires higher carbon skeleton provided through higher photosynthetic rate 327
(Tang et al 2012 Tang et al 2013) Our results showed that chlorophyll content 328
intercellular CO2 concentration and photosynthetic rate were significantly higher in the H than 329
the L genotype at both seedling and flowering stages (Table 2) Moreover the NO3- 330
assimilating enzymes were strongly induced by NO3- providing sufficient capacity for N 331
assimilation (Smirnoff et al 1985 Andrews et al 1986) Nitrate reductase (NR) and 332
glutamine synthetase (GS) activities in roots of the H genotype were significantly lower than 333
in the L genotype both at seedling and flowering stages (Fig S7) In contrast NR activities in 334
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12
shoots of the H genotype were significantly higher than in the L genotype at both stages (Fig 335
S7AB) and GS activity in the H genotype was also higher than those of the L genotype at 336
seedling stage (Fig S7C) 337
Experiments comparing A thaliana wild type (col-0 Ws) with mutants defective in 338
vacuolar proton-pumps (vha-a2 vha-a3avp1) and transport channel (clca-2) yielded similar 339
results that is increases in NO3- shootroot ratio essentially contributed to enhanced NUE 340
(Fig 6) The NO3- shootroot ratios in A thaliana mutants were higher than that in the wild 341
type (Fig 5GH) Generally the efficiency of inorganic N (NO3-) assimilation into organic N 342
is higher in shoots than in roots (Smirnoff et al 1985 Andrews et al 1986) therefore the 343
NO3- concentration in A thaliana mutants decreased (Fig 6CD) leading to higher NUE 344
compared with the wild type (Fig 6AB) 345
346
BnNRT15BnNRT18 in B napus affects NO3- long-distance transport from roots to 347
shoots 348
Both B napus and A thaliana are members of Cruciferae Family The amino acid 349
sequence identity between BnNRT15 and AtNRT15 was 90 (Fig S8A) the amino acid 350
sequence similarity between BnNRT18 and AtNRT18 was 908 (Fig S8B) Comparisons of 351
nucleotide and amino acid sequences (Harper et al2012) of these genes 352
(httpbrassicanbiacukcgi-binmicroarray_databasecgi) showed that BnNRT15 353
(EV220114) and BnNRT18 (EV116423) of B napus are respectively highly homologous 354
with AtNRT15 and AtNRT18 and the two genes are mainly expressed in roots of both species 355
showing similar organ-specificity (Lin et al 2008 Li et al 2010) 356
NO3- long-distance transport from roots to shoots is therefore regulated by NRT15 and 357
NRT18 as reported before (Lin et al 2008 Li et al 2010) The [15N-traced] SR ratios and 358
the [NO3-] SR ratios were significantly lower in nrt15-3 mutants relative to the wild type 359
(col-0) while [15N] SR and [NO3-] SR ratios showed significant increase in nrt18-2mutant 360
relative to col-0 (Fig S9CD) A previous study showed that expression of NRT15 is 361
down-regulated by ACC and MeJA treatments while the expression of NRT18 is strongly 362
up-regulated in A thaliana (Zhang et al 2014) Consequently NO3- accumulated in plant 363
roots probably as an adaptive measure for abiotic stresses (Chen et al 2012) Both the H- 364
and L- B napus genotypes showed significantly lower expression of BnNRT15 in roots when 365
treated with ACC (Fig S10A) but the expression of BnNRT18 remained higher than in the 366
control plants (Fig S10B) This resulted in significantly lower [15N] SR ratios following 367
ACC treatment (Fig S10CDE) These data indicate that BnNRT15 and BnNRT18 play 368
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13
similar functions for NO3- long-distance transport between root and shoot in both B napus 369
and A thaliana (Lin et al 2008 Li et al 2010) 370
371
372
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14
Discussion 373
Nitrate long-distance transport from root to shoot is regulated by cytosolic NO3- in roots 374
NO3- long-distance transport from root to shoot is controlled by NRT gene family For 375
instance xylem NO3- loading and unloading are mainly controlled by NRT15 and NRT18 376
respectively (Lin et al 2008 Li et al 2010) Whereas the NO3- short-distance transport 377
between the vacuole and cytosol is controlled by tonoplast proton-pumps (V-ATPase and 378
V-PPase) (Krebs et al 2010) and by activity of the CLCa channel in the tonoplast membrane 379
(Angeli et al 2006 Wege et al 2014) tonoplast proton-pumps provide the energy required 380
for NO3- accumulation into the vacuole through CLCa (Gaxiola et al 2001 Brux et al 2008 381
Krebs et al 2010) The expression of NRT15 is up-regulated by NO3- in plant culture 382
solution (Lin et al 2008) whereas regulation of NRT18 expression is contrary to that of 383
NRT15 under both control and abiotic stress conditions (Li et al 2010 Chen et al 2012 384
Zhang et al 2014) 385
Despite the progress made in characterizing the genes involved in transport and 386
metabolism of NO3- in plants the relationship between long- and short-distance transport and 387
how these two pathways are regulated have not been thoroughly investigated We hypothesize 388
that NRT15 expression and its role in long-distance transport is regulated by NO3- in plant 389
tissue as was observed in culture solution (Lin et al 2008) and our data support this 390
hypothesis Both BnNRT15 and AtNRT15 expression were up-regulated by increasing 391
cytosolic NO3- in roots (Fig 4 Fig 5) The increase in cytosolic NO3
- was achieved through (i) 392
use of A thaliana mutants defective in proton-pumps (V-ATPase and V-PPase) or CLCa 393
activities in the tonoplast and (ii) inhibition of B napus tonoplast proton-pumps In both 394
model plants the influx of NO3- into vacuoles was substantially reduced resulting in higher 395
concentrations of NO3- in the cytosol (Fig 4A-D Fig 5A-D Fig S6A-C) Under those 396
conditions expression of BnNRT15 and AtNRT15 was up-regulated by the enhanced 397
cytosolic NO3- while expression of BnNRT18 and AtNRT18 was down-regulated (Fig 4EF 398
Fig 5EF FigS6DE) As a consequence more NO3- was loaded into the xylem sap and 399
transported from root to shoot (Fig S5AB FigS6F) This is also reflected as increased 400
shootroot ratios of N traced by [15N] and [NO3-] (Fig 4GH Fig 5GH Fig S6GH) 401
Interestingly the expression of both BnNRT15 and AtNRT15 is up-regulated by 402
increased cytosolic NO3- concentration in roots but were not affected by NO3
- sequestered 403
into vacuoles (Fig 4 Fig 5 Fig S6) This is because vacuolar NO3- is functionally separated 404
by the tonoplast and cannot be assimilated by enzymes that are localized in the cytosol 405
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15
Therefore NO3--inducible genes such as NR and NRT15 were not influenced by vacuolar 406
NO3- concentration (Martinoia et al 1981 Martinoia et al 2000 Zhang et al 2012) In 407
addition NO3- concentration in the cytosol is controlled by its short-distance transport through 408
the tonoplast which is mediated through the tonoplast proton-pumps providing the required 409
energy for its sequestration into vacuoles against concentration gradient (Gaxiola et al 2001 410
Brux et al 2008 Krebs et al 2010) The gradient of H+ between the inside of the vacuole 411
and the cytosol is maintained by an 2NO3-1H+ antiporter mechanism that facilitatesNO3
- 412
transport through CLCa (Angeli et al 2006 Wege et al 2014) Our data provide clear 413
evidence that NO3- long-distance transport from root to shoot is regulated by cytosolic NO3
- 414
concentration in roots 415
Regulatory mechanisms that control the expression of NRT18 and NRT15 as mediated 416
by NO3- are still unknown however previous studies showed that their expression is also 417
affected by abiotic stresses whereby NRT15 is down-regulated and NRT18 is up-regulated 418
(Li et al 2010 Zhang et al 2014) AtNRT18 expression in A thaliana nrt15 mutants is 419
highly up-regulated (Chen et al 2012) as was also observed here (Fig 3C) Moreover 420
AtNRT15 expression was enhanced by NO3- in culture solution and was not affected in 421
nrt18-2 mutant (Fig 3AB) On the other hand AtNRT18 expression was not affected by 422
cytosolic NO3- but down-regulated when AtNRT15 expression was up-regulated by NO3
-(Fig 423
S6DE) Analogous results were also obtained with B napus genotypes (Fig 3EF) Based on 424
these results we argue that NRT18 expression is not directly influenced by cytosolic NO3- but 425
rather by NRT15 expression (Fig 4 Fig 5 and Fig S6) suggesting NRT18 is probably 426
acting downstream of NRT15 Apparently the regulatory mechanisms of this NRT gene 427
family still awaits further studies to be elucidated 428
429
Response of NUE to NO3- long-distance transport 430
NO3- assimilation efficiency is higher in shoot than in root tissues and can be further 431
enhanced by carbon assimilation (Smirnoff et al 1985 Tang et al 2013) Therefore 432
translocation of higher proportion of NO3- from roots to shoots likely contributes to better 433
crop growth and higher NUE (Andrews et al 1986 Tang et al 2012) The present study 434
provides direct evidence for these results using contrasting B napus genotype identified 435
before (Zhang et al 2009 Han et al 2015a) Our data confirmed the genetic variation in 436
NUE between these two genotypes (Table 1) 437
We measured NO3- distribution in root and shoot of these genotypes to elucidate the 438
physiological mechanisms contributing to variation in NUE The data were consistent with 439
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16
our hypothesis that concentration of NO3- in the xylem sap and shoot would be significantly 440
higher in the H genotype than in the L genotype (Fig 2EFFig S2EF Fig S4) The NO3- 441
long-distance transport from root to shoot was affected by cytosolic NO3- in roots of both A 442
thaliana and B napus genotypes (Fig 4-5 Fig S6) The activities of the tonoplast 443
proton-pumps in roots were significantly lower in the H genotype than in the L genotype (Fig 444
1AB) Consequently less NO3- was sequestered into the vacuole and more NO3
- was retained 445
in cytosol (Fig 1CD) BnNRT15 expression was then up-regulated resulting in greater 446
loading into the xylem while BnNRT18 was down-regulated (Fig 2AB) probably to 447
suppress NO3- unloading from xylem sap Consequently a higher proportion of NO3
- was 448
transported from root to shoots in the H genotype(Fig 2C-D Fig S4) 449
Moreover photosynthetic carbon fixation (Table 2) and activities of both NR and GS 450
were higher in shoots of the H genotype (Fig S7) both of which promote higher N 451
assimilation These results agreed with previous reports (Smirnoff et al 1985 Andrews et al 452
1986 Tang et al 2012 Tang et al 2013) where higher NO3- transport from root to shoot 453
higher photosynthetic rate and N assimilation efficiency in shoots were suggested as essential 454
mechanisms for high-NUE in crop plants These qualities endowed the B napus H genotype 455
Xiangyou15 with higher NUE as compared with the L genotype 814 456
NO3- long-distance transport in B napus is not only regulated by BnNRT15 and 457
BnNRT18 in root tissues (Fig 4 Fig 5 Fig S6) but also by stomatal conductance and 458
transpiration by indirectly controlling xylem sap flow and long-distance transport 459
(Dechorgnat et al 2011 Krapp et al 2014) Stomatal conductance and transpiration rates of 460
the H genotype were significantly higher than those of the L genotype (Table S1) a result 461
consistent with other studies describing characteristics of high-NUE plants (Daniel-Vedele et 462
al 1998 Wilkinson et al 2007 Dechorgnat et al 2011 Krapp et al 2014) 463
464
Response of NUE to NO3- short-distance transport 465
NUE is also strongly controlled by NO3- short-distance distribution between the vacuole 466
and cytosol (Han et al 2015a) A lower proportion of NO3- accumulating in vacuoles and a 467
higher proportion retained in the cytosol will contribute to better NUE in crop plants (Han et 468
al 2015b) The energy required for vacuolar NO3- sequestration is provided by the tonoplast 469
proton-pumps (Gaxiola et al 2001 Brux et al 2008 Krebs et al 2010) while the 470
distribution channel CLCa enables transport of NO3- across the vacuolar membrane (Angeli et 471
al 2006 Wege et al 2014) The activities of the tonoplast proton-pumps (V-ATPase and 472
V-PPase) in leaves of the H genotype were significantly lower than those of the L genotype at 473
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17
flowering stages (Han et al 2015aHan et al 2015b) thus we expected similar differences in 474
root tissues Our results confirmed that the activities of tonoplast proton-pumps in root tissues 475
of B napus H genotype and A thaliana mutants were lower than those in the L genotype and 476
A thaliana wild type leading to less NO3- influx into vacuoles and more in the cytosol (Fig 1 477
Fig 5A-D) Moreover the enzymes catalyzing N assimilation are located in the cytoplasm 478
where NO3- could be assimilated (Martinoia et al 1981 Han et al 2015a) or immediately 479
loaded into xylem sap for long-distance transport from roots to shoots (Lin et al 2008) 480
Consequently the NO3- concentration in root tissues of the H genotype and A thaliana 481
mutants defective in tonoplast proton-pumps was significantly lower than that in the L 482
genotype (Fig 2E) and in A thaliana wild type (Fig 6C) Therefore the higher proportion of 483
NO3- transported to shoot and the lower concentrations retained in root vacuoles contributed 484
to the higher NUE in Xiangyou15 and A thaliana mutants (vha-a2 vha-a3 and avp1) (Table 485
1Fig 6A) Further studies are needed to assess the impacts of regulating vacuolar 486
sequestration of NO3- and rates of long-distance transport for enhancing NUE in other crop 487
species and genotypes 488
These data suggested that NO3- distribution between vacuoles and cytosol within cells 489
regulates NO3- long-distance transport from root to shoot and ultimately affects NUE NO3
- 490
loading into the xylem sap is an active transport process (Lin et al 2008) therefore not 491
regulated by absolute concentration of NO3- in the cytosol but rather by genes involved in 492
NO3- long-distance transport (NRT15 and NRT18) Apparently the expression of NRT15 the 493
gene responsible for xylem loading is regulated by NO3-concentration in the cytosol 494
Therefore both NO3- short-distance transport across the tonoplast and its long-distance 495
transport from root to shoot are important determinants of NUE in both B napus and A 496
thaliana (Table 1 and Fig 6) 497
498
Roles of NRT15 and NRT18 in NO3- long-distance transport in B napus and A 499
thaliana 500
Our results (Figs S9 S10) showed that the proportion of NO3- distributed from root to 501
shoot is strongly controlled by NRT15 in accord with previous studies (Lin et al 2008 Li et 502
al 2010) The data suggest that the high-NUE genotypes possess lower activities of tonoplast 503
proton-pumps (V-ATPase and V-PPase) resulting in less NO3- accumulation in vacuoles and 504
more NO3- retention in cytosol (Fig 7) The higher NO3
- in the cytosol then up-regulated 505
NRT15 which then down-regulates NRT18 As a result more NO3- is loaded into the xylem 506
system by NRT15 mechanism and less NO3- is unloaded via NRT18 (Fig 7) Differential 507
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18
regulation of these NRT genes in high-NUE plants increased transport of NO3- from root to 508
shoot through the xylem vascular tissues (Fig 7) This coordinated NO3- long- and 509
short-distance transport likely fine-tunes NO3- allocation and modulates the balance of NO3
- 510
distribution between roots and shoots (Fig 7) 511
Our research also revealed differences between A thaliana and B napus in the 512
expression of the wild-type NRT genes in response to NO3- in the protoplast Moreover using 513
A thaliananrt15-3 mutants we uncovered possible regulatory interactions between a 514
functioning (wild-type) NRT15 gene and NRT18 expression in response to NO3- 515
concentration Because mutants defective in nrt15 are not available for B napus we could 516
not confirm that the respective NRT15 and NRT18 systems behave in an analogous way in 517
these plant species Based on Harper et al (2012) research database our homology 518
comparisons and gene function annotations provided strong evidence for the roles of 519
BnNRT15 and BnNRT18 in B napus Finally our studies were conducted using two model 520
dicotyledonous plant species for which we had ample data on responses to specific inhibitors 521
(B napus) or specific mutants were available with results forming the bases for the model 522
presented in Figure 7 Further research using genetically diverse plant species (eg 523
monocotyledonous crops such as rice wheat and corn) will help validate this model and its 524
broader application across species The information generated in this study could have future 525
application for improving NUE in commercial crops with consequent reduction in nitrogen 526
use and benefits to the environment 527
528
Materials and Methods 529
Plant material 530
The two oilseed rape (Bnapus) cultivars used in this study have been characterized 531
before as high- (Xiangyou15 referred to as H genotype hereafter) and low- (814 referred to 532
as L genotype) nitrogen use efficiency (NUE) genotypes (Zhang et al 2009 Han et al 2015b) 533
Here we define NUE as the total biomass per unit of N uptake by the plant with total 534
biomass includes roots shoots and grains The two genotypes were provided by the Hunan 535
Sub-Center of Improvement Center of National Oil Crops Hunan China 536
The Arabidopsis thaliana wild-type Columbia-0 (col-0) was used as control for 537
V-ATPase (vha-a2 and vha-a3) V-PPase (avp1) nrt15-3 and nrt18-2 mutants whereas the A 538
thaliana wild-type Wassilewskija (Ws) was used as control for clca-2 mutants A thaliana 539
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
19
mutants and transgenic lines (nrt15-3 and nrt18-2) have been described previously (Chen et 540
al 2012 Zhang et al 2014) The mutant lines vha-a2 (Salk_142642) and vha-a3 541
(Salk_122135) described by Krebs et al (2010) were obtained from the Arabidopsis 542
Biological Resources Center (ABRC) The mutant line avp1 (GK-005004) described in Li et 543
al (2005) was obtained from the European Arabidopsis Stock Centre while the mutant line 544
clca-2 (FST 171A06) described in Angeli et al (2006) was from the Institute National de la 545
Recherche Agronomique (INRA) collection in France together with the T-DNA mutants used 546
for genotyping to select pure mutant lines 547
548
Growth conditions 549
B napus plants were grown hydroponically in ceramic pots(20cmtimes15cm) filled with a 550
nutrient solution in the greenhouse under natural light as described in Han et al (2015b) The 551
pots were arranged in a completely randomized design with six biological replications The 552
nutrient solution was replaced every 3 days and its pH adjusted to 55 daily Experiments were 553
conducted at the field station of Hunan Agricultural University Southern China 554
A thaliana plants were grown in a nutrient solution in plastic pots as described in Arteca 555
et al (2000) and Gong et al (2003) The solution was changed every 3 days with pH adjusted 556
daily to 58 Pots were arranged in a completely randomized design with six biological 557
replications The nutrient solution used for both species consisted of 125mM KNO3 558
0625mM KH2PO4 05mM MgSO4 05mM Ca (NO3)2middot4H2O 0025mM Fe-EDTA 025 559
mlL-1 micronutrients (stock solution concentrations 70mM B 14mM Mn 1mM Zn 05mM 560
Cu and 02mM Mo) The experiments were conducted at Hunan Agricultural University in a 561
phytotron set at 70 relative humidity16h-light8h-dark cycle and constant temperature of 562
22degC 563
564
Experimental treatments and phenotyping 565
All measurements made on B napus plants were conducted either at the seedling stage 566
(2 months after transplanting) or at flowering (5 months after transplanting) The whole root 567
tissue was harvested and used for analyses at either stage For shoot measurements the fourth 568
leaf from the bottom was used for measurements made at seedling stage whereas the 12th leaf 569
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
from the bottom was harvested for measurements made at flowering stage For A thaliana 570
whole root and shoot of four-week-old plants were sampled and used for different assays 571
572
Vacuolar proton-pumps inhibitor treatments 573
Inhibition of vacuolar proton pumps of B napus (Xiangyou15) was conducted at the seedling 574
stage in hydroponic culture as described in Han et al (2015a) with minor modifications 575
Plants grown in hydroponic solution were used as control Inhibitor treatment was conducted 576
using several chemicals Bafi(25 nmol L-1 Bafilomycin A1) an inhibitor of V-ATPase 577
DCCD(10 μmol L-1DCCD + 50 mmolL-1 Na2SO3) specifically inhibits V-PPase and 578
Bafi+DCCD in a 11 ratio as inhibitor of both V-ATPase and V-PPase The inhibitors were 579
applied in the hydroponic solutions for 24 h Fresh plant tissue samples from all treatments 580
were collected for different assays 581
582
NO3- induced gene expression 583
Four-week old A thaliana mutants (nrt15-3nrt18-2) were grown in hydroponics with 584
225mM (NH4)2succinate for 3 days and shifted to hydroponics with 45mM NO3- for 12 h 585
then root tissues were collected to assess relative expression of AtNRT15 and AtNRT18 586
genes Seedlings of B napus (Xiangyou15) plants grown hydroponically were treated with 587
5mM (NH4)2succinate for 3 d and shifted to fresh hydroponic solution containing 15 mM 588
NO3- for 12 h Root tissue was then collected to assess the relative expression of BnNRT15 589
and BnNRT18 genes using quantitative RT-PCR 590
591
ACC treatment 592
Hydroponically grown seedlings of B napus were subjected to ACC treatment as described in 593
Zhang et al (2014) with minor modifications Seedlings of the two genotypes Xiangyou15 594
and 814 were transferred to a nutrient solution containing 002mM L-1 ACC for 6 h or grown 595
in normal hydroponic solution as control Root tissues were then collected to assay relative 596
expression of BnNRT15 and BnNRT18 using quantitative RT-PCR Another set of B napus 597
plants were grown in ACC-containing hydroponic solution but supplemented with 15NO3- (22 598
15N) replacing the unlabeled NO3- for 1 h Root and shoot tissues were collected separately 599
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21
for 15N measurement using a continuous-flow isotope ratio mass spectrometer coupled with a 600
C-N elemental analyzer (ANCA-MS PDZ Europa) 601
602
Biomass and N concentration 603
Grain yield dry biomass and N concentration were determined in plant samples at 604
harvest Plants were harvested (including senescing leaves) then dried in an oven for 30 min 605
at 105degC then at 70degC to a constant weight and weighed N concentration was determined 606
using the Kjeldahl method (Shi et al 2010 Han et al 2015b)02g of dried samples were 607
digested with H2SO4-H2O2 in a 100-ml Kjeldahl digestion flask after which N concentration 608
was determined using a Foss Auto Analyser Unit (Kjeldahl 8400) 609
610
V-ATPase and V-PPase activities 611
Roots (10 g) of B napus were collected at seedling and flowering stages and that of A 612
thaliana (05 g) sampled at seedling stage then used for determining the activities of 613
V-ATPase and V-PPase (Han et al 2015b) Activities of these two energy pumps within 614
microsomal membranes were followed colorimetrically based on released Pi as described by 615
Zhu et al (2001) and Krebs et al (2010) 616
617
Assay of NO3- flux in vacuoles 618
Roots of both species were collected similarly as that used for V-ATPase and V-PPase 619
activities then used for vacuole isolation and measurement of NO3- flux in vacuoles as 620
described by Robert et al (2007) with minor modification (Han et al 2015b) Net fluxes of 621
NO3- in vacuoles were measured non-invasively using SIET (scanning ion-selective electrode 622
technique SIET system BIO-003A Younger USA Science and Technology Corporation 623
USA) An NO3--selective microelectrode used for the assay of NO3
- flux was vibrated in the 624
measuring solution between two positions 1 μm and 11 μm from the vacuole surface 625
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles Background 626
signal was recorded by vibrating the electrode in the measuring solution in the absence of 627
vacuoles Ion flux was calculated by Fickrsquos law of diffusion J = -D(dcdx) where J represents 628
the ion flux (picomolescm-2s-1) dcdx is the ion concentration gradient and D is the ion 629
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
diffusion constant in a particular medium The direction of the flux was derived from Fickrsquos 630
law of diffusion that relates the concentration gradient 631
632
Isolation of intact protoplasts and vacuoles for determining NO3- concentration 633
Similar to the above assays root tissues (10 g) were collected from B napus at seedling 634
and flowering stages and from A thaliana (05 g) seedlings and used to isolate intact 635
protoplasts and vacuoles as described by Robert et al (2007) with minor modifications as 636
outlined in Huang et al (2012) and Han et al(2015b) The purified protoplasts were divided 637
into two equal aliquots with one of them used for isolation of vacuoles The purified 638
protoplasts and vacuoles were then sub-sampled and used to determine NO3- concentrations 639
(Vogeli-Lange and Wagner 1990) and for enzyme activity assays (Ma et al 2005) 640
NO3-concentrations in protoplasts and vacuoles were measured by a continuous-flow 641
auto-analyzer (Auto Analyser 3 Bran and Luebbe Germany) as described previously (Han et 642
al 2015b)The activities of acid phosphatase (ACP) and cytochrome oxidase (COX) were 643
determined using plant ACP colorimetry- and COX-assay kits (GenMedSci Inc) following 644
the manufacturerrsquos instructions ACP activity specific to vacuoles was determined and used to 645
normalize NO3- accumulation We measured NO3
- in the protoplast outside the vacuole which 646
includes the cytosol and organelles eg mitochondria and Golgi Apparatus (Robert et 647
al2007) Since most of the NO3- in the protoplast outside the vacuole is located in the cytosol 648
(Krebs et al 2010) we refer to NO3- distribution between vacuoles and cytosol rather than 649
vacuole versus protoplast 650
651
Quantitative RT-PCR 652
Total RNA extracted from B napus roots was prepared using Trizol reagent (Invitrogen) 653
cDNAs were synthesized using M-MLV reverse transcriptase (Promega) following 654
manufacturerrsquos protocol The relative expression of BnNRT15 (EV220114) BnNRT18 655
(EV116423) and Bnactin (AF1118121) genes in plant roots were determined by quantitative 656
RT-PCR and run on a LightCycler instrument (Roche) with the SYBR Green Real-Time PCR 657
Master Mix Kit (TOYOBO Japan) under the following conditions 95degC for 2min then 45 658
cycles of 95degC for 10s 60degC for 10s and 72degC for 20s The primer sequences of BnNRT15 659
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
BnNRT18 and Bnactin genes were obtained from httpbrassicanbiacuk as described in 660
Harper et al (2012) and expression levels were normalized to Bnactin as control 661
Total RNA extracted from the 4-week-old A thaliana roots under the treatments 662
conditions described above were prepared using Trizol reagent (Invitrogen) The cDNAs were 663
synthesized using M-MLV reverse transcriptase (Promega) based on the manufacturerrsquos 664
protocol and as described in Li et al (2010)The primer sequences of quantitative RT-PCR 665
used in these assays are listed in Supplemental Table 2 and were described previously (Chen 666
et al 2012 Zhang et al 2014) and their expression was normalized to Atactin2 as control 667
668
Determination of nitrate distribution using 15NO3- 669
Hydroponically grown B napus (seedling and flowering stages) and A thaliana 670
(seedling) were transferred to 01mM CaSO4 solution for 1min then to their respective 671
hydroponic nutrient solutions with15NO3- (22 excess) replacing unlabeled NO3
- for 1h 672
Plants were then transferred to 01mM CaSO4 solution for 1min after which roots were 673
washed with deionized water Root and shoot samples were separated and dried at 105degC for 674
30min followed by 70degC for 3 d then grounded and used for assaying 15N content using a 675
continuous-flow isotope ratio mass spectrometer coupled with a carbon-nitrogen elemental 676
analyzer (ANCA-MS PDZ Europa) 677
678
Xylem sap collection and assay of nitrate concentration 679
We used the method of Tang et al (2012) for collecting xylem sap Plants were cut 680
leaving1cm segments with intact roots at seedling and flowering stages for B napus and at 681
seedling stage for A thaliana Roots were immediately immersed in their respective nutrition 682
solutions Weighed cotton was put on the cut surface to absorb extruding xylem sap for 1 h 683
The cotton was then wrapped in plastic film and the volume of xylem sap was calculated as 684
the weight gain of the cotton Xylem sap was then squeezed from the cotton using a syringe 685
and used for subsequent assay of nitrate concentration using a continuous-flow auto-analyzer 686
(Auto Analyser 3 Bran and Luebbe Germany) 687
To determine nitrate concentration samples (10 g fresh root and shoot at seedling and 688
flowering of B napus and 05 g fresh root and shoot of 4-week-old A thaliana) were frozen 689
in liquid N2 and ground with a mortar and pestle the powder was then transferred to a beaker 690
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
containing 70 ml deionized water and boiled for 30 min to extract nitrate then cooled and 691
made to 100 ml volume and 02 g activated carbon added to eliminate the effect of 692
chlorophyll The mixture was filtered and nitrate in the filtrate was determined using a 693
continuous-flow auto-analyser (Atuo-Analyser 3 Bran and Luebbe Germany) 694
695
Measurements of photosynthesis 696
All measurements of photosynthesis and related parameters were conducted at 1000 h 697
using intact leaves The 4th leaf from the bottom in B napus at seedling stage and the 12th leaf 698
from the bottom at flowering stage were used for determining chlorophyll concentration 699
photosynthetic rate intercellular CO2 concentration transpiration rate and stomatal 700
conductance Chlorophyll concentration was determined by SPAD-502 (Minolta Camera Co 701
Ltd Japan) (Luo et al 2006 Yandeau-Nelson et al 2011) Photosynthetic rate intercellular 702
CO2 concentration transpiration rate and stomatal conductance were measured using LI-6400 703
Portable Photosynthesis System (Li-Cor Biosciences Co Ltd USA) set at flow rate of 500 704
μmol s-1 photosynthetic photon flux density of 1000 μmol m-2 s-1 relative humidity of 65 705
CO2 concentration of 400ppm and ambient temperatures 706
707
NR and GS activities 708
Leaves with similar age as those used for photosynthesis in B napus were used for the 709
assay of nitrate reductase (NR) and glutamine synthetase (GS) activities NR activity was 710
measured by the modified method of Fan et al (2007) Fresh samples (05g) were frozen in 711
liquid N2 ground into powder in the presence of acid-washed sand and homogenized with 4 712
ml of extraction buffer (0025mol L-1phosphate buffer pH87 1211g L-1 cysteine 0372g L-1 713
EDTA) Homogenates were centrifuged at 30000timesg for 15 min at 4degC and the supernatants 714
were treated with sulfanilamide and α-naphthylamine reagents for colorimetric (540nm) 715
determination of nitrite as described by Fan et al (2007) 716
GS activity was determined using a modified reverseγ-glutamyltransferase method 717
(Wang et al2014) which measures the GS-catalyzed formation of glutamyl-γ-hydroxamate 718
from glutamine and hydroxylamine Fresh samples were frozen for 30 min at -20degC then 719
ground in 10ml Tris-HCl buffer and acid-washed sand The homogenates were filtered 720
through two layers of gauze and centrifuged at 8000timesg for 15 min at 4degC About 12ml of the 721
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
supernatant was added in a reaction mixture and treated as described (Wang et al2014) After 722
the GS-catalyzed reaction was stopped and the solution centrifuged glutamyl-γ723
-hydroxamate was quantified colorimetrically in the supernatant (at 485 nm) and the 724
concentration was determined using a standard curve 725
726
Statistical analyses 727
We used the SPSS software (Statistical Product and Service Solutions V130 USA) for 728
ANOVA and mean separation of main-effects and interactions using Duncanrsquos multiple range 729
test at Plt005Values are presented as means and SD of three or six replicates from three 730
independent experiments Different letters or an asterisk () associated with specific data (eg 731
at the top of histogram bars in figures or within tables) denote significant differences at 732
Plt005 733
734
Supplemental Data 735
Supplemental Figure 1 B napus with higher NUE showed lower vacuolar sequestration 736
capacity (VSC) for NO3-in roots at flowering stage 737
Supplemental Figure 2 B napus with higher NUE showed enhanced long-distance transport 738
of NO3- from roots to shoots at flowering stage 739
Supplemental Figure 3 The two B napus (H and L genotypes) showed the same total N per 740
plant at seedling stage (A) and flowering stage (B) 741
Supplemental Figure 4 B napus with higher NUE showed increased NO3- concentration in 742
the xylem sap at seedling and flowering stages 743
Supplemental Figure 5 NO3- concentration in the xylem sap as affected by inhibitor 744
treatments in B napus and in the energy pumpsrsquo mutants of A thaliana (col-0 vha-a2 vha-a3 745
avp1) 746
Supplemental Figure 6 Reduced VSC for NO3- in roots drives long-distance transport of 747
NO3- from roots to shoots in the A thaliana wild type (Ws) and mutant (clca-2) 748
Supplemental Figure 7 Differences of NR and GS activities between the two B napus (H 749
and L genotypes) at seedling and flowering stages 750
Supplemental Figure 8 Amino acid sequences of BnNRT15 and BnNRT18 751
Supplemental Figure 9 Functions of AtNRT15 and AtNRT18 genes in root tissues of A 752
thaliana in controlling NO3- long-distance transport from root to shoots 753
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
Supplemental Figure 10 B napus BnNRT15 and BnNRT18 genes in roots were 754
coordinately modulated to facilitate NO3- long distance transport from roots to shoots 755
Supplemental Table 1 Differences in stomatal conductance and transpiration rate between 756
the two B napus genotypes 757
Supplemental Table 2 Sequences of primera used for qRT-PCR 758
759
Acknowledgements 760
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 761
Shanghai Institutes for Biological Sciences) for providing nrt15-3 and nrt18-2 seeds 762
This study was supported by the National Natural Science Foundation of China (Grant 763
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 764
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 765
The Ministry of Education in China Open Novel Science Foundation of Hunan province 766
(13K062) the National Key Laboratory of Plant Molecular Genetics and the Twelfth 767
Five-Year National Science and technology support program (2012BAD15BO4) 768
769
Figure 1 B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 770
NO3- in roots at the seedling stage 771
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 772
genotype 814 Specific activities of the tonoplast proton pumps are expressed as μmol Pi 773
released mg-1 protein h-1 NO3- fluxes are expressed as pmol NO3
- cm-2 S-1 Mature vacuoles 774
were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 775
measuring solution between the two positions 1 μm and 11 μm from the vacuole surface 776
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles recording the 777
stable reading data The background was recorded by vibrating the electrode in measuring 778
solution without vacuoles 779
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 780
for NO3- content and NO3
- accumulation normalized against the specific activity of the 781
vacuole acid phosphatase (ACP) as described in Materials and Methods and plotted as μmol 782
NO3- per μmol p-nitrophenol the end product of ACP assay 783
Proton pump activities in root tissues between H and L genotypes are shown at the seedling 784
stage (A) Different letters at the top of the histogram bars denote significant differences of 785
V-ATPase in root tissues between H and L genotypes (Plt005) an asterisk () at the top of the 786
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
histogram bars indicates significant difference in V-PPase activity in root tissues between H 787
and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788
Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789
genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790
bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791
bars on the figures indicate SD (n=6) 792
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793
seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794
to the total NO3- in protoplasts 795
NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796
total NO3-in the cytosol and was calculated as total NO3
- in protoplasts ndash total NO3- in 797
vacuole Different letters at the top of histogram bars denote significant differences of total 798
NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799
(n=6) 800
801
Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802
roots to shoots at the seedling stage 803
H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804
Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805
the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806
as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807
the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808
the histogram bars denote significant differences at Plt005 809
Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810
in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811
genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812
of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813
SD (n=3) different letters at the top of the histogram bars denote significant differences at 814
Plt005 level 815
The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816
at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817
genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818
different letters at the top of the histogram bars denote significant differences at Plt005 819
820
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
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31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
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Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
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b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
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Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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Parsed CitationsAndrews M (1986) The partitioning of nitrate assimilation between root and shoot of higher plants Plant Cell Environment 9511-519
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12
shoots of the H genotype were significantly higher than in the L genotype at both stages (Fig 335
S7AB) and GS activity in the H genotype was also higher than those of the L genotype at 336
seedling stage (Fig S7C) 337
Experiments comparing A thaliana wild type (col-0 Ws) with mutants defective in 338
vacuolar proton-pumps (vha-a2 vha-a3avp1) and transport channel (clca-2) yielded similar 339
results that is increases in NO3- shootroot ratio essentially contributed to enhanced NUE 340
(Fig 6) The NO3- shootroot ratios in A thaliana mutants were higher than that in the wild 341
type (Fig 5GH) Generally the efficiency of inorganic N (NO3-) assimilation into organic N 342
is higher in shoots than in roots (Smirnoff et al 1985 Andrews et al 1986) therefore the 343
NO3- concentration in A thaliana mutants decreased (Fig 6CD) leading to higher NUE 344
compared with the wild type (Fig 6AB) 345
346
BnNRT15BnNRT18 in B napus affects NO3- long-distance transport from roots to 347
shoots 348
Both B napus and A thaliana are members of Cruciferae Family The amino acid 349
sequence identity between BnNRT15 and AtNRT15 was 90 (Fig S8A) the amino acid 350
sequence similarity between BnNRT18 and AtNRT18 was 908 (Fig S8B) Comparisons of 351
nucleotide and amino acid sequences (Harper et al2012) of these genes 352
(httpbrassicanbiacukcgi-binmicroarray_databasecgi) showed that BnNRT15 353
(EV220114) and BnNRT18 (EV116423) of B napus are respectively highly homologous 354
with AtNRT15 and AtNRT18 and the two genes are mainly expressed in roots of both species 355
showing similar organ-specificity (Lin et al 2008 Li et al 2010) 356
NO3- long-distance transport from roots to shoots is therefore regulated by NRT15 and 357
NRT18 as reported before (Lin et al 2008 Li et al 2010) The [15N-traced] SR ratios and 358
the [NO3-] SR ratios were significantly lower in nrt15-3 mutants relative to the wild type 359
(col-0) while [15N] SR and [NO3-] SR ratios showed significant increase in nrt18-2mutant 360
relative to col-0 (Fig S9CD) A previous study showed that expression of NRT15 is 361
down-regulated by ACC and MeJA treatments while the expression of NRT18 is strongly 362
up-regulated in A thaliana (Zhang et al 2014) Consequently NO3- accumulated in plant 363
roots probably as an adaptive measure for abiotic stresses (Chen et al 2012) Both the H- 364
and L- B napus genotypes showed significantly lower expression of BnNRT15 in roots when 365
treated with ACC (Fig S10A) but the expression of BnNRT18 remained higher than in the 366
control plants (Fig S10B) This resulted in significantly lower [15N] SR ratios following 367
ACC treatment (Fig S10CDE) These data indicate that BnNRT15 and BnNRT18 play 368
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13
similar functions for NO3- long-distance transport between root and shoot in both B napus 369
and A thaliana (Lin et al 2008 Li et al 2010) 370
371
372
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14
Discussion 373
Nitrate long-distance transport from root to shoot is regulated by cytosolic NO3- in roots 374
NO3- long-distance transport from root to shoot is controlled by NRT gene family For 375
instance xylem NO3- loading and unloading are mainly controlled by NRT15 and NRT18 376
respectively (Lin et al 2008 Li et al 2010) Whereas the NO3- short-distance transport 377
between the vacuole and cytosol is controlled by tonoplast proton-pumps (V-ATPase and 378
V-PPase) (Krebs et al 2010) and by activity of the CLCa channel in the tonoplast membrane 379
(Angeli et al 2006 Wege et al 2014) tonoplast proton-pumps provide the energy required 380
for NO3- accumulation into the vacuole through CLCa (Gaxiola et al 2001 Brux et al 2008 381
Krebs et al 2010) The expression of NRT15 is up-regulated by NO3- in plant culture 382
solution (Lin et al 2008) whereas regulation of NRT18 expression is contrary to that of 383
NRT15 under both control and abiotic stress conditions (Li et al 2010 Chen et al 2012 384
Zhang et al 2014) 385
Despite the progress made in characterizing the genes involved in transport and 386
metabolism of NO3- in plants the relationship between long- and short-distance transport and 387
how these two pathways are regulated have not been thoroughly investigated We hypothesize 388
that NRT15 expression and its role in long-distance transport is regulated by NO3- in plant 389
tissue as was observed in culture solution (Lin et al 2008) and our data support this 390
hypothesis Both BnNRT15 and AtNRT15 expression were up-regulated by increasing 391
cytosolic NO3- in roots (Fig 4 Fig 5) The increase in cytosolic NO3
- was achieved through (i) 392
use of A thaliana mutants defective in proton-pumps (V-ATPase and V-PPase) or CLCa 393
activities in the tonoplast and (ii) inhibition of B napus tonoplast proton-pumps In both 394
model plants the influx of NO3- into vacuoles was substantially reduced resulting in higher 395
concentrations of NO3- in the cytosol (Fig 4A-D Fig 5A-D Fig S6A-C) Under those 396
conditions expression of BnNRT15 and AtNRT15 was up-regulated by the enhanced 397
cytosolic NO3- while expression of BnNRT18 and AtNRT18 was down-regulated (Fig 4EF 398
Fig 5EF FigS6DE) As a consequence more NO3- was loaded into the xylem sap and 399
transported from root to shoot (Fig S5AB FigS6F) This is also reflected as increased 400
shootroot ratios of N traced by [15N] and [NO3-] (Fig 4GH Fig 5GH Fig S6GH) 401
Interestingly the expression of both BnNRT15 and AtNRT15 is up-regulated by 402
increased cytosolic NO3- concentration in roots but were not affected by NO3
- sequestered 403
into vacuoles (Fig 4 Fig 5 Fig S6) This is because vacuolar NO3- is functionally separated 404
by the tonoplast and cannot be assimilated by enzymes that are localized in the cytosol 405
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15
Therefore NO3--inducible genes such as NR and NRT15 were not influenced by vacuolar 406
NO3- concentration (Martinoia et al 1981 Martinoia et al 2000 Zhang et al 2012) In 407
addition NO3- concentration in the cytosol is controlled by its short-distance transport through 408
the tonoplast which is mediated through the tonoplast proton-pumps providing the required 409
energy for its sequestration into vacuoles against concentration gradient (Gaxiola et al 2001 410
Brux et al 2008 Krebs et al 2010) The gradient of H+ between the inside of the vacuole 411
and the cytosol is maintained by an 2NO3-1H+ antiporter mechanism that facilitatesNO3
- 412
transport through CLCa (Angeli et al 2006 Wege et al 2014) Our data provide clear 413
evidence that NO3- long-distance transport from root to shoot is regulated by cytosolic NO3
- 414
concentration in roots 415
Regulatory mechanisms that control the expression of NRT18 and NRT15 as mediated 416
by NO3- are still unknown however previous studies showed that their expression is also 417
affected by abiotic stresses whereby NRT15 is down-regulated and NRT18 is up-regulated 418
(Li et al 2010 Zhang et al 2014) AtNRT18 expression in A thaliana nrt15 mutants is 419
highly up-regulated (Chen et al 2012) as was also observed here (Fig 3C) Moreover 420
AtNRT15 expression was enhanced by NO3- in culture solution and was not affected in 421
nrt18-2 mutant (Fig 3AB) On the other hand AtNRT18 expression was not affected by 422
cytosolic NO3- but down-regulated when AtNRT15 expression was up-regulated by NO3
-(Fig 423
S6DE) Analogous results were also obtained with B napus genotypes (Fig 3EF) Based on 424
these results we argue that NRT18 expression is not directly influenced by cytosolic NO3- but 425
rather by NRT15 expression (Fig 4 Fig 5 and Fig S6) suggesting NRT18 is probably 426
acting downstream of NRT15 Apparently the regulatory mechanisms of this NRT gene 427
family still awaits further studies to be elucidated 428
429
Response of NUE to NO3- long-distance transport 430
NO3- assimilation efficiency is higher in shoot than in root tissues and can be further 431
enhanced by carbon assimilation (Smirnoff et al 1985 Tang et al 2013) Therefore 432
translocation of higher proportion of NO3- from roots to shoots likely contributes to better 433
crop growth and higher NUE (Andrews et al 1986 Tang et al 2012) The present study 434
provides direct evidence for these results using contrasting B napus genotype identified 435
before (Zhang et al 2009 Han et al 2015a) Our data confirmed the genetic variation in 436
NUE between these two genotypes (Table 1) 437
We measured NO3- distribution in root and shoot of these genotypes to elucidate the 438
physiological mechanisms contributing to variation in NUE The data were consistent with 439
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16
our hypothesis that concentration of NO3- in the xylem sap and shoot would be significantly 440
higher in the H genotype than in the L genotype (Fig 2EFFig S2EF Fig S4) The NO3- 441
long-distance transport from root to shoot was affected by cytosolic NO3- in roots of both A 442
thaliana and B napus genotypes (Fig 4-5 Fig S6) The activities of the tonoplast 443
proton-pumps in roots were significantly lower in the H genotype than in the L genotype (Fig 444
1AB) Consequently less NO3- was sequestered into the vacuole and more NO3
- was retained 445
in cytosol (Fig 1CD) BnNRT15 expression was then up-regulated resulting in greater 446
loading into the xylem while BnNRT18 was down-regulated (Fig 2AB) probably to 447
suppress NO3- unloading from xylem sap Consequently a higher proportion of NO3
- was 448
transported from root to shoots in the H genotype(Fig 2C-D Fig S4) 449
Moreover photosynthetic carbon fixation (Table 2) and activities of both NR and GS 450
were higher in shoots of the H genotype (Fig S7) both of which promote higher N 451
assimilation These results agreed with previous reports (Smirnoff et al 1985 Andrews et al 452
1986 Tang et al 2012 Tang et al 2013) where higher NO3- transport from root to shoot 453
higher photosynthetic rate and N assimilation efficiency in shoots were suggested as essential 454
mechanisms for high-NUE in crop plants These qualities endowed the B napus H genotype 455
Xiangyou15 with higher NUE as compared with the L genotype 814 456
NO3- long-distance transport in B napus is not only regulated by BnNRT15 and 457
BnNRT18 in root tissues (Fig 4 Fig 5 Fig S6) but also by stomatal conductance and 458
transpiration by indirectly controlling xylem sap flow and long-distance transport 459
(Dechorgnat et al 2011 Krapp et al 2014) Stomatal conductance and transpiration rates of 460
the H genotype were significantly higher than those of the L genotype (Table S1) a result 461
consistent with other studies describing characteristics of high-NUE plants (Daniel-Vedele et 462
al 1998 Wilkinson et al 2007 Dechorgnat et al 2011 Krapp et al 2014) 463
464
Response of NUE to NO3- short-distance transport 465
NUE is also strongly controlled by NO3- short-distance distribution between the vacuole 466
and cytosol (Han et al 2015a) A lower proportion of NO3- accumulating in vacuoles and a 467
higher proportion retained in the cytosol will contribute to better NUE in crop plants (Han et 468
al 2015b) The energy required for vacuolar NO3- sequestration is provided by the tonoplast 469
proton-pumps (Gaxiola et al 2001 Brux et al 2008 Krebs et al 2010) while the 470
distribution channel CLCa enables transport of NO3- across the vacuolar membrane (Angeli et 471
al 2006 Wege et al 2014) The activities of the tonoplast proton-pumps (V-ATPase and 472
V-PPase) in leaves of the H genotype were significantly lower than those of the L genotype at 473
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17
flowering stages (Han et al 2015aHan et al 2015b) thus we expected similar differences in 474
root tissues Our results confirmed that the activities of tonoplast proton-pumps in root tissues 475
of B napus H genotype and A thaliana mutants were lower than those in the L genotype and 476
A thaliana wild type leading to less NO3- influx into vacuoles and more in the cytosol (Fig 1 477
Fig 5A-D) Moreover the enzymes catalyzing N assimilation are located in the cytoplasm 478
where NO3- could be assimilated (Martinoia et al 1981 Han et al 2015a) or immediately 479
loaded into xylem sap for long-distance transport from roots to shoots (Lin et al 2008) 480
Consequently the NO3- concentration in root tissues of the H genotype and A thaliana 481
mutants defective in tonoplast proton-pumps was significantly lower than that in the L 482
genotype (Fig 2E) and in A thaliana wild type (Fig 6C) Therefore the higher proportion of 483
NO3- transported to shoot and the lower concentrations retained in root vacuoles contributed 484
to the higher NUE in Xiangyou15 and A thaliana mutants (vha-a2 vha-a3 and avp1) (Table 485
1Fig 6A) Further studies are needed to assess the impacts of regulating vacuolar 486
sequestration of NO3- and rates of long-distance transport for enhancing NUE in other crop 487
species and genotypes 488
These data suggested that NO3- distribution between vacuoles and cytosol within cells 489
regulates NO3- long-distance transport from root to shoot and ultimately affects NUE NO3
- 490
loading into the xylem sap is an active transport process (Lin et al 2008) therefore not 491
regulated by absolute concentration of NO3- in the cytosol but rather by genes involved in 492
NO3- long-distance transport (NRT15 and NRT18) Apparently the expression of NRT15 the 493
gene responsible for xylem loading is regulated by NO3-concentration in the cytosol 494
Therefore both NO3- short-distance transport across the tonoplast and its long-distance 495
transport from root to shoot are important determinants of NUE in both B napus and A 496
thaliana (Table 1 and Fig 6) 497
498
Roles of NRT15 and NRT18 in NO3- long-distance transport in B napus and A 499
thaliana 500
Our results (Figs S9 S10) showed that the proportion of NO3- distributed from root to 501
shoot is strongly controlled by NRT15 in accord with previous studies (Lin et al 2008 Li et 502
al 2010) The data suggest that the high-NUE genotypes possess lower activities of tonoplast 503
proton-pumps (V-ATPase and V-PPase) resulting in less NO3- accumulation in vacuoles and 504
more NO3- retention in cytosol (Fig 7) The higher NO3
- in the cytosol then up-regulated 505
NRT15 which then down-regulates NRT18 As a result more NO3- is loaded into the xylem 506
system by NRT15 mechanism and less NO3- is unloaded via NRT18 (Fig 7) Differential 507
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18
regulation of these NRT genes in high-NUE plants increased transport of NO3- from root to 508
shoot through the xylem vascular tissues (Fig 7) This coordinated NO3- long- and 509
short-distance transport likely fine-tunes NO3- allocation and modulates the balance of NO3
- 510
distribution between roots and shoots (Fig 7) 511
Our research also revealed differences between A thaliana and B napus in the 512
expression of the wild-type NRT genes in response to NO3- in the protoplast Moreover using 513
A thaliananrt15-3 mutants we uncovered possible regulatory interactions between a 514
functioning (wild-type) NRT15 gene and NRT18 expression in response to NO3- 515
concentration Because mutants defective in nrt15 are not available for B napus we could 516
not confirm that the respective NRT15 and NRT18 systems behave in an analogous way in 517
these plant species Based on Harper et al (2012) research database our homology 518
comparisons and gene function annotations provided strong evidence for the roles of 519
BnNRT15 and BnNRT18 in B napus Finally our studies were conducted using two model 520
dicotyledonous plant species for which we had ample data on responses to specific inhibitors 521
(B napus) or specific mutants were available with results forming the bases for the model 522
presented in Figure 7 Further research using genetically diverse plant species (eg 523
monocotyledonous crops such as rice wheat and corn) will help validate this model and its 524
broader application across species The information generated in this study could have future 525
application for improving NUE in commercial crops with consequent reduction in nitrogen 526
use and benefits to the environment 527
528
Materials and Methods 529
Plant material 530
The two oilseed rape (Bnapus) cultivars used in this study have been characterized 531
before as high- (Xiangyou15 referred to as H genotype hereafter) and low- (814 referred to 532
as L genotype) nitrogen use efficiency (NUE) genotypes (Zhang et al 2009 Han et al 2015b) 533
Here we define NUE as the total biomass per unit of N uptake by the plant with total 534
biomass includes roots shoots and grains The two genotypes were provided by the Hunan 535
Sub-Center of Improvement Center of National Oil Crops Hunan China 536
The Arabidopsis thaliana wild-type Columbia-0 (col-0) was used as control for 537
V-ATPase (vha-a2 and vha-a3) V-PPase (avp1) nrt15-3 and nrt18-2 mutants whereas the A 538
thaliana wild-type Wassilewskija (Ws) was used as control for clca-2 mutants A thaliana 539
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
19
mutants and transgenic lines (nrt15-3 and nrt18-2) have been described previously (Chen et 540
al 2012 Zhang et al 2014) The mutant lines vha-a2 (Salk_142642) and vha-a3 541
(Salk_122135) described by Krebs et al (2010) were obtained from the Arabidopsis 542
Biological Resources Center (ABRC) The mutant line avp1 (GK-005004) described in Li et 543
al (2005) was obtained from the European Arabidopsis Stock Centre while the mutant line 544
clca-2 (FST 171A06) described in Angeli et al (2006) was from the Institute National de la 545
Recherche Agronomique (INRA) collection in France together with the T-DNA mutants used 546
for genotyping to select pure mutant lines 547
548
Growth conditions 549
B napus plants were grown hydroponically in ceramic pots(20cmtimes15cm) filled with a 550
nutrient solution in the greenhouse under natural light as described in Han et al (2015b) The 551
pots were arranged in a completely randomized design with six biological replications The 552
nutrient solution was replaced every 3 days and its pH adjusted to 55 daily Experiments were 553
conducted at the field station of Hunan Agricultural University Southern China 554
A thaliana plants were grown in a nutrient solution in plastic pots as described in Arteca 555
et al (2000) and Gong et al (2003) The solution was changed every 3 days with pH adjusted 556
daily to 58 Pots were arranged in a completely randomized design with six biological 557
replications The nutrient solution used for both species consisted of 125mM KNO3 558
0625mM KH2PO4 05mM MgSO4 05mM Ca (NO3)2middot4H2O 0025mM Fe-EDTA 025 559
mlL-1 micronutrients (stock solution concentrations 70mM B 14mM Mn 1mM Zn 05mM 560
Cu and 02mM Mo) The experiments were conducted at Hunan Agricultural University in a 561
phytotron set at 70 relative humidity16h-light8h-dark cycle and constant temperature of 562
22degC 563
564
Experimental treatments and phenotyping 565
All measurements made on B napus plants were conducted either at the seedling stage 566
(2 months after transplanting) or at flowering (5 months after transplanting) The whole root 567
tissue was harvested and used for analyses at either stage For shoot measurements the fourth 568
leaf from the bottom was used for measurements made at seedling stage whereas the 12th leaf 569
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
from the bottom was harvested for measurements made at flowering stage For A thaliana 570
whole root and shoot of four-week-old plants were sampled and used for different assays 571
572
Vacuolar proton-pumps inhibitor treatments 573
Inhibition of vacuolar proton pumps of B napus (Xiangyou15) was conducted at the seedling 574
stage in hydroponic culture as described in Han et al (2015a) with minor modifications 575
Plants grown in hydroponic solution were used as control Inhibitor treatment was conducted 576
using several chemicals Bafi(25 nmol L-1 Bafilomycin A1) an inhibitor of V-ATPase 577
DCCD(10 μmol L-1DCCD + 50 mmolL-1 Na2SO3) specifically inhibits V-PPase and 578
Bafi+DCCD in a 11 ratio as inhibitor of both V-ATPase and V-PPase The inhibitors were 579
applied in the hydroponic solutions for 24 h Fresh plant tissue samples from all treatments 580
were collected for different assays 581
582
NO3- induced gene expression 583
Four-week old A thaliana mutants (nrt15-3nrt18-2) were grown in hydroponics with 584
225mM (NH4)2succinate for 3 days and shifted to hydroponics with 45mM NO3- for 12 h 585
then root tissues were collected to assess relative expression of AtNRT15 and AtNRT18 586
genes Seedlings of B napus (Xiangyou15) plants grown hydroponically were treated with 587
5mM (NH4)2succinate for 3 d and shifted to fresh hydroponic solution containing 15 mM 588
NO3- for 12 h Root tissue was then collected to assess the relative expression of BnNRT15 589
and BnNRT18 genes using quantitative RT-PCR 590
591
ACC treatment 592
Hydroponically grown seedlings of B napus were subjected to ACC treatment as described in 593
Zhang et al (2014) with minor modifications Seedlings of the two genotypes Xiangyou15 594
and 814 were transferred to a nutrient solution containing 002mM L-1 ACC for 6 h or grown 595
in normal hydroponic solution as control Root tissues were then collected to assay relative 596
expression of BnNRT15 and BnNRT18 using quantitative RT-PCR Another set of B napus 597
plants were grown in ACC-containing hydroponic solution but supplemented with 15NO3- (22 598
15N) replacing the unlabeled NO3- for 1 h Root and shoot tissues were collected separately 599
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21
for 15N measurement using a continuous-flow isotope ratio mass spectrometer coupled with a 600
C-N elemental analyzer (ANCA-MS PDZ Europa) 601
602
Biomass and N concentration 603
Grain yield dry biomass and N concentration were determined in plant samples at 604
harvest Plants were harvested (including senescing leaves) then dried in an oven for 30 min 605
at 105degC then at 70degC to a constant weight and weighed N concentration was determined 606
using the Kjeldahl method (Shi et al 2010 Han et al 2015b)02g of dried samples were 607
digested with H2SO4-H2O2 in a 100-ml Kjeldahl digestion flask after which N concentration 608
was determined using a Foss Auto Analyser Unit (Kjeldahl 8400) 609
610
V-ATPase and V-PPase activities 611
Roots (10 g) of B napus were collected at seedling and flowering stages and that of A 612
thaliana (05 g) sampled at seedling stage then used for determining the activities of 613
V-ATPase and V-PPase (Han et al 2015b) Activities of these two energy pumps within 614
microsomal membranes were followed colorimetrically based on released Pi as described by 615
Zhu et al (2001) and Krebs et al (2010) 616
617
Assay of NO3- flux in vacuoles 618
Roots of both species were collected similarly as that used for V-ATPase and V-PPase 619
activities then used for vacuole isolation and measurement of NO3- flux in vacuoles as 620
described by Robert et al (2007) with minor modification (Han et al 2015b) Net fluxes of 621
NO3- in vacuoles were measured non-invasively using SIET (scanning ion-selective electrode 622
technique SIET system BIO-003A Younger USA Science and Technology Corporation 623
USA) An NO3--selective microelectrode used for the assay of NO3
- flux was vibrated in the 624
measuring solution between two positions 1 μm and 11 μm from the vacuole surface 625
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles Background 626
signal was recorded by vibrating the electrode in the measuring solution in the absence of 627
vacuoles Ion flux was calculated by Fickrsquos law of diffusion J = -D(dcdx) where J represents 628
the ion flux (picomolescm-2s-1) dcdx is the ion concentration gradient and D is the ion 629
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
diffusion constant in a particular medium The direction of the flux was derived from Fickrsquos 630
law of diffusion that relates the concentration gradient 631
632
Isolation of intact protoplasts and vacuoles for determining NO3- concentration 633
Similar to the above assays root tissues (10 g) were collected from B napus at seedling 634
and flowering stages and from A thaliana (05 g) seedlings and used to isolate intact 635
protoplasts and vacuoles as described by Robert et al (2007) with minor modifications as 636
outlined in Huang et al (2012) and Han et al(2015b) The purified protoplasts were divided 637
into two equal aliquots with one of them used for isolation of vacuoles The purified 638
protoplasts and vacuoles were then sub-sampled and used to determine NO3- concentrations 639
(Vogeli-Lange and Wagner 1990) and for enzyme activity assays (Ma et al 2005) 640
NO3-concentrations in protoplasts and vacuoles were measured by a continuous-flow 641
auto-analyzer (Auto Analyser 3 Bran and Luebbe Germany) as described previously (Han et 642
al 2015b)The activities of acid phosphatase (ACP) and cytochrome oxidase (COX) were 643
determined using plant ACP colorimetry- and COX-assay kits (GenMedSci Inc) following 644
the manufacturerrsquos instructions ACP activity specific to vacuoles was determined and used to 645
normalize NO3- accumulation We measured NO3
- in the protoplast outside the vacuole which 646
includes the cytosol and organelles eg mitochondria and Golgi Apparatus (Robert et 647
al2007) Since most of the NO3- in the protoplast outside the vacuole is located in the cytosol 648
(Krebs et al 2010) we refer to NO3- distribution between vacuoles and cytosol rather than 649
vacuole versus protoplast 650
651
Quantitative RT-PCR 652
Total RNA extracted from B napus roots was prepared using Trizol reagent (Invitrogen) 653
cDNAs were synthesized using M-MLV reverse transcriptase (Promega) following 654
manufacturerrsquos protocol The relative expression of BnNRT15 (EV220114) BnNRT18 655
(EV116423) and Bnactin (AF1118121) genes in plant roots were determined by quantitative 656
RT-PCR and run on a LightCycler instrument (Roche) with the SYBR Green Real-Time PCR 657
Master Mix Kit (TOYOBO Japan) under the following conditions 95degC for 2min then 45 658
cycles of 95degC for 10s 60degC for 10s and 72degC for 20s The primer sequences of BnNRT15 659
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
BnNRT18 and Bnactin genes were obtained from httpbrassicanbiacuk as described in 660
Harper et al (2012) and expression levels were normalized to Bnactin as control 661
Total RNA extracted from the 4-week-old A thaliana roots under the treatments 662
conditions described above were prepared using Trizol reagent (Invitrogen) The cDNAs were 663
synthesized using M-MLV reverse transcriptase (Promega) based on the manufacturerrsquos 664
protocol and as described in Li et al (2010)The primer sequences of quantitative RT-PCR 665
used in these assays are listed in Supplemental Table 2 and were described previously (Chen 666
et al 2012 Zhang et al 2014) and their expression was normalized to Atactin2 as control 667
668
Determination of nitrate distribution using 15NO3- 669
Hydroponically grown B napus (seedling and flowering stages) and A thaliana 670
(seedling) were transferred to 01mM CaSO4 solution for 1min then to their respective 671
hydroponic nutrient solutions with15NO3- (22 excess) replacing unlabeled NO3
- for 1h 672
Plants were then transferred to 01mM CaSO4 solution for 1min after which roots were 673
washed with deionized water Root and shoot samples were separated and dried at 105degC for 674
30min followed by 70degC for 3 d then grounded and used for assaying 15N content using a 675
continuous-flow isotope ratio mass spectrometer coupled with a carbon-nitrogen elemental 676
analyzer (ANCA-MS PDZ Europa) 677
678
Xylem sap collection and assay of nitrate concentration 679
We used the method of Tang et al (2012) for collecting xylem sap Plants were cut 680
leaving1cm segments with intact roots at seedling and flowering stages for B napus and at 681
seedling stage for A thaliana Roots were immediately immersed in their respective nutrition 682
solutions Weighed cotton was put on the cut surface to absorb extruding xylem sap for 1 h 683
The cotton was then wrapped in plastic film and the volume of xylem sap was calculated as 684
the weight gain of the cotton Xylem sap was then squeezed from the cotton using a syringe 685
and used for subsequent assay of nitrate concentration using a continuous-flow auto-analyzer 686
(Auto Analyser 3 Bran and Luebbe Germany) 687
To determine nitrate concentration samples (10 g fresh root and shoot at seedling and 688
flowering of B napus and 05 g fresh root and shoot of 4-week-old A thaliana) were frozen 689
in liquid N2 and ground with a mortar and pestle the powder was then transferred to a beaker 690
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
containing 70 ml deionized water and boiled for 30 min to extract nitrate then cooled and 691
made to 100 ml volume and 02 g activated carbon added to eliminate the effect of 692
chlorophyll The mixture was filtered and nitrate in the filtrate was determined using a 693
continuous-flow auto-analyser (Atuo-Analyser 3 Bran and Luebbe Germany) 694
695
Measurements of photosynthesis 696
All measurements of photosynthesis and related parameters were conducted at 1000 h 697
using intact leaves The 4th leaf from the bottom in B napus at seedling stage and the 12th leaf 698
from the bottom at flowering stage were used for determining chlorophyll concentration 699
photosynthetic rate intercellular CO2 concentration transpiration rate and stomatal 700
conductance Chlorophyll concentration was determined by SPAD-502 (Minolta Camera Co 701
Ltd Japan) (Luo et al 2006 Yandeau-Nelson et al 2011) Photosynthetic rate intercellular 702
CO2 concentration transpiration rate and stomatal conductance were measured using LI-6400 703
Portable Photosynthesis System (Li-Cor Biosciences Co Ltd USA) set at flow rate of 500 704
μmol s-1 photosynthetic photon flux density of 1000 μmol m-2 s-1 relative humidity of 65 705
CO2 concentration of 400ppm and ambient temperatures 706
707
NR and GS activities 708
Leaves with similar age as those used for photosynthesis in B napus were used for the 709
assay of nitrate reductase (NR) and glutamine synthetase (GS) activities NR activity was 710
measured by the modified method of Fan et al (2007) Fresh samples (05g) were frozen in 711
liquid N2 ground into powder in the presence of acid-washed sand and homogenized with 4 712
ml of extraction buffer (0025mol L-1phosphate buffer pH87 1211g L-1 cysteine 0372g L-1 713
EDTA) Homogenates were centrifuged at 30000timesg for 15 min at 4degC and the supernatants 714
were treated with sulfanilamide and α-naphthylamine reagents for colorimetric (540nm) 715
determination of nitrite as described by Fan et al (2007) 716
GS activity was determined using a modified reverseγ-glutamyltransferase method 717
(Wang et al2014) which measures the GS-catalyzed formation of glutamyl-γ-hydroxamate 718
from glutamine and hydroxylamine Fresh samples were frozen for 30 min at -20degC then 719
ground in 10ml Tris-HCl buffer and acid-washed sand The homogenates were filtered 720
through two layers of gauze and centrifuged at 8000timesg for 15 min at 4degC About 12ml of the 721
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
supernatant was added in a reaction mixture and treated as described (Wang et al2014) After 722
the GS-catalyzed reaction was stopped and the solution centrifuged glutamyl-γ723
-hydroxamate was quantified colorimetrically in the supernatant (at 485 nm) and the 724
concentration was determined using a standard curve 725
726
Statistical analyses 727
We used the SPSS software (Statistical Product and Service Solutions V130 USA) for 728
ANOVA and mean separation of main-effects and interactions using Duncanrsquos multiple range 729
test at Plt005Values are presented as means and SD of three or six replicates from three 730
independent experiments Different letters or an asterisk () associated with specific data (eg 731
at the top of histogram bars in figures or within tables) denote significant differences at 732
Plt005 733
734
Supplemental Data 735
Supplemental Figure 1 B napus with higher NUE showed lower vacuolar sequestration 736
capacity (VSC) for NO3-in roots at flowering stage 737
Supplemental Figure 2 B napus with higher NUE showed enhanced long-distance transport 738
of NO3- from roots to shoots at flowering stage 739
Supplemental Figure 3 The two B napus (H and L genotypes) showed the same total N per 740
plant at seedling stage (A) and flowering stage (B) 741
Supplemental Figure 4 B napus with higher NUE showed increased NO3- concentration in 742
the xylem sap at seedling and flowering stages 743
Supplemental Figure 5 NO3- concentration in the xylem sap as affected by inhibitor 744
treatments in B napus and in the energy pumpsrsquo mutants of A thaliana (col-0 vha-a2 vha-a3 745
avp1) 746
Supplemental Figure 6 Reduced VSC for NO3- in roots drives long-distance transport of 747
NO3- from roots to shoots in the A thaliana wild type (Ws) and mutant (clca-2) 748
Supplemental Figure 7 Differences of NR and GS activities between the two B napus (H 749
and L genotypes) at seedling and flowering stages 750
Supplemental Figure 8 Amino acid sequences of BnNRT15 and BnNRT18 751
Supplemental Figure 9 Functions of AtNRT15 and AtNRT18 genes in root tissues of A 752
thaliana in controlling NO3- long-distance transport from root to shoots 753
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
Supplemental Figure 10 B napus BnNRT15 and BnNRT18 genes in roots were 754
coordinately modulated to facilitate NO3- long distance transport from roots to shoots 755
Supplemental Table 1 Differences in stomatal conductance and transpiration rate between 756
the two B napus genotypes 757
Supplemental Table 2 Sequences of primera used for qRT-PCR 758
759
Acknowledgements 760
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 761
Shanghai Institutes for Biological Sciences) for providing nrt15-3 and nrt18-2 seeds 762
This study was supported by the National Natural Science Foundation of China (Grant 763
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 764
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 765
The Ministry of Education in China Open Novel Science Foundation of Hunan province 766
(13K062) the National Key Laboratory of Plant Molecular Genetics and the Twelfth 767
Five-Year National Science and technology support program (2012BAD15BO4) 768
769
Figure 1 B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 770
NO3- in roots at the seedling stage 771
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 772
genotype 814 Specific activities of the tonoplast proton pumps are expressed as μmol Pi 773
released mg-1 protein h-1 NO3- fluxes are expressed as pmol NO3
- cm-2 S-1 Mature vacuoles 774
were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 775
measuring solution between the two positions 1 μm and 11 μm from the vacuole surface 776
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles recording the 777
stable reading data The background was recorded by vibrating the electrode in measuring 778
solution without vacuoles 779
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 780
for NO3- content and NO3
- accumulation normalized against the specific activity of the 781
vacuole acid phosphatase (ACP) as described in Materials and Methods and plotted as μmol 782
NO3- per μmol p-nitrophenol the end product of ACP assay 783
Proton pump activities in root tissues between H and L genotypes are shown at the seedling 784
stage (A) Different letters at the top of the histogram bars denote significant differences of 785
V-ATPase in root tissues between H and L genotypes (Plt005) an asterisk () at the top of the 786
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
histogram bars indicates significant difference in V-PPase activity in root tissues between H 787
and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788
Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789
genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790
bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791
bars on the figures indicate SD (n=6) 792
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793
seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794
to the total NO3- in protoplasts 795
NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796
total NO3-in the cytosol and was calculated as total NO3
- in protoplasts ndash total NO3- in 797
vacuole Different letters at the top of histogram bars denote significant differences of total 798
NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799
(n=6) 800
801
Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802
roots to shoots at the seedling stage 803
H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804
Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805
the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806
as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807
the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808
the histogram bars denote significant differences at Plt005 809
Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810
in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811
genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812
of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813
SD (n=3) different letters at the top of the histogram bars denote significant differences at 814
Plt005 level 815
The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816
at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817
genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818
different letters at the top of the histogram bars denote significant differences at Plt005 819
820
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
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30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
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31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
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Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
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b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
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Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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similar functions for NO3- long-distance transport between root and shoot in both B napus 369
and A thaliana (Lin et al 2008 Li et al 2010) 370
371
372
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Discussion 373
Nitrate long-distance transport from root to shoot is regulated by cytosolic NO3- in roots 374
NO3- long-distance transport from root to shoot is controlled by NRT gene family For 375
instance xylem NO3- loading and unloading are mainly controlled by NRT15 and NRT18 376
respectively (Lin et al 2008 Li et al 2010) Whereas the NO3- short-distance transport 377
between the vacuole and cytosol is controlled by tonoplast proton-pumps (V-ATPase and 378
V-PPase) (Krebs et al 2010) and by activity of the CLCa channel in the tonoplast membrane 379
(Angeli et al 2006 Wege et al 2014) tonoplast proton-pumps provide the energy required 380
for NO3- accumulation into the vacuole through CLCa (Gaxiola et al 2001 Brux et al 2008 381
Krebs et al 2010) The expression of NRT15 is up-regulated by NO3- in plant culture 382
solution (Lin et al 2008) whereas regulation of NRT18 expression is contrary to that of 383
NRT15 under both control and abiotic stress conditions (Li et al 2010 Chen et al 2012 384
Zhang et al 2014) 385
Despite the progress made in characterizing the genes involved in transport and 386
metabolism of NO3- in plants the relationship between long- and short-distance transport and 387
how these two pathways are regulated have not been thoroughly investigated We hypothesize 388
that NRT15 expression and its role in long-distance transport is regulated by NO3- in plant 389
tissue as was observed in culture solution (Lin et al 2008) and our data support this 390
hypothesis Both BnNRT15 and AtNRT15 expression were up-regulated by increasing 391
cytosolic NO3- in roots (Fig 4 Fig 5) The increase in cytosolic NO3
- was achieved through (i) 392
use of A thaliana mutants defective in proton-pumps (V-ATPase and V-PPase) or CLCa 393
activities in the tonoplast and (ii) inhibition of B napus tonoplast proton-pumps In both 394
model plants the influx of NO3- into vacuoles was substantially reduced resulting in higher 395
concentrations of NO3- in the cytosol (Fig 4A-D Fig 5A-D Fig S6A-C) Under those 396
conditions expression of BnNRT15 and AtNRT15 was up-regulated by the enhanced 397
cytosolic NO3- while expression of BnNRT18 and AtNRT18 was down-regulated (Fig 4EF 398
Fig 5EF FigS6DE) As a consequence more NO3- was loaded into the xylem sap and 399
transported from root to shoot (Fig S5AB FigS6F) This is also reflected as increased 400
shootroot ratios of N traced by [15N] and [NO3-] (Fig 4GH Fig 5GH Fig S6GH) 401
Interestingly the expression of both BnNRT15 and AtNRT15 is up-regulated by 402
increased cytosolic NO3- concentration in roots but were not affected by NO3
- sequestered 403
into vacuoles (Fig 4 Fig 5 Fig S6) This is because vacuolar NO3- is functionally separated 404
by the tonoplast and cannot be assimilated by enzymes that are localized in the cytosol 405
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15
Therefore NO3--inducible genes such as NR and NRT15 were not influenced by vacuolar 406
NO3- concentration (Martinoia et al 1981 Martinoia et al 2000 Zhang et al 2012) In 407
addition NO3- concentration in the cytosol is controlled by its short-distance transport through 408
the tonoplast which is mediated through the tonoplast proton-pumps providing the required 409
energy for its sequestration into vacuoles against concentration gradient (Gaxiola et al 2001 410
Brux et al 2008 Krebs et al 2010) The gradient of H+ between the inside of the vacuole 411
and the cytosol is maintained by an 2NO3-1H+ antiporter mechanism that facilitatesNO3
- 412
transport through CLCa (Angeli et al 2006 Wege et al 2014) Our data provide clear 413
evidence that NO3- long-distance transport from root to shoot is regulated by cytosolic NO3
- 414
concentration in roots 415
Regulatory mechanisms that control the expression of NRT18 and NRT15 as mediated 416
by NO3- are still unknown however previous studies showed that their expression is also 417
affected by abiotic stresses whereby NRT15 is down-regulated and NRT18 is up-regulated 418
(Li et al 2010 Zhang et al 2014) AtNRT18 expression in A thaliana nrt15 mutants is 419
highly up-regulated (Chen et al 2012) as was also observed here (Fig 3C) Moreover 420
AtNRT15 expression was enhanced by NO3- in culture solution and was not affected in 421
nrt18-2 mutant (Fig 3AB) On the other hand AtNRT18 expression was not affected by 422
cytosolic NO3- but down-regulated when AtNRT15 expression was up-regulated by NO3
-(Fig 423
S6DE) Analogous results were also obtained with B napus genotypes (Fig 3EF) Based on 424
these results we argue that NRT18 expression is not directly influenced by cytosolic NO3- but 425
rather by NRT15 expression (Fig 4 Fig 5 and Fig S6) suggesting NRT18 is probably 426
acting downstream of NRT15 Apparently the regulatory mechanisms of this NRT gene 427
family still awaits further studies to be elucidated 428
429
Response of NUE to NO3- long-distance transport 430
NO3- assimilation efficiency is higher in shoot than in root tissues and can be further 431
enhanced by carbon assimilation (Smirnoff et al 1985 Tang et al 2013) Therefore 432
translocation of higher proportion of NO3- from roots to shoots likely contributes to better 433
crop growth and higher NUE (Andrews et al 1986 Tang et al 2012) The present study 434
provides direct evidence for these results using contrasting B napus genotype identified 435
before (Zhang et al 2009 Han et al 2015a) Our data confirmed the genetic variation in 436
NUE between these two genotypes (Table 1) 437
We measured NO3- distribution in root and shoot of these genotypes to elucidate the 438
physiological mechanisms contributing to variation in NUE The data were consistent with 439
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16
our hypothesis that concentration of NO3- in the xylem sap and shoot would be significantly 440
higher in the H genotype than in the L genotype (Fig 2EFFig S2EF Fig S4) The NO3- 441
long-distance transport from root to shoot was affected by cytosolic NO3- in roots of both A 442
thaliana and B napus genotypes (Fig 4-5 Fig S6) The activities of the tonoplast 443
proton-pumps in roots were significantly lower in the H genotype than in the L genotype (Fig 444
1AB) Consequently less NO3- was sequestered into the vacuole and more NO3
- was retained 445
in cytosol (Fig 1CD) BnNRT15 expression was then up-regulated resulting in greater 446
loading into the xylem while BnNRT18 was down-regulated (Fig 2AB) probably to 447
suppress NO3- unloading from xylem sap Consequently a higher proportion of NO3
- was 448
transported from root to shoots in the H genotype(Fig 2C-D Fig S4) 449
Moreover photosynthetic carbon fixation (Table 2) and activities of both NR and GS 450
were higher in shoots of the H genotype (Fig S7) both of which promote higher N 451
assimilation These results agreed with previous reports (Smirnoff et al 1985 Andrews et al 452
1986 Tang et al 2012 Tang et al 2013) where higher NO3- transport from root to shoot 453
higher photosynthetic rate and N assimilation efficiency in shoots were suggested as essential 454
mechanisms for high-NUE in crop plants These qualities endowed the B napus H genotype 455
Xiangyou15 with higher NUE as compared with the L genotype 814 456
NO3- long-distance transport in B napus is not only regulated by BnNRT15 and 457
BnNRT18 in root tissues (Fig 4 Fig 5 Fig S6) but also by stomatal conductance and 458
transpiration by indirectly controlling xylem sap flow and long-distance transport 459
(Dechorgnat et al 2011 Krapp et al 2014) Stomatal conductance and transpiration rates of 460
the H genotype were significantly higher than those of the L genotype (Table S1) a result 461
consistent with other studies describing characteristics of high-NUE plants (Daniel-Vedele et 462
al 1998 Wilkinson et al 2007 Dechorgnat et al 2011 Krapp et al 2014) 463
464
Response of NUE to NO3- short-distance transport 465
NUE is also strongly controlled by NO3- short-distance distribution between the vacuole 466
and cytosol (Han et al 2015a) A lower proportion of NO3- accumulating in vacuoles and a 467
higher proportion retained in the cytosol will contribute to better NUE in crop plants (Han et 468
al 2015b) The energy required for vacuolar NO3- sequestration is provided by the tonoplast 469
proton-pumps (Gaxiola et al 2001 Brux et al 2008 Krebs et al 2010) while the 470
distribution channel CLCa enables transport of NO3- across the vacuolar membrane (Angeli et 471
al 2006 Wege et al 2014) The activities of the tonoplast proton-pumps (V-ATPase and 472
V-PPase) in leaves of the H genotype were significantly lower than those of the L genotype at 473
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17
flowering stages (Han et al 2015aHan et al 2015b) thus we expected similar differences in 474
root tissues Our results confirmed that the activities of tonoplast proton-pumps in root tissues 475
of B napus H genotype and A thaliana mutants were lower than those in the L genotype and 476
A thaliana wild type leading to less NO3- influx into vacuoles and more in the cytosol (Fig 1 477
Fig 5A-D) Moreover the enzymes catalyzing N assimilation are located in the cytoplasm 478
where NO3- could be assimilated (Martinoia et al 1981 Han et al 2015a) or immediately 479
loaded into xylem sap for long-distance transport from roots to shoots (Lin et al 2008) 480
Consequently the NO3- concentration in root tissues of the H genotype and A thaliana 481
mutants defective in tonoplast proton-pumps was significantly lower than that in the L 482
genotype (Fig 2E) and in A thaliana wild type (Fig 6C) Therefore the higher proportion of 483
NO3- transported to shoot and the lower concentrations retained in root vacuoles contributed 484
to the higher NUE in Xiangyou15 and A thaliana mutants (vha-a2 vha-a3 and avp1) (Table 485
1Fig 6A) Further studies are needed to assess the impacts of regulating vacuolar 486
sequestration of NO3- and rates of long-distance transport for enhancing NUE in other crop 487
species and genotypes 488
These data suggested that NO3- distribution between vacuoles and cytosol within cells 489
regulates NO3- long-distance transport from root to shoot and ultimately affects NUE NO3
- 490
loading into the xylem sap is an active transport process (Lin et al 2008) therefore not 491
regulated by absolute concentration of NO3- in the cytosol but rather by genes involved in 492
NO3- long-distance transport (NRT15 and NRT18) Apparently the expression of NRT15 the 493
gene responsible for xylem loading is regulated by NO3-concentration in the cytosol 494
Therefore both NO3- short-distance transport across the tonoplast and its long-distance 495
transport from root to shoot are important determinants of NUE in both B napus and A 496
thaliana (Table 1 and Fig 6) 497
498
Roles of NRT15 and NRT18 in NO3- long-distance transport in B napus and A 499
thaliana 500
Our results (Figs S9 S10) showed that the proportion of NO3- distributed from root to 501
shoot is strongly controlled by NRT15 in accord with previous studies (Lin et al 2008 Li et 502
al 2010) The data suggest that the high-NUE genotypes possess lower activities of tonoplast 503
proton-pumps (V-ATPase and V-PPase) resulting in less NO3- accumulation in vacuoles and 504
more NO3- retention in cytosol (Fig 7) The higher NO3
- in the cytosol then up-regulated 505
NRT15 which then down-regulates NRT18 As a result more NO3- is loaded into the xylem 506
system by NRT15 mechanism and less NO3- is unloaded via NRT18 (Fig 7) Differential 507
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18
regulation of these NRT genes in high-NUE plants increased transport of NO3- from root to 508
shoot through the xylem vascular tissues (Fig 7) This coordinated NO3- long- and 509
short-distance transport likely fine-tunes NO3- allocation and modulates the balance of NO3
- 510
distribution between roots and shoots (Fig 7) 511
Our research also revealed differences between A thaliana and B napus in the 512
expression of the wild-type NRT genes in response to NO3- in the protoplast Moreover using 513
A thaliananrt15-3 mutants we uncovered possible regulatory interactions between a 514
functioning (wild-type) NRT15 gene and NRT18 expression in response to NO3- 515
concentration Because mutants defective in nrt15 are not available for B napus we could 516
not confirm that the respective NRT15 and NRT18 systems behave in an analogous way in 517
these plant species Based on Harper et al (2012) research database our homology 518
comparisons and gene function annotations provided strong evidence for the roles of 519
BnNRT15 and BnNRT18 in B napus Finally our studies were conducted using two model 520
dicotyledonous plant species for which we had ample data on responses to specific inhibitors 521
(B napus) or specific mutants were available with results forming the bases for the model 522
presented in Figure 7 Further research using genetically diverse plant species (eg 523
monocotyledonous crops such as rice wheat and corn) will help validate this model and its 524
broader application across species The information generated in this study could have future 525
application for improving NUE in commercial crops with consequent reduction in nitrogen 526
use and benefits to the environment 527
528
Materials and Methods 529
Plant material 530
The two oilseed rape (Bnapus) cultivars used in this study have been characterized 531
before as high- (Xiangyou15 referred to as H genotype hereafter) and low- (814 referred to 532
as L genotype) nitrogen use efficiency (NUE) genotypes (Zhang et al 2009 Han et al 2015b) 533
Here we define NUE as the total biomass per unit of N uptake by the plant with total 534
biomass includes roots shoots and grains The two genotypes were provided by the Hunan 535
Sub-Center of Improvement Center of National Oil Crops Hunan China 536
The Arabidopsis thaliana wild-type Columbia-0 (col-0) was used as control for 537
V-ATPase (vha-a2 and vha-a3) V-PPase (avp1) nrt15-3 and nrt18-2 mutants whereas the A 538
thaliana wild-type Wassilewskija (Ws) was used as control for clca-2 mutants A thaliana 539
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19
mutants and transgenic lines (nrt15-3 and nrt18-2) have been described previously (Chen et 540
al 2012 Zhang et al 2014) The mutant lines vha-a2 (Salk_142642) and vha-a3 541
(Salk_122135) described by Krebs et al (2010) were obtained from the Arabidopsis 542
Biological Resources Center (ABRC) The mutant line avp1 (GK-005004) described in Li et 543
al (2005) was obtained from the European Arabidopsis Stock Centre while the mutant line 544
clca-2 (FST 171A06) described in Angeli et al (2006) was from the Institute National de la 545
Recherche Agronomique (INRA) collection in France together with the T-DNA mutants used 546
for genotyping to select pure mutant lines 547
548
Growth conditions 549
B napus plants were grown hydroponically in ceramic pots(20cmtimes15cm) filled with a 550
nutrient solution in the greenhouse under natural light as described in Han et al (2015b) The 551
pots were arranged in a completely randomized design with six biological replications The 552
nutrient solution was replaced every 3 days and its pH adjusted to 55 daily Experiments were 553
conducted at the field station of Hunan Agricultural University Southern China 554
A thaliana plants were grown in a nutrient solution in plastic pots as described in Arteca 555
et al (2000) and Gong et al (2003) The solution was changed every 3 days with pH adjusted 556
daily to 58 Pots were arranged in a completely randomized design with six biological 557
replications The nutrient solution used for both species consisted of 125mM KNO3 558
0625mM KH2PO4 05mM MgSO4 05mM Ca (NO3)2middot4H2O 0025mM Fe-EDTA 025 559
mlL-1 micronutrients (stock solution concentrations 70mM B 14mM Mn 1mM Zn 05mM 560
Cu and 02mM Mo) The experiments were conducted at Hunan Agricultural University in a 561
phytotron set at 70 relative humidity16h-light8h-dark cycle and constant temperature of 562
22degC 563
564
Experimental treatments and phenotyping 565
All measurements made on B napus plants were conducted either at the seedling stage 566
(2 months after transplanting) or at flowering (5 months after transplanting) The whole root 567
tissue was harvested and used for analyses at either stage For shoot measurements the fourth 568
leaf from the bottom was used for measurements made at seedling stage whereas the 12th leaf 569
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
from the bottom was harvested for measurements made at flowering stage For A thaliana 570
whole root and shoot of four-week-old plants were sampled and used for different assays 571
572
Vacuolar proton-pumps inhibitor treatments 573
Inhibition of vacuolar proton pumps of B napus (Xiangyou15) was conducted at the seedling 574
stage in hydroponic culture as described in Han et al (2015a) with minor modifications 575
Plants grown in hydroponic solution were used as control Inhibitor treatment was conducted 576
using several chemicals Bafi(25 nmol L-1 Bafilomycin A1) an inhibitor of V-ATPase 577
DCCD(10 μmol L-1DCCD + 50 mmolL-1 Na2SO3) specifically inhibits V-PPase and 578
Bafi+DCCD in a 11 ratio as inhibitor of both V-ATPase and V-PPase The inhibitors were 579
applied in the hydroponic solutions for 24 h Fresh plant tissue samples from all treatments 580
were collected for different assays 581
582
NO3- induced gene expression 583
Four-week old A thaliana mutants (nrt15-3nrt18-2) were grown in hydroponics with 584
225mM (NH4)2succinate for 3 days and shifted to hydroponics with 45mM NO3- for 12 h 585
then root tissues were collected to assess relative expression of AtNRT15 and AtNRT18 586
genes Seedlings of B napus (Xiangyou15) plants grown hydroponically were treated with 587
5mM (NH4)2succinate for 3 d and shifted to fresh hydroponic solution containing 15 mM 588
NO3- for 12 h Root tissue was then collected to assess the relative expression of BnNRT15 589
and BnNRT18 genes using quantitative RT-PCR 590
591
ACC treatment 592
Hydroponically grown seedlings of B napus were subjected to ACC treatment as described in 593
Zhang et al (2014) with minor modifications Seedlings of the two genotypes Xiangyou15 594
and 814 were transferred to a nutrient solution containing 002mM L-1 ACC for 6 h or grown 595
in normal hydroponic solution as control Root tissues were then collected to assay relative 596
expression of BnNRT15 and BnNRT18 using quantitative RT-PCR Another set of B napus 597
plants were grown in ACC-containing hydroponic solution but supplemented with 15NO3- (22 598
15N) replacing the unlabeled NO3- for 1 h Root and shoot tissues were collected separately 599
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21
for 15N measurement using a continuous-flow isotope ratio mass spectrometer coupled with a 600
C-N elemental analyzer (ANCA-MS PDZ Europa) 601
602
Biomass and N concentration 603
Grain yield dry biomass and N concentration were determined in plant samples at 604
harvest Plants were harvested (including senescing leaves) then dried in an oven for 30 min 605
at 105degC then at 70degC to a constant weight and weighed N concentration was determined 606
using the Kjeldahl method (Shi et al 2010 Han et al 2015b)02g of dried samples were 607
digested with H2SO4-H2O2 in a 100-ml Kjeldahl digestion flask after which N concentration 608
was determined using a Foss Auto Analyser Unit (Kjeldahl 8400) 609
610
V-ATPase and V-PPase activities 611
Roots (10 g) of B napus were collected at seedling and flowering stages and that of A 612
thaliana (05 g) sampled at seedling stage then used for determining the activities of 613
V-ATPase and V-PPase (Han et al 2015b) Activities of these two energy pumps within 614
microsomal membranes were followed colorimetrically based on released Pi as described by 615
Zhu et al (2001) and Krebs et al (2010) 616
617
Assay of NO3- flux in vacuoles 618
Roots of both species were collected similarly as that used for V-ATPase and V-PPase 619
activities then used for vacuole isolation and measurement of NO3- flux in vacuoles as 620
described by Robert et al (2007) with minor modification (Han et al 2015b) Net fluxes of 621
NO3- in vacuoles were measured non-invasively using SIET (scanning ion-selective electrode 622
technique SIET system BIO-003A Younger USA Science and Technology Corporation 623
USA) An NO3--selective microelectrode used for the assay of NO3
- flux was vibrated in the 624
measuring solution between two positions 1 μm and 11 μm from the vacuole surface 625
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles Background 626
signal was recorded by vibrating the electrode in the measuring solution in the absence of 627
vacuoles Ion flux was calculated by Fickrsquos law of diffusion J = -D(dcdx) where J represents 628
the ion flux (picomolescm-2s-1) dcdx is the ion concentration gradient and D is the ion 629
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
diffusion constant in a particular medium The direction of the flux was derived from Fickrsquos 630
law of diffusion that relates the concentration gradient 631
632
Isolation of intact protoplasts and vacuoles for determining NO3- concentration 633
Similar to the above assays root tissues (10 g) were collected from B napus at seedling 634
and flowering stages and from A thaliana (05 g) seedlings and used to isolate intact 635
protoplasts and vacuoles as described by Robert et al (2007) with minor modifications as 636
outlined in Huang et al (2012) and Han et al(2015b) The purified protoplasts were divided 637
into two equal aliquots with one of them used for isolation of vacuoles The purified 638
protoplasts and vacuoles were then sub-sampled and used to determine NO3- concentrations 639
(Vogeli-Lange and Wagner 1990) and for enzyme activity assays (Ma et al 2005) 640
NO3-concentrations in protoplasts and vacuoles were measured by a continuous-flow 641
auto-analyzer (Auto Analyser 3 Bran and Luebbe Germany) as described previously (Han et 642
al 2015b)The activities of acid phosphatase (ACP) and cytochrome oxidase (COX) were 643
determined using plant ACP colorimetry- and COX-assay kits (GenMedSci Inc) following 644
the manufacturerrsquos instructions ACP activity specific to vacuoles was determined and used to 645
normalize NO3- accumulation We measured NO3
- in the protoplast outside the vacuole which 646
includes the cytosol and organelles eg mitochondria and Golgi Apparatus (Robert et 647
al2007) Since most of the NO3- in the protoplast outside the vacuole is located in the cytosol 648
(Krebs et al 2010) we refer to NO3- distribution between vacuoles and cytosol rather than 649
vacuole versus protoplast 650
651
Quantitative RT-PCR 652
Total RNA extracted from B napus roots was prepared using Trizol reagent (Invitrogen) 653
cDNAs were synthesized using M-MLV reverse transcriptase (Promega) following 654
manufacturerrsquos protocol The relative expression of BnNRT15 (EV220114) BnNRT18 655
(EV116423) and Bnactin (AF1118121) genes in plant roots were determined by quantitative 656
RT-PCR and run on a LightCycler instrument (Roche) with the SYBR Green Real-Time PCR 657
Master Mix Kit (TOYOBO Japan) under the following conditions 95degC for 2min then 45 658
cycles of 95degC for 10s 60degC for 10s and 72degC for 20s The primer sequences of BnNRT15 659
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
BnNRT18 and Bnactin genes were obtained from httpbrassicanbiacuk as described in 660
Harper et al (2012) and expression levels were normalized to Bnactin as control 661
Total RNA extracted from the 4-week-old A thaliana roots under the treatments 662
conditions described above were prepared using Trizol reagent (Invitrogen) The cDNAs were 663
synthesized using M-MLV reverse transcriptase (Promega) based on the manufacturerrsquos 664
protocol and as described in Li et al (2010)The primer sequences of quantitative RT-PCR 665
used in these assays are listed in Supplemental Table 2 and were described previously (Chen 666
et al 2012 Zhang et al 2014) and their expression was normalized to Atactin2 as control 667
668
Determination of nitrate distribution using 15NO3- 669
Hydroponically grown B napus (seedling and flowering stages) and A thaliana 670
(seedling) were transferred to 01mM CaSO4 solution for 1min then to their respective 671
hydroponic nutrient solutions with15NO3- (22 excess) replacing unlabeled NO3
- for 1h 672
Plants were then transferred to 01mM CaSO4 solution for 1min after which roots were 673
washed with deionized water Root and shoot samples were separated and dried at 105degC for 674
30min followed by 70degC for 3 d then grounded and used for assaying 15N content using a 675
continuous-flow isotope ratio mass spectrometer coupled with a carbon-nitrogen elemental 676
analyzer (ANCA-MS PDZ Europa) 677
678
Xylem sap collection and assay of nitrate concentration 679
We used the method of Tang et al (2012) for collecting xylem sap Plants were cut 680
leaving1cm segments with intact roots at seedling and flowering stages for B napus and at 681
seedling stage for A thaliana Roots were immediately immersed in their respective nutrition 682
solutions Weighed cotton was put on the cut surface to absorb extruding xylem sap for 1 h 683
The cotton was then wrapped in plastic film and the volume of xylem sap was calculated as 684
the weight gain of the cotton Xylem sap was then squeezed from the cotton using a syringe 685
and used for subsequent assay of nitrate concentration using a continuous-flow auto-analyzer 686
(Auto Analyser 3 Bran and Luebbe Germany) 687
To determine nitrate concentration samples (10 g fresh root and shoot at seedling and 688
flowering of B napus and 05 g fresh root and shoot of 4-week-old A thaliana) were frozen 689
in liquid N2 and ground with a mortar and pestle the powder was then transferred to a beaker 690
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
containing 70 ml deionized water and boiled for 30 min to extract nitrate then cooled and 691
made to 100 ml volume and 02 g activated carbon added to eliminate the effect of 692
chlorophyll The mixture was filtered and nitrate in the filtrate was determined using a 693
continuous-flow auto-analyser (Atuo-Analyser 3 Bran and Luebbe Germany) 694
695
Measurements of photosynthesis 696
All measurements of photosynthesis and related parameters were conducted at 1000 h 697
using intact leaves The 4th leaf from the bottom in B napus at seedling stage and the 12th leaf 698
from the bottom at flowering stage were used for determining chlorophyll concentration 699
photosynthetic rate intercellular CO2 concentration transpiration rate and stomatal 700
conductance Chlorophyll concentration was determined by SPAD-502 (Minolta Camera Co 701
Ltd Japan) (Luo et al 2006 Yandeau-Nelson et al 2011) Photosynthetic rate intercellular 702
CO2 concentration transpiration rate and stomatal conductance were measured using LI-6400 703
Portable Photosynthesis System (Li-Cor Biosciences Co Ltd USA) set at flow rate of 500 704
μmol s-1 photosynthetic photon flux density of 1000 μmol m-2 s-1 relative humidity of 65 705
CO2 concentration of 400ppm and ambient temperatures 706
707
NR and GS activities 708
Leaves with similar age as those used for photosynthesis in B napus were used for the 709
assay of nitrate reductase (NR) and glutamine synthetase (GS) activities NR activity was 710
measured by the modified method of Fan et al (2007) Fresh samples (05g) were frozen in 711
liquid N2 ground into powder in the presence of acid-washed sand and homogenized with 4 712
ml of extraction buffer (0025mol L-1phosphate buffer pH87 1211g L-1 cysteine 0372g L-1 713
EDTA) Homogenates were centrifuged at 30000timesg for 15 min at 4degC and the supernatants 714
were treated with sulfanilamide and α-naphthylamine reagents for colorimetric (540nm) 715
determination of nitrite as described by Fan et al (2007) 716
GS activity was determined using a modified reverseγ-glutamyltransferase method 717
(Wang et al2014) which measures the GS-catalyzed formation of glutamyl-γ-hydroxamate 718
from glutamine and hydroxylamine Fresh samples were frozen for 30 min at -20degC then 719
ground in 10ml Tris-HCl buffer and acid-washed sand The homogenates were filtered 720
through two layers of gauze and centrifuged at 8000timesg for 15 min at 4degC About 12ml of the 721
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
supernatant was added in a reaction mixture and treated as described (Wang et al2014) After 722
the GS-catalyzed reaction was stopped and the solution centrifuged glutamyl-γ723
-hydroxamate was quantified colorimetrically in the supernatant (at 485 nm) and the 724
concentration was determined using a standard curve 725
726
Statistical analyses 727
We used the SPSS software (Statistical Product and Service Solutions V130 USA) for 728
ANOVA and mean separation of main-effects and interactions using Duncanrsquos multiple range 729
test at Plt005Values are presented as means and SD of three or six replicates from three 730
independent experiments Different letters or an asterisk () associated with specific data (eg 731
at the top of histogram bars in figures or within tables) denote significant differences at 732
Plt005 733
734
Supplemental Data 735
Supplemental Figure 1 B napus with higher NUE showed lower vacuolar sequestration 736
capacity (VSC) for NO3-in roots at flowering stage 737
Supplemental Figure 2 B napus with higher NUE showed enhanced long-distance transport 738
of NO3- from roots to shoots at flowering stage 739
Supplemental Figure 3 The two B napus (H and L genotypes) showed the same total N per 740
plant at seedling stage (A) and flowering stage (B) 741
Supplemental Figure 4 B napus with higher NUE showed increased NO3- concentration in 742
the xylem sap at seedling and flowering stages 743
Supplemental Figure 5 NO3- concentration in the xylem sap as affected by inhibitor 744
treatments in B napus and in the energy pumpsrsquo mutants of A thaliana (col-0 vha-a2 vha-a3 745
avp1) 746
Supplemental Figure 6 Reduced VSC for NO3- in roots drives long-distance transport of 747
NO3- from roots to shoots in the A thaliana wild type (Ws) and mutant (clca-2) 748
Supplemental Figure 7 Differences of NR and GS activities between the two B napus (H 749
and L genotypes) at seedling and flowering stages 750
Supplemental Figure 8 Amino acid sequences of BnNRT15 and BnNRT18 751
Supplemental Figure 9 Functions of AtNRT15 and AtNRT18 genes in root tissues of A 752
thaliana in controlling NO3- long-distance transport from root to shoots 753
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
Supplemental Figure 10 B napus BnNRT15 and BnNRT18 genes in roots were 754
coordinately modulated to facilitate NO3- long distance transport from roots to shoots 755
Supplemental Table 1 Differences in stomatal conductance and transpiration rate between 756
the two B napus genotypes 757
Supplemental Table 2 Sequences of primera used for qRT-PCR 758
759
Acknowledgements 760
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 761
Shanghai Institutes for Biological Sciences) for providing nrt15-3 and nrt18-2 seeds 762
This study was supported by the National Natural Science Foundation of China (Grant 763
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 764
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 765
The Ministry of Education in China Open Novel Science Foundation of Hunan province 766
(13K062) the National Key Laboratory of Plant Molecular Genetics and the Twelfth 767
Five-Year National Science and technology support program (2012BAD15BO4) 768
769
Figure 1 B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 770
NO3- in roots at the seedling stage 771
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 772
genotype 814 Specific activities of the tonoplast proton pumps are expressed as μmol Pi 773
released mg-1 protein h-1 NO3- fluxes are expressed as pmol NO3
- cm-2 S-1 Mature vacuoles 774
were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 775
measuring solution between the two positions 1 μm and 11 μm from the vacuole surface 776
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles recording the 777
stable reading data The background was recorded by vibrating the electrode in measuring 778
solution without vacuoles 779
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 780
for NO3- content and NO3
- accumulation normalized against the specific activity of the 781
vacuole acid phosphatase (ACP) as described in Materials and Methods and plotted as μmol 782
NO3- per μmol p-nitrophenol the end product of ACP assay 783
Proton pump activities in root tissues between H and L genotypes are shown at the seedling 784
stage (A) Different letters at the top of the histogram bars denote significant differences of 785
V-ATPase in root tissues between H and L genotypes (Plt005) an asterisk () at the top of the 786
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
histogram bars indicates significant difference in V-PPase activity in root tissues between H 787
and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788
Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789
genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790
bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791
bars on the figures indicate SD (n=6) 792
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793
seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794
to the total NO3- in protoplasts 795
NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796
total NO3-in the cytosol and was calculated as total NO3
- in protoplasts ndash total NO3- in 797
vacuole Different letters at the top of histogram bars denote significant differences of total 798
NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799
(n=6) 800
801
Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802
roots to shoots at the seedling stage 803
H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804
Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805
the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806
as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807
the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808
the histogram bars denote significant differences at Plt005 809
Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810
in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811
genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812
of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813
SD (n=3) different letters at the top of the histogram bars denote significant differences at 814
Plt005 level 815
The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816
at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817
genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818
different letters at the top of the histogram bars denote significant differences at Plt005 819
820
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
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31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
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Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
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b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
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Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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Discussion 373
Nitrate long-distance transport from root to shoot is regulated by cytosolic NO3- in roots 374
NO3- long-distance transport from root to shoot is controlled by NRT gene family For 375
instance xylem NO3- loading and unloading are mainly controlled by NRT15 and NRT18 376
respectively (Lin et al 2008 Li et al 2010) Whereas the NO3- short-distance transport 377
between the vacuole and cytosol is controlled by tonoplast proton-pumps (V-ATPase and 378
V-PPase) (Krebs et al 2010) and by activity of the CLCa channel in the tonoplast membrane 379
(Angeli et al 2006 Wege et al 2014) tonoplast proton-pumps provide the energy required 380
for NO3- accumulation into the vacuole through CLCa (Gaxiola et al 2001 Brux et al 2008 381
Krebs et al 2010) The expression of NRT15 is up-regulated by NO3- in plant culture 382
solution (Lin et al 2008) whereas regulation of NRT18 expression is contrary to that of 383
NRT15 under both control and abiotic stress conditions (Li et al 2010 Chen et al 2012 384
Zhang et al 2014) 385
Despite the progress made in characterizing the genes involved in transport and 386
metabolism of NO3- in plants the relationship between long- and short-distance transport and 387
how these two pathways are regulated have not been thoroughly investigated We hypothesize 388
that NRT15 expression and its role in long-distance transport is regulated by NO3- in plant 389
tissue as was observed in culture solution (Lin et al 2008) and our data support this 390
hypothesis Both BnNRT15 and AtNRT15 expression were up-regulated by increasing 391
cytosolic NO3- in roots (Fig 4 Fig 5) The increase in cytosolic NO3
- was achieved through (i) 392
use of A thaliana mutants defective in proton-pumps (V-ATPase and V-PPase) or CLCa 393
activities in the tonoplast and (ii) inhibition of B napus tonoplast proton-pumps In both 394
model plants the influx of NO3- into vacuoles was substantially reduced resulting in higher 395
concentrations of NO3- in the cytosol (Fig 4A-D Fig 5A-D Fig S6A-C) Under those 396
conditions expression of BnNRT15 and AtNRT15 was up-regulated by the enhanced 397
cytosolic NO3- while expression of BnNRT18 and AtNRT18 was down-regulated (Fig 4EF 398
Fig 5EF FigS6DE) As a consequence more NO3- was loaded into the xylem sap and 399
transported from root to shoot (Fig S5AB FigS6F) This is also reflected as increased 400
shootroot ratios of N traced by [15N] and [NO3-] (Fig 4GH Fig 5GH Fig S6GH) 401
Interestingly the expression of both BnNRT15 and AtNRT15 is up-regulated by 402
increased cytosolic NO3- concentration in roots but were not affected by NO3
- sequestered 403
into vacuoles (Fig 4 Fig 5 Fig S6) This is because vacuolar NO3- is functionally separated 404
by the tonoplast and cannot be assimilated by enzymes that are localized in the cytosol 405
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15
Therefore NO3--inducible genes such as NR and NRT15 were not influenced by vacuolar 406
NO3- concentration (Martinoia et al 1981 Martinoia et al 2000 Zhang et al 2012) In 407
addition NO3- concentration in the cytosol is controlled by its short-distance transport through 408
the tonoplast which is mediated through the tonoplast proton-pumps providing the required 409
energy for its sequestration into vacuoles against concentration gradient (Gaxiola et al 2001 410
Brux et al 2008 Krebs et al 2010) The gradient of H+ between the inside of the vacuole 411
and the cytosol is maintained by an 2NO3-1H+ antiporter mechanism that facilitatesNO3
- 412
transport through CLCa (Angeli et al 2006 Wege et al 2014) Our data provide clear 413
evidence that NO3- long-distance transport from root to shoot is regulated by cytosolic NO3
- 414
concentration in roots 415
Regulatory mechanisms that control the expression of NRT18 and NRT15 as mediated 416
by NO3- are still unknown however previous studies showed that their expression is also 417
affected by abiotic stresses whereby NRT15 is down-regulated and NRT18 is up-regulated 418
(Li et al 2010 Zhang et al 2014) AtNRT18 expression in A thaliana nrt15 mutants is 419
highly up-regulated (Chen et al 2012) as was also observed here (Fig 3C) Moreover 420
AtNRT15 expression was enhanced by NO3- in culture solution and was not affected in 421
nrt18-2 mutant (Fig 3AB) On the other hand AtNRT18 expression was not affected by 422
cytosolic NO3- but down-regulated when AtNRT15 expression was up-regulated by NO3
-(Fig 423
S6DE) Analogous results were also obtained with B napus genotypes (Fig 3EF) Based on 424
these results we argue that NRT18 expression is not directly influenced by cytosolic NO3- but 425
rather by NRT15 expression (Fig 4 Fig 5 and Fig S6) suggesting NRT18 is probably 426
acting downstream of NRT15 Apparently the regulatory mechanisms of this NRT gene 427
family still awaits further studies to be elucidated 428
429
Response of NUE to NO3- long-distance transport 430
NO3- assimilation efficiency is higher in shoot than in root tissues and can be further 431
enhanced by carbon assimilation (Smirnoff et al 1985 Tang et al 2013) Therefore 432
translocation of higher proportion of NO3- from roots to shoots likely contributes to better 433
crop growth and higher NUE (Andrews et al 1986 Tang et al 2012) The present study 434
provides direct evidence for these results using contrasting B napus genotype identified 435
before (Zhang et al 2009 Han et al 2015a) Our data confirmed the genetic variation in 436
NUE between these two genotypes (Table 1) 437
We measured NO3- distribution in root and shoot of these genotypes to elucidate the 438
physiological mechanisms contributing to variation in NUE The data were consistent with 439
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16
our hypothesis that concentration of NO3- in the xylem sap and shoot would be significantly 440
higher in the H genotype than in the L genotype (Fig 2EFFig S2EF Fig S4) The NO3- 441
long-distance transport from root to shoot was affected by cytosolic NO3- in roots of both A 442
thaliana and B napus genotypes (Fig 4-5 Fig S6) The activities of the tonoplast 443
proton-pumps in roots were significantly lower in the H genotype than in the L genotype (Fig 444
1AB) Consequently less NO3- was sequestered into the vacuole and more NO3
- was retained 445
in cytosol (Fig 1CD) BnNRT15 expression was then up-regulated resulting in greater 446
loading into the xylem while BnNRT18 was down-regulated (Fig 2AB) probably to 447
suppress NO3- unloading from xylem sap Consequently a higher proportion of NO3
- was 448
transported from root to shoots in the H genotype(Fig 2C-D Fig S4) 449
Moreover photosynthetic carbon fixation (Table 2) and activities of both NR and GS 450
were higher in shoots of the H genotype (Fig S7) both of which promote higher N 451
assimilation These results agreed with previous reports (Smirnoff et al 1985 Andrews et al 452
1986 Tang et al 2012 Tang et al 2013) where higher NO3- transport from root to shoot 453
higher photosynthetic rate and N assimilation efficiency in shoots were suggested as essential 454
mechanisms for high-NUE in crop plants These qualities endowed the B napus H genotype 455
Xiangyou15 with higher NUE as compared with the L genotype 814 456
NO3- long-distance transport in B napus is not only regulated by BnNRT15 and 457
BnNRT18 in root tissues (Fig 4 Fig 5 Fig S6) but also by stomatal conductance and 458
transpiration by indirectly controlling xylem sap flow and long-distance transport 459
(Dechorgnat et al 2011 Krapp et al 2014) Stomatal conductance and transpiration rates of 460
the H genotype were significantly higher than those of the L genotype (Table S1) a result 461
consistent with other studies describing characteristics of high-NUE plants (Daniel-Vedele et 462
al 1998 Wilkinson et al 2007 Dechorgnat et al 2011 Krapp et al 2014) 463
464
Response of NUE to NO3- short-distance transport 465
NUE is also strongly controlled by NO3- short-distance distribution between the vacuole 466
and cytosol (Han et al 2015a) A lower proportion of NO3- accumulating in vacuoles and a 467
higher proportion retained in the cytosol will contribute to better NUE in crop plants (Han et 468
al 2015b) The energy required for vacuolar NO3- sequestration is provided by the tonoplast 469
proton-pumps (Gaxiola et al 2001 Brux et al 2008 Krebs et al 2010) while the 470
distribution channel CLCa enables transport of NO3- across the vacuolar membrane (Angeli et 471
al 2006 Wege et al 2014) The activities of the tonoplast proton-pumps (V-ATPase and 472
V-PPase) in leaves of the H genotype were significantly lower than those of the L genotype at 473
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17
flowering stages (Han et al 2015aHan et al 2015b) thus we expected similar differences in 474
root tissues Our results confirmed that the activities of tonoplast proton-pumps in root tissues 475
of B napus H genotype and A thaliana mutants were lower than those in the L genotype and 476
A thaliana wild type leading to less NO3- influx into vacuoles and more in the cytosol (Fig 1 477
Fig 5A-D) Moreover the enzymes catalyzing N assimilation are located in the cytoplasm 478
where NO3- could be assimilated (Martinoia et al 1981 Han et al 2015a) or immediately 479
loaded into xylem sap for long-distance transport from roots to shoots (Lin et al 2008) 480
Consequently the NO3- concentration in root tissues of the H genotype and A thaliana 481
mutants defective in tonoplast proton-pumps was significantly lower than that in the L 482
genotype (Fig 2E) and in A thaliana wild type (Fig 6C) Therefore the higher proportion of 483
NO3- transported to shoot and the lower concentrations retained in root vacuoles contributed 484
to the higher NUE in Xiangyou15 and A thaliana mutants (vha-a2 vha-a3 and avp1) (Table 485
1Fig 6A) Further studies are needed to assess the impacts of regulating vacuolar 486
sequestration of NO3- and rates of long-distance transport for enhancing NUE in other crop 487
species and genotypes 488
These data suggested that NO3- distribution between vacuoles and cytosol within cells 489
regulates NO3- long-distance transport from root to shoot and ultimately affects NUE NO3
- 490
loading into the xylem sap is an active transport process (Lin et al 2008) therefore not 491
regulated by absolute concentration of NO3- in the cytosol but rather by genes involved in 492
NO3- long-distance transport (NRT15 and NRT18) Apparently the expression of NRT15 the 493
gene responsible for xylem loading is regulated by NO3-concentration in the cytosol 494
Therefore both NO3- short-distance transport across the tonoplast and its long-distance 495
transport from root to shoot are important determinants of NUE in both B napus and A 496
thaliana (Table 1 and Fig 6) 497
498
Roles of NRT15 and NRT18 in NO3- long-distance transport in B napus and A 499
thaliana 500
Our results (Figs S9 S10) showed that the proportion of NO3- distributed from root to 501
shoot is strongly controlled by NRT15 in accord with previous studies (Lin et al 2008 Li et 502
al 2010) The data suggest that the high-NUE genotypes possess lower activities of tonoplast 503
proton-pumps (V-ATPase and V-PPase) resulting in less NO3- accumulation in vacuoles and 504
more NO3- retention in cytosol (Fig 7) The higher NO3
- in the cytosol then up-regulated 505
NRT15 which then down-regulates NRT18 As a result more NO3- is loaded into the xylem 506
system by NRT15 mechanism and less NO3- is unloaded via NRT18 (Fig 7) Differential 507
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18
regulation of these NRT genes in high-NUE plants increased transport of NO3- from root to 508
shoot through the xylem vascular tissues (Fig 7) This coordinated NO3- long- and 509
short-distance transport likely fine-tunes NO3- allocation and modulates the balance of NO3
- 510
distribution between roots and shoots (Fig 7) 511
Our research also revealed differences between A thaliana and B napus in the 512
expression of the wild-type NRT genes in response to NO3- in the protoplast Moreover using 513
A thaliananrt15-3 mutants we uncovered possible regulatory interactions between a 514
functioning (wild-type) NRT15 gene and NRT18 expression in response to NO3- 515
concentration Because mutants defective in nrt15 are not available for B napus we could 516
not confirm that the respective NRT15 and NRT18 systems behave in an analogous way in 517
these plant species Based on Harper et al (2012) research database our homology 518
comparisons and gene function annotations provided strong evidence for the roles of 519
BnNRT15 and BnNRT18 in B napus Finally our studies were conducted using two model 520
dicotyledonous plant species for which we had ample data on responses to specific inhibitors 521
(B napus) or specific mutants were available with results forming the bases for the model 522
presented in Figure 7 Further research using genetically diverse plant species (eg 523
monocotyledonous crops such as rice wheat and corn) will help validate this model and its 524
broader application across species The information generated in this study could have future 525
application for improving NUE in commercial crops with consequent reduction in nitrogen 526
use and benefits to the environment 527
528
Materials and Methods 529
Plant material 530
The two oilseed rape (Bnapus) cultivars used in this study have been characterized 531
before as high- (Xiangyou15 referred to as H genotype hereafter) and low- (814 referred to 532
as L genotype) nitrogen use efficiency (NUE) genotypes (Zhang et al 2009 Han et al 2015b) 533
Here we define NUE as the total biomass per unit of N uptake by the plant with total 534
biomass includes roots shoots and grains The two genotypes were provided by the Hunan 535
Sub-Center of Improvement Center of National Oil Crops Hunan China 536
The Arabidopsis thaliana wild-type Columbia-0 (col-0) was used as control for 537
V-ATPase (vha-a2 and vha-a3) V-PPase (avp1) nrt15-3 and nrt18-2 mutants whereas the A 538
thaliana wild-type Wassilewskija (Ws) was used as control for clca-2 mutants A thaliana 539
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19
mutants and transgenic lines (nrt15-3 and nrt18-2) have been described previously (Chen et 540
al 2012 Zhang et al 2014) The mutant lines vha-a2 (Salk_142642) and vha-a3 541
(Salk_122135) described by Krebs et al (2010) were obtained from the Arabidopsis 542
Biological Resources Center (ABRC) The mutant line avp1 (GK-005004) described in Li et 543
al (2005) was obtained from the European Arabidopsis Stock Centre while the mutant line 544
clca-2 (FST 171A06) described in Angeli et al (2006) was from the Institute National de la 545
Recherche Agronomique (INRA) collection in France together with the T-DNA mutants used 546
for genotyping to select pure mutant lines 547
548
Growth conditions 549
B napus plants were grown hydroponically in ceramic pots(20cmtimes15cm) filled with a 550
nutrient solution in the greenhouse under natural light as described in Han et al (2015b) The 551
pots were arranged in a completely randomized design with six biological replications The 552
nutrient solution was replaced every 3 days and its pH adjusted to 55 daily Experiments were 553
conducted at the field station of Hunan Agricultural University Southern China 554
A thaliana plants were grown in a nutrient solution in plastic pots as described in Arteca 555
et al (2000) and Gong et al (2003) The solution was changed every 3 days with pH adjusted 556
daily to 58 Pots were arranged in a completely randomized design with six biological 557
replications The nutrient solution used for both species consisted of 125mM KNO3 558
0625mM KH2PO4 05mM MgSO4 05mM Ca (NO3)2middot4H2O 0025mM Fe-EDTA 025 559
mlL-1 micronutrients (stock solution concentrations 70mM B 14mM Mn 1mM Zn 05mM 560
Cu and 02mM Mo) The experiments were conducted at Hunan Agricultural University in a 561
phytotron set at 70 relative humidity16h-light8h-dark cycle and constant temperature of 562
22degC 563
564
Experimental treatments and phenotyping 565
All measurements made on B napus plants were conducted either at the seedling stage 566
(2 months after transplanting) or at flowering (5 months after transplanting) The whole root 567
tissue was harvested and used for analyses at either stage For shoot measurements the fourth 568
leaf from the bottom was used for measurements made at seedling stage whereas the 12th leaf 569
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20
from the bottom was harvested for measurements made at flowering stage For A thaliana 570
whole root and shoot of four-week-old plants were sampled and used for different assays 571
572
Vacuolar proton-pumps inhibitor treatments 573
Inhibition of vacuolar proton pumps of B napus (Xiangyou15) was conducted at the seedling 574
stage in hydroponic culture as described in Han et al (2015a) with minor modifications 575
Plants grown in hydroponic solution were used as control Inhibitor treatment was conducted 576
using several chemicals Bafi(25 nmol L-1 Bafilomycin A1) an inhibitor of V-ATPase 577
DCCD(10 μmol L-1DCCD + 50 mmolL-1 Na2SO3) specifically inhibits V-PPase and 578
Bafi+DCCD in a 11 ratio as inhibitor of both V-ATPase and V-PPase The inhibitors were 579
applied in the hydroponic solutions for 24 h Fresh plant tissue samples from all treatments 580
were collected for different assays 581
582
NO3- induced gene expression 583
Four-week old A thaliana mutants (nrt15-3nrt18-2) were grown in hydroponics with 584
225mM (NH4)2succinate for 3 days and shifted to hydroponics with 45mM NO3- for 12 h 585
then root tissues were collected to assess relative expression of AtNRT15 and AtNRT18 586
genes Seedlings of B napus (Xiangyou15) plants grown hydroponically were treated with 587
5mM (NH4)2succinate for 3 d and shifted to fresh hydroponic solution containing 15 mM 588
NO3- for 12 h Root tissue was then collected to assess the relative expression of BnNRT15 589
and BnNRT18 genes using quantitative RT-PCR 590
591
ACC treatment 592
Hydroponically grown seedlings of B napus were subjected to ACC treatment as described in 593
Zhang et al (2014) with minor modifications Seedlings of the two genotypes Xiangyou15 594
and 814 were transferred to a nutrient solution containing 002mM L-1 ACC for 6 h or grown 595
in normal hydroponic solution as control Root tissues were then collected to assay relative 596
expression of BnNRT15 and BnNRT18 using quantitative RT-PCR Another set of B napus 597
plants were grown in ACC-containing hydroponic solution but supplemented with 15NO3- (22 598
15N) replacing the unlabeled NO3- for 1 h Root and shoot tissues were collected separately 599
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
21
for 15N measurement using a continuous-flow isotope ratio mass spectrometer coupled with a 600
C-N elemental analyzer (ANCA-MS PDZ Europa) 601
602
Biomass and N concentration 603
Grain yield dry biomass and N concentration were determined in plant samples at 604
harvest Plants were harvested (including senescing leaves) then dried in an oven for 30 min 605
at 105degC then at 70degC to a constant weight and weighed N concentration was determined 606
using the Kjeldahl method (Shi et al 2010 Han et al 2015b)02g of dried samples were 607
digested with H2SO4-H2O2 in a 100-ml Kjeldahl digestion flask after which N concentration 608
was determined using a Foss Auto Analyser Unit (Kjeldahl 8400) 609
610
V-ATPase and V-PPase activities 611
Roots (10 g) of B napus were collected at seedling and flowering stages and that of A 612
thaliana (05 g) sampled at seedling stage then used for determining the activities of 613
V-ATPase and V-PPase (Han et al 2015b) Activities of these two energy pumps within 614
microsomal membranes were followed colorimetrically based on released Pi as described by 615
Zhu et al (2001) and Krebs et al (2010) 616
617
Assay of NO3- flux in vacuoles 618
Roots of both species were collected similarly as that used for V-ATPase and V-PPase 619
activities then used for vacuole isolation and measurement of NO3- flux in vacuoles as 620
described by Robert et al (2007) with minor modification (Han et al 2015b) Net fluxes of 621
NO3- in vacuoles were measured non-invasively using SIET (scanning ion-selective electrode 622
technique SIET system BIO-003A Younger USA Science and Technology Corporation 623
USA) An NO3--selective microelectrode used for the assay of NO3
- flux was vibrated in the 624
measuring solution between two positions 1 μm and 11 μm from the vacuole surface 625
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles Background 626
signal was recorded by vibrating the electrode in the measuring solution in the absence of 627
vacuoles Ion flux was calculated by Fickrsquos law of diffusion J = -D(dcdx) where J represents 628
the ion flux (picomolescm-2s-1) dcdx is the ion concentration gradient and D is the ion 629
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
diffusion constant in a particular medium The direction of the flux was derived from Fickrsquos 630
law of diffusion that relates the concentration gradient 631
632
Isolation of intact protoplasts and vacuoles for determining NO3- concentration 633
Similar to the above assays root tissues (10 g) were collected from B napus at seedling 634
and flowering stages and from A thaliana (05 g) seedlings and used to isolate intact 635
protoplasts and vacuoles as described by Robert et al (2007) with minor modifications as 636
outlined in Huang et al (2012) and Han et al(2015b) The purified protoplasts were divided 637
into two equal aliquots with one of them used for isolation of vacuoles The purified 638
protoplasts and vacuoles were then sub-sampled and used to determine NO3- concentrations 639
(Vogeli-Lange and Wagner 1990) and for enzyme activity assays (Ma et al 2005) 640
NO3-concentrations in protoplasts and vacuoles were measured by a continuous-flow 641
auto-analyzer (Auto Analyser 3 Bran and Luebbe Germany) as described previously (Han et 642
al 2015b)The activities of acid phosphatase (ACP) and cytochrome oxidase (COX) were 643
determined using plant ACP colorimetry- and COX-assay kits (GenMedSci Inc) following 644
the manufacturerrsquos instructions ACP activity specific to vacuoles was determined and used to 645
normalize NO3- accumulation We measured NO3
- in the protoplast outside the vacuole which 646
includes the cytosol and organelles eg mitochondria and Golgi Apparatus (Robert et 647
al2007) Since most of the NO3- in the protoplast outside the vacuole is located in the cytosol 648
(Krebs et al 2010) we refer to NO3- distribution between vacuoles and cytosol rather than 649
vacuole versus protoplast 650
651
Quantitative RT-PCR 652
Total RNA extracted from B napus roots was prepared using Trizol reagent (Invitrogen) 653
cDNAs were synthesized using M-MLV reverse transcriptase (Promega) following 654
manufacturerrsquos protocol The relative expression of BnNRT15 (EV220114) BnNRT18 655
(EV116423) and Bnactin (AF1118121) genes in plant roots were determined by quantitative 656
RT-PCR and run on a LightCycler instrument (Roche) with the SYBR Green Real-Time PCR 657
Master Mix Kit (TOYOBO Japan) under the following conditions 95degC for 2min then 45 658
cycles of 95degC for 10s 60degC for 10s and 72degC for 20s The primer sequences of BnNRT15 659
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
BnNRT18 and Bnactin genes were obtained from httpbrassicanbiacuk as described in 660
Harper et al (2012) and expression levels were normalized to Bnactin as control 661
Total RNA extracted from the 4-week-old A thaliana roots under the treatments 662
conditions described above were prepared using Trizol reagent (Invitrogen) The cDNAs were 663
synthesized using M-MLV reverse transcriptase (Promega) based on the manufacturerrsquos 664
protocol and as described in Li et al (2010)The primer sequences of quantitative RT-PCR 665
used in these assays are listed in Supplemental Table 2 and were described previously (Chen 666
et al 2012 Zhang et al 2014) and their expression was normalized to Atactin2 as control 667
668
Determination of nitrate distribution using 15NO3- 669
Hydroponically grown B napus (seedling and flowering stages) and A thaliana 670
(seedling) were transferred to 01mM CaSO4 solution for 1min then to their respective 671
hydroponic nutrient solutions with15NO3- (22 excess) replacing unlabeled NO3
- for 1h 672
Plants were then transferred to 01mM CaSO4 solution for 1min after which roots were 673
washed with deionized water Root and shoot samples were separated and dried at 105degC for 674
30min followed by 70degC for 3 d then grounded and used for assaying 15N content using a 675
continuous-flow isotope ratio mass spectrometer coupled with a carbon-nitrogen elemental 676
analyzer (ANCA-MS PDZ Europa) 677
678
Xylem sap collection and assay of nitrate concentration 679
We used the method of Tang et al (2012) for collecting xylem sap Plants were cut 680
leaving1cm segments with intact roots at seedling and flowering stages for B napus and at 681
seedling stage for A thaliana Roots were immediately immersed in their respective nutrition 682
solutions Weighed cotton was put on the cut surface to absorb extruding xylem sap for 1 h 683
The cotton was then wrapped in plastic film and the volume of xylem sap was calculated as 684
the weight gain of the cotton Xylem sap was then squeezed from the cotton using a syringe 685
and used for subsequent assay of nitrate concentration using a continuous-flow auto-analyzer 686
(Auto Analyser 3 Bran and Luebbe Germany) 687
To determine nitrate concentration samples (10 g fresh root and shoot at seedling and 688
flowering of B napus and 05 g fresh root and shoot of 4-week-old A thaliana) were frozen 689
in liquid N2 and ground with a mortar and pestle the powder was then transferred to a beaker 690
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
containing 70 ml deionized water and boiled for 30 min to extract nitrate then cooled and 691
made to 100 ml volume and 02 g activated carbon added to eliminate the effect of 692
chlorophyll The mixture was filtered and nitrate in the filtrate was determined using a 693
continuous-flow auto-analyser (Atuo-Analyser 3 Bran and Luebbe Germany) 694
695
Measurements of photosynthesis 696
All measurements of photosynthesis and related parameters were conducted at 1000 h 697
using intact leaves The 4th leaf from the bottom in B napus at seedling stage and the 12th leaf 698
from the bottom at flowering stage were used for determining chlorophyll concentration 699
photosynthetic rate intercellular CO2 concentration transpiration rate and stomatal 700
conductance Chlorophyll concentration was determined by SPAD-502 (Minolta Camera Co 701
Ltd Japan) (Luo et al 2006 Yandeau-Nelson et al 2011) Photosynthetic rate intercellular 702
CO2 concentration transpiration rate and stomatal conductance were measured using LI-6400 703
Portable Photosynthesis System (Li-Cor Biosciences Co Ltd USA) set at flow rate of 500 704
μmol s-1 photosynthetic photon flux density of 1000 μmol m-2 s-1 relative humidity of 65 705
CO2 concentration of 400ppm and ambient temperatures 706
707
NR and GS activities 708
Leaves with similar age as those used for photosynthesis in B napus were used for the 709
assay of nitrate reductase (NR) and glutamine synthetase (GS) activities NR activity was 710
measured by the modified method of Fan et al (2007) Fresh samples (05g) were frozen in 711
liquid N2 ground into powder in the presence of acid-washed sand and homogenized with 4 712
ml of extraction buffer (0025mol L-1phosphate buffer pH87 1211g L-1 cysteine 0372g L-1 713
EDTA) Homogenates were centrifuged at 30000timesg for 15 min at 4degC and the supernatants 714
were treated with sulfanilamide and α-naphthylamine reagents for colorimetric (540nm) 715
determination of nitrite as described by Fan et al (2007) 716
GS activity was determined using a modified reverseγ-glutamyltransferase method 717
(Wang et al2014) which measures the GS-catalyzed formation of glutamyl-γ-hydroxamate 718
from glutamine and hydroxylamine Fresh samples were frozen for 30 min at -20degC then 719
ground in 10ml Tris-HCl buffer and acid-washed sand The homogenates were filtered 720
through two layers of gauze and centrifuged at 8000timesg for 15 min at 4degC About 12ml of the 721
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
supernatant was added in a reaction mixture and treated as described (Wang et al2014) After 722
the GS-catalyzed reaction was stopped and the solution centrifuged glutamyl-γ723
-hydroxamate was quantified colorimetrically in the supernatant (at 485 nm) and the 724
concentration was determined using a standard curve 725
726
Statistical analyses 727
We used the SPSS software (Statistical Product and Service Solutions V130 USA) for 728
ANOVA and mean separation of main-effects and interactions using Duncanrsquos multiple range 729
test at Plt005Values are presented as means and SD of three or six replicates from three 730
independent experiments Different letters or an asterisk () associated with specific data (eg 731
at the top of histogram bars in figures or within tables) denote significant differences at 732
Plt005 733
734
Supplemental Data 735
Supplemental Figure 1 B napus with higher NUE showed lower vacuolar sequestration 736
capacity (VSC) for NO3-in roots at flowering stage 737
Supplemental Figure 2 B napus with higher NUE showed enhanced long-distance transport 738
of NO3- from roots to shoots at flowering stage 739
Supplemental Figure 3 The two B napus (H and L genotypes) showed the same total N per 740
plant at seedling stage (A) and flowering stage (B) 741
Supplemental Figure 4 B napus with higher NUE showed increased NO3- concentration in 742
the xylem sap at seedling and flowering stages 743
Supplemental Figure 5 NO3- concentration in the xylem sap as affected by inhibitor 744
treatments in B napus and in the energy pumpsrsquo mutants of A thaliana (col-0 vha-a2 vha-a3 745
avp1) 746
Supplemental Figure 6 Reduced VSC for NO3- in roots drives long-distance transport of 747
NO3- from roots to shoots in the A thaliana wild type (Ws) and mutant (clca-2) 748
Supplemental Figure 7 Differences of NR and GS activities between the two B napus (H 749
and L genotypes) at seedling and flowering stages 750
Supplemental Figure 8 Amino acid sequences of BnNRT15 and BnNRT18 751
Supplemental Figure 9 Functions of AtNRT15 and AtNRT18 genes in root tissues of A 752
thaliana in controlling NO3- long-distance transport from root to shoots 753
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
Supplemental Figure 10 B napus BnNRT15 and BnNRT18 genes in roots were 754
coordinately modulated to facilitate NO3- long distance transport from roots to shoots 755
Supplemental Table 1 Differences in stomatal conductance and transpiration rate between 756
the two B napus genotypes 757
Supplemental Table 2 Sequences of primera used for qRT-PCR 758
759
Acknowledgements 760
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 761
Shanghai Institutes for Biological Sciences) for providing nrt15-3 and nrt18-2 seeds 762
This study was supported by the National Natural Science Foundation of China (Grant 763
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 764
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 765
The Ministry of Education in China Open Novel Science Foundation of Hunan province 766
(13K062) the National Key Laboratory of Plant Molecular Genetics and the Twelfth 767
Five-Year National Science and technology support program (2012BAD15BO4) 768
769
Figure 1 B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 770
NO3- in roots at the seedling stage 771
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 772
genotype 814 Specific activities of the tonoplast proton pumps are expressed as μmol Pi 773
released mg-1 protein h-1 NO3- fluxes are expressed as pmol NO3
- cm-2 S-1 Mature vacuoles 774
were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 775
measuring solution between the two positions 1 μm and 11 μm from the vacuole surface 776
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles recording the 777
stable reading data The background was recorded by vibrating the electrode in measuring 778
solution without vacuoles 779
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 780
for NO3- content and NO3
- accumulation normalized against the specific activity of the 781
vacuole acid phosphatase (ACP) as described in Materials and Methods and plotted as μmol 782
NO3- per μmol p-nitrophenol the end product of ACP assay 783
Proton pump activities in root tissues between H and L genotypes are shown at the seedling 784
stage (A) Different letters at the top of the histogram bars denote significant differences of 785
V-ATPase in root tissues between H and L genotypes (Plt005) an asterisk () at the top of the 786
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
histogram bars indicates significant difference in V-PPase activity in root tissues between H 787
and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788
Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789
genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790
bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791
bars on the figures indicate SD (n=6) 792
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793
seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794
to the total NO3- in protoplasts 795
NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796
total NO3-in the cytosol and was calculated as total NO3
- in protoplasts ndash total NO3- in 797
vacuole Different letters at the top of histogram bars denote significant differences of total 798
NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799
(n=6) 800
801
Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802
roots to shoots at the seedling stage 803
H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804
Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805
the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806
as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807
the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808
the histogram bars denote significant differences at Plt005 809
Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810
in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811
genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812
of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813
SD (n=3) different letters at the top of the histogram bars denote significant differences at 814
Plt005 level 815
The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816
at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817
genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818
different letters at the top of the histogram bars denote significant differences at Plt005 819
820
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
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b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
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Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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15
Therefore NO3--inducible genes such as NR and NRT15 were not influenced by vacuolar 406
NO3- concentration (Martinoia et al 1981 Martinoia et al 2000 Zhang et al 2012) In 407
addition NO3- concentration in the cytosol is controlled by its short-distance transport through 408
the tonoplast which is mediated through the tonoplast proton-pumps providing the required 409
energy for its sequestration into vacuoles against concentration gradient (Gaxiola et al 2001 410
Brux et al 2008 Krebs et al 2010) The gradient of H+ between the inside of the vacuole 411
and the cytosol is maintained by an 2NO3-1H+ antiporter mechanism that facilitatesNO3
- 412
transport through CLCa (Angeli et al 2006 Wege et al 2014) Our data provide clear 413
evidence that NO3- long-distance transport from root to shoot is regulated by cytosolic NO3
- 414
concentration in roots 415
Regulatory mechanisms that control the expression of NRT18 and NRT15 as mediated 416
by NO3- are still unknown however previous studies showed that their expression is also 417
affected by abiotic stresses whereby NRT15 is down-regulated and NRT18 is up-regulated 418
(Li et al 2010 Zhang et al 2014) AtNRT18 expression in A thaliana nrt15 mutants is 419
highly up-regulated (Chen et al 2012) as was also observed here (Fig 3C) Moreover 420
AtNRT15 expression was enhanced by NO3- in culture solution and was not affected in 421
nrt18-2 mutant (Fig 3AB) On the other hand AtNRT18 expression was not affected by 422
cytosolic NO3- but down-regulated when AtNRT15 expression was up-regulated by NO3
-(Fig 423
S6DE) Analogous results were also obtained with B napus genotypes (Fig 3EF) Based on 424
these results we argue that NRT18 expression is not directly influenced by cytosolic NO3- but 425
rather by NRT15 expression (Fig 4 Fig 5 and Fig S6) suggesting NRT18 is probably 426
acting downstream of NRT15 Apparently the regulatory mechanisms of this NRT gene 427
family still awaits further studies to be elucidated 428
429
Response of NUE to NO3- long-distance transport 430
NO3- assimilation efficiency is higher in shoot than in root tissues and can be further 431
enhanced by carbon assimilation (Smirnoff et al 1985 Tang et al 2013) Therefore 432
translocation of higher proportion of NO3- from roots to shoots likely contributes to better 433
crop growth and higher NUE (Andrews et al 1986 Tang et al 2012) The present study 434
provides direct evidence for these results using contrasting B napus genotype identified 435
before (Zhang et al 2009 Han et al 2015a) Our data confirmed the genetic variation in 436
NUE between these two genotypes (Table 1) 437
We measured NO3- distribution in root and shoot of these genotypes to elucidate the 438
physiological mechanisms contributing to variation in NUE The data were consistent with 439
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16
our hypothesis that concentration of NO3- in the xylem sap and shoot would be significantly 440
higher in the H genotype than in the L genotype (Fig 2EFFig S2EF Fig S4) The NO3- 441
long-distance transport from root to shoot was affected by cytosolic NO3- in roots of both A 442
thaliana and B napus genotypes (Fig 4-5 Fig S6) The activities of the tonoplast 443
proton-pumps in roots were significantly lower in the H genotype than in the L genotype (Fig 444
1AB) Consequently less NO3- was sequestered into the vacuole and more NO3
- was retained 445
in cytosol (Fig 1CD) BnNRT15 expression was then up-regulated resulting in greater 446
loading into the xylem while BnNRT18 was down-regulated (Fig 2AB) probably to 447
suppress NO3- unloading from xylem sap Consequently a higher proportion of NO3
- was 448
transported from root to shoots in the H genotype(Fig 2C-D Fig S4) 449
Moreover photosynthetic carbon fixation (Table 2) and activities of both NR and GS 450
were higher in shoots of the H genotype (Fig S7) both of which promote higher N 451
assimilation These results agreed with previous reports (Smirnoff et al 1985 Andrews et al 452
1986 Tang et al 2012 Tang et al 2013) where higher NO3- transport from root to shoot 453
higher photosynthetic rate and N assimilation efficiency in shoots were suggested as essential 454
mechanisms for high-NUE in crop plants These qualities endowed the B napus H genotype 455
Xiangyou15 with higher NUE as compared with the L genotype 814 456
NO3- long-distance transport in B napus is not only regulated by BnNRT15 and 457
BnNRT18 in root tissues (Fig 4 Fig 5 Fig S6) but also by stomatal conductance and 458
transpiration by indirectly controlling xylem sap flow and long-distance transport 459
(Dechorgnat et al 2011 Krapp et al 2014) Stomatal conductance and transpiration rates of 460
the H genotype were significantly higher than those of the L genotype (Table S1) a result 461
consistent with other studies describing characteristics of high-NUE plants (Daniel-Vedele et 462
al 1998 Wilkinson et al 2007 Dechorgnat et al 2011 Krapp et al 2014) 463
464
Response of NUE to NO3- short-distance transport 465
NUE is also strongly controlled by NO3- short-distance distribution between the vacuole 466
and cytosol (Han et al 2015a) A lower proportion of NO3- accumulating in vacuoles and a 467
higher proportion retained in the cytosol will contribute to better NUE in crop plants (Han et 468
al 2015b) The energy required for vacuolar NO3- sequestration is provided by the tonoplast 469
proton-pumps (Gaxiola et al 2001 Brux et al 2008 Krebs et al 2010) while the 470
distribution channel CLCa enables transport of NO3- across the vacuolar membrane (Angeli et 471
al 2006 Wege et al 2014) The activities of the tonoplast proton-pumps (V-ATPase and 472
V-PPase) in leaves of the H genotype were significantly lower than those of the L genotype at 473
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17
flowering stages (Han et al 2015aHan et al 2015b) thus we expected similar differences in 474
root tissues Our results confirmed that the activities of tonoplast proton-pumps in root tissues 475
of B napus H genotype and A thaliana mutants were lower than those in the L genotype and 476
A thaliana wild type leading to less NO3- influx into vacuoles and more in the cytosol (Fig 1 477
Fig 5A-D) Moreover the enzymes catalyzing N assimilation are located in the cytoplasm 478
where NO3- could be assimilated (Martinoia et al 1981 Han et al 2015a) or immediately 479
loaded into xylem sap for long-distance transport from roots to shoots (Lin et al 2008) 480
Consequently the NO3- concentration in root tissues of the H genotype and A thaliana 481
mutants defective in tonoplast proton-pumps was significantly lower than that in the L 482
genotype (Fig 2E) and in A thaliana wild type (Fig 6C) Therefore the higher proportion of 483
NO3- transported to shoot and the lower concentrations retained in root vacuoles contributed 484
to the higher NUE in Xiangyou15 and A thaliana mutants (vha-a2 vha-a3 and avp1) (Table 485
1Fig 6A) Further studies are needed to assess the impacts of regulating vacuolar 486
sequestration of NO3- and rates of long-distance transport for enhancing NUE in other crop 487
species and genotypes 488
These data suggested that NO3- distribution between vacuoles and cytosol within cells 489
regulates NO3- long-distance transport from root to shoot and ultimately affects NUE NO3
- 490
loading into the xylem sap is an active transport process (Lin et al 2008) therefore not 491
regulated by absolute concentration of NO3- in the cytosol but rather by genes involved in 492
NO3- long-distance transport (NRT15 and NRT18) Apparently the expression of NRT15 the 493
gene responsible for xylem loading is regulated by NO3-concentration in the cytosol 494
Therefore both NO3- short-distance transport across the tonoplast and its long-distance 495
transport from root to shoot are important determinants of NUE in both B napus and A 496
thaliana (Table 1 and Fig 6) 497
498
Roles of NRT15 and NRT18 in NO3- long-distance transport in B napus and A 499
thaliana 500
Our results (Figs S9 S10) showed that the proportion of NO3- distributed from root to 501
shoot is strongly controlled by NRT15 in accord with previous studies (Lin et al 2008 Li et 502
al 2010) The data suggest that the high-NUE genotypes possess lower activities of tonoplast 503
proton-pumps (V-ATPase and V-PPase) resulting in less NO3- accumulation in vacuoles and 504
more NO3- retention in cytosol (Fig 7) The higher NO3
- in the cytosol then up-regulated 505
NRT15 which then down-regulates NRT18 As a result more NO3- is loaded into the xylem 506
system by NRT15 mechanism and less NO3- is unloaded via NRT18 (Fig 7) Differential 507
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18
regulation of these NRT genes in high-NUE plants increased transport of NO3- from root to 508
shoot through the xylem vascular tissues (Fig 7) This coordinated NO3- long- and 509
short-distance transport likely fine-tunes NO3- allocation and modulates the balance of NO3
- 510
distribution between roots and shoots (Fig 7) 511
Our research also revealed differences between A thaliana and B napus in the 512
expression of the wild-type NRT genes in response to NO3- in the protoplast Moreover using 513
A thaliananrt15-3 mutants we uncovered possible regulatory interactions between a 514
functioning (wild-type) NRT15 gene and NRT18 expression in response to NO3- 515
concentration Because mutants defective in nrt15 are not available for B napus we could 516
not confirm that the respective NRT15 and NRT18 systems behave in an analogous way in 517
these plant species Based on Harper et al (2012) research database our homology 518
comparisons and gene function annotations provided strong evidence for the roles of 519
BnNRT15 and BnNRT18 in B napus Finally our studies were conducted using two model 520
dicotyledonous plant species for which we had ample data on responses to specific inhibitors 521
(B napus) or specific mutants were available with results forming the bases for the model 522
presented in Figure 7 Further research using genetically diverse plant species (eg 523
monocotyledonous crops such as rice wheat and corn) will help validate this model and its 524
broader application across species The information generated in this study could have future 525
application for improving NUE in commercial crops with consequent reduction in nitrogen 526
use and benefits to the environment 527
528
Materials and Methods 529
Plant material 530
The two oilseed rape (Bnapus) cultivars used in this study have been characterized 531
before as high- (Xiangyou15 referred to as H genotype hereafter) and low- (814 referred to 532
as L genotype) nitrogen use efficiency (NUE) genotypes (Zhang et al 2009 Han et al 2015b) 533
Here we define NUE as the total biomass per unit of N uptake by the plant with total 534
biomass includes roots shoots and grains The two genotypes were provided by the Hunan 535
Sub-Center of Improvement Center of National Oil Crops Hunan China 536
The Arabidopsis thaliana wild-type Columbia-0 (col-0) was used as control for 537
V-ATPase (vha-a2 and vha-a3) V-PPase (avp1) nrt15-3 and nrt18-2 mutants whereas the A 538
thaliana wild-type Wassilewskija (Ws) was used as control for clca-2 mutants A thaliana 539
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19
mutants and transgenic lines (nrt15-3 and nrt18-2) have been described previously (Chen et 540
al 2012 Zhang et al 2014) The mutant lines vha-a2 (Salk_142642) and vha-a3 541
(Salk_122135) described by Krebs et al (2010) were obtained from the Arabidopsis 542
Biological Resources Center (ABRC) The mutant line avp1 (GK-005004) described in Li et 543
al (2005) was obtained from the European Arabidopsis Stock Centre while the mutant line 544
clca-2 (FST 171A06) described in Angeli et al (2006) was from the Institute National de la 545
Recherche Agronomique (INRA) collection in France together with the T-DNA mutants used 546
for genotyping to select pure mutant lines 547
548
Growth conditions 549
B napus plants were grown hydroponically in ceramic pots(20cmtimes15cm) filled with a 550
nutrient solution in the greenhouse under natural light as described in Han et al (2015b) The 551
pots were arranged in a completely randomized design with six biological replications The 552
nutrient solution was replaced every 3 days and its pH adjusted to 55 daily Experiments were 553
conducted at the field station of Hunan Agricultural University Southern China 554
A thaliana plants were grown in a nutrient solution in plastic pots as described in Arteca 555
et al (2000) and Gong et al (2003) The solution was changed every 3 days with pH adjusted 556
daily to 58 Pots were arranged in a completely randomized design with six biological 557
replications The nutrient solution used for both species consisted of 125mM KNO3 558
0625mM KH2PO4 05mM MgSO4 05mM Ca (NO3)2middot4H2O 0025mM Fe-EDTA 025 559
mlL-1 micronutrients (stock solution concentrations 70mM B 14mM Mn 1mM Zn 05mM 560
Cu and 02mM Mo) The experiments were conducted at Hunan Agricultural University in a 561
phytotron set at 70 relative humidity16h-light8h-dark cycle and constant temperature of 562
22degC 563
564
Experimental treatments and phenotyping 565
All measurements made on B napus plants were conducted either at the seedling stage 566
(2 months after transplanting) or at flowering (5 months after transplanting) The whole root 567
tissue was harvested and used for analyses at either stage For shoot measurements the fourth 568
leaf from the bottom was used for measurements made at seedling stage whereas the 12th leaf 569
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20
from the bottom was harvested for measurements made at flowering stage For A thaliana 570
whole root and shoot of four-week-old plants were sampled and used for different assays 571
572
Vacuolar proton-pumps inhibitor treatments 573
Inhibition of vacuolar proton pumps of B napus (Xiangyou15) was conducted at the seedling 574
stage in hydroponic culture as described in Han et al (2015a) with minor modifications 575
Plants grown in hydroponic solution were used as control Inhibitor treatment was conducted 576
using several chemicals Bafi(25 nmol L-1 Bafilomycin A1) an inhibitor of V-ATPase 577
DCCD(10 μmol L-1DCCD + 50 mmolL-1 Na2SO3) specifically inhibits V-PPase and 578
Bafi+DCCD in a 11 ratio as inhibitor of both V-ATPase and V-PPase The inhibitors were 579
applied in the hydroponic solutions for 24 h Fresh plant tissue samples from all treatments 580
were collected for different assays 581
582
NO3- induced gene expression 583
Four-week old A thaliana mutants (nrt15-3nrt18-2) were grown in hydroponics with 584
225mM (NH4)2succinate for 3 days and shifted to hydroponics with 45mM NO3- for 12 h 585
then root tissues were collected to assess relative expression of AtNRT15 and AtNRT18 586
genes Seedlings of B napus (Xiangyou15) plants grown hydroponically were treated with 587
5mM (NH4)2succinate for 3 d and shifted to fresh hydroponic solution containing 15 mM 588
NO3- for 12 h Root tissue was then collected to assess the relative expression of BnNRT15 589
and BnNRT18 genes using quantitative RT-PCR 590
591
ACC treatment 592
Hydroponically grown seedlings of B napus were subjected to ACC treatment as described in 593
Zhang et al (2014) with minor modifications Seedlings of the two genotypes Xiangyou15 594
and 814 were transferred to a nutrient solution containing 002mM L-1 ACC for 6 h or grown 595
in normal hydroponic solution as control Root tissues were then collected to assay relative 596
expression of BnNRT15 and BnNRT18 using quantitative RT-PCR Another set of B napus 597
plants were grown in ACC-containing hydroponic solution but supplemented with 15NO3- (22 598
15N) replacing the unlabeled NO3- for 1 h Root and shoot tissues were collected separately 599
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21
for 15N measurement using a continuous-flow isotope ratio mass spectrometer coupled with a 600
C-N elemental analyzer (ANCA-MS PDZ Europa) 601
602
Biomass and N concentration 603
Grain yield dry biomass and N concentration were determined in plant samples at 604
harvest Plants were harvested (including senescing leaves) then dried in an oven for 30 min 605
at 105degC then at 70degC to a constant weight and weighed N concentration was determined 606
using the Kjeldahl method (Shi et al 2010 Han et al 2015b)02g of dried samples were 607
digested with H2SO4-H2O2 in a 100-ml Kjeldahl digestion flask after which N concentration 608
was determined using a Foss Auto Analyser Unit (Kjeldahl 8400) 609
610
V-ATPase and V-PPase activities 611
Roots (10 g) of B napus were collected at seedling and flowering stages and that of A 612
thaliana (05 g) sampled at seedling stage then used for determining the activities of 613
V-ATPase and V-PPase (Han et al 2015b) Activities of these two energy pumps within 614
microsomal membranes were followed colorimetrically based on released Pi as described by 615
Zhu et al (2001) and Krebs et al (2010) 616
617
Assay of NO3- flux in vacuoles 618
Roots of both species were collected similarly as that used for V-ATPase and V-PPase 619
activities then used for vacuole isolation and measurement of NO3- flux in vacuoles as 620
described by Robert et al (2007) with minor modification (Han et al 2015b) Net fluxes of 621
NO3- in vacuoles were measured non-invasively using SIET (scanning ion-selective electrode 622
technique SIET system BIO-003A Younger USA Science and Technology Corporation 623
USA) An NO3--selective microelectrode used for the assay of NO3
- flux was vibrated in the 624
measuring solution between two positions 1 μm and 11 μm from the vacuole surface 625
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles Background 626
signal was recorded by vibrating the electrode in the measuring solution in the absence of 627
vacuoles Ion flux was calculated by Fickrsquos law of diffusion J = -D(dcdx) where J represents 628
the ion flux (picomolescm-2s-1) dcdx is the ion concentration gradient and D is the ion 629
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22
diffusion constant in a particular medium The direction of the flux was derived from Fickrsquos 630
law of diffusion that relates the concentration gradient 631
632
Isolation of intact protoplasts and vacuoles for determining NO3- concentration 633
Similar to the above assays root tissues (10 g) were collected from B napus at seedling 634
and flowering stages and from A thaliana (05 g) seedlings and used to isolate intact 635
protoplasts and vacuoles as described by Robert et al (2007) with minor modifications as 636
outlined in Huang et al (2012) and Han et al(2015b) The purified protoplasts were divided 637
into two equal aliquots with one of them used for isolation of vacuoles The purified 638
protoplasts and vacuoles were then sub-sampled and used to determine NO3- concentrations 639
(Vogeli-Lange and Wagner 1990) and for enzyme activity assays (Ma et al 2005) 640
NO3-concentrations in protoplasts and vacuoles were measured by a continuous-flow 641
auto-analyzer (Auto Analyser 3 Bran and Luebbe Germany) as described previously (Han et 642
al 2015b)The activities of acid phosphatase (ACP) and cytochrome oxidase (COX) were 643
determined using plant ACP colorimetry- and COX-assay kits (GenMedSci Inc) following 644
the manufacturerrsquos instructions ACP activity specific to vacuoles was determined and used to 645
normalize NO3- accumulation We measured NO3
- in the protoplast outside the vacuole which 646
includes the cytosol and organelles eg mitochondria and Golgi Apparatus (Robert et 647
al2007) Since most of the NO3- in the protoplast outside the vacuole is located in the cytosol 648
(Krebs et al 2010) we refer to NO3- distribution between vacuoles and cytosol rather than 649
vacuole versus protoplast 650
651
Quantitative RT-PCR 652
Total RNA extracted from B napus roots was prepared using Trizol reagent (Invitrogen) 653
cDNAs were synthesized using M-MLV reverse transcriptase (Promega) following 654
manufacturerrsquos protocol The relative expression of BnNRT15 (EV220114) BnNRT18 655
(EV116423) and Bnactin (AF1118121) genes in plant roots were determined by quantitative 656
RT-PCR and run on a LightCycler instrument (Roche) with the SYBR Green Real-Time PCR 657
Master Mix Kit (TOYOBO Japan) under the following conditions 95degC for 2min then 45 658
cycles of 95degC for 10s 60degC for 10s and 72degC for 20s The primer sequences of BnNRT15 659
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
BnNRT18 and Bnactin genes were obtained from httpbrassicanbiacuk as described in 660
Harper et al (2012) and expression levels were normalized to Bnactin as control 661
Total RNA extracted from the 4-week-old A thaliana roots under the treatments 662
conditions described above were prepared using Trizol reagent (Invitrogen) The cDNAs were 663
synthesized using M-MLV reverse transcriptase (Promega) based on the manufacturerrsquos 664
protocol and as described in Li et al (2010)The primer sequences of quantitative RT-PCR 665
used in these assays are listed in Supplemental Table 2 and were described previously (Chen 666
et al 2012 Zhang et al 2014) and their expression was normalized to Atactin2 as control 667
668
Determination of nitrate distribution using 15NO3- 669
Hydroponically grown B napus (seedling and flowering stages) and A thaliana 670
(seedling) were transferred to 01mM CaSO4 solution for 1min then to their respective 671
hydroponic nutrient solutions with15NO3- (22 excess) replacing unlabeled NO3
- for 1h 672
Plants were then transferred to 01mM CaSO4 solution for 1min after which roots were 673
washed with deionized water Root and shoot samples were separated and dried at 105degC for 674
30min followed by 70degC for 3 d then grounded and used for assaying 15N content using a 675
continuous-flow isotope ratio mass spectrometer coupled with a carbon-nitrogen elemental 676
analyzer (ANCA-MS PDZ Europa) 677
678
Xylem sap collection and assay of nitrate concentration 679
We used the method of Tang et al (2012) for collecting xylem sap Plants were cut 680
leaving1cm segments with intact roots at seedling and flowering stages for B napus and at 681
seedling stage for A thaliana Roots were immediately immersed in their respective nutrition 682
solutions Weighed cotton was put on the cut surface to absorb extruding xylem sap for 1 h 683
The cotton was then wrapped in plastic film and the volume of xylem sap was calculated as 684
the weight gain of the cotton Xylem sap was then squeezed from the cotton using a syringe 685
and used for subsequent assay of nitrate concentration using a continuous-flow auto-analyzer 686
(Auto Analyser 3 Bran and Luebbe Germany) 687
To determine nitrate concentration samples (10 g fresh root and shoot at seedling and 688
flowering of B napus and 05 g fresh root and shoot of 4-week-old A thaliana) were frozen 689
in liquid N2 and ground with a mortar and pestle the powder was then transferred to a beaker 690
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
containing 70 ml deionized water and boiled for 30 min to extract nitrate then cooled and 691
made to 100 ml volume and 02 g activated carbon added to eliminate the effect of 692
chlorophyll The mixture was filtered and nitrate in the filtrate was determined using a 693
continuous-flow auto-analyser (Atuo-Analyser 3 Bran and Luebbe Germany) 694
695
Measurements of photosynthesis 696
All measurements of photosynthesis and related parameters were conducted at 1000 h 697
using intact leaves The 4th leaf from the bottom in B napus at seedling stage and the 12th leaf 698
from the bottom at flowering stage were used for determining chlorophyll concentration 699
photosynthetic rate intercellular CO2 concentration transpiration rate and stomatal 700
conductance Chlorophyll concentration was determined by SPAD-502 (Minolta Camera Co 701
Ltd Japan) (Luo et al 2006 Yandeau-Nelson et al 2011) Photosynthetic rate intercellular 702
CO2 concentration transpiration rate and stomatal conductance were measured using LI-6400 703
Portable Photosynthesis System (Li-Cor Biosciences Co Ltd USA) set at flow rate of 500 704
μmol s-1 photosynthetic photon flux density of 1000 μmol m-2 s-1 relative humidity of 65 705
CO2 concentration of 400ppm and ambient temperatures 706
707
NR and GS activities 708
Leaves with similar age as those used for photosynthesis in B napus were used for the 709
assay of nitrate reductase (NR) and glutamine synthetase (GS) activities NR activity was 710
measured by the modified method of Fan et al (2007) Fresh samples (05g) were frozen in 711
liquid N2 ground into powder in the presence of acid-washed sand and homogenized with 4 712
ml of extraction buffer (0025mol L-1phosphate buffer pH87 1211g L-1 cysteine 0372g L-1 713
EDTA) Homogenates were centrifuged at 30000timesg for 15 min at 4degC and the supernatants 714
were treated with sulfanilamide and α-naphthylamine reagents for colorimetric (540nm) 715
determination of nitrite as described by Fan et al (2007) 716
GS activity was determined using a modified reverseγ-glutamyltransferase method 717
(Wang et al2014) which measures the GS-catalyzed formation of glutamyl-γ-hydroxamate 718
from glutamine and hydroxylamine Fresh samples were frozen for 30 min at -20degC then 719
ground in 10ml Tris-HCl buffer and acid-washed sand The homogenates were filtered 720
through two layers of gauze and centrifuged at 8000timesg for 15 min at 4degC About 12ml of the 721
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
supernatant was added in a reaction mixture and treated as described (Wang et al2014) After 722
the GS-catalyzed reaction was stopped and the solution centrifuged glutamyl-γ723
-hydroxamate was quantified colorimetrically in the supernatant (at 485 nm) and the 724
concentration was determined using a standard curve 725
726
Statistical analyses 727
We used the SPSS software (Statistical Product and Service Solutions V130 USA) for 728
ANOVA and mean separation of main-effects and interactions using Duncanrsquos multiple range 729
test at Plt005Values are presented as means and SD of three or six replicates from three 730
independent experiments Different letters or an asterisk () associated with specific data (eg 731
at the top of histogram bars in figures or within tables) denote significant differences at 732
Plt005 733
734
Supplemental Data 735
Supplemental Figure 1 B napus with higher NUE showed lower vacuolar sequestration 736
capacity (VSC) for NO3-in roots at flowering stage 737
Supplemental Figure 2 B napus with higher NUE showed enhanced long-distance transport 738
of NO3- from roots to shoots at flowering stage 739
Supplemental Figure 3 The two B napus (H and L genotypes) showed the same total N per 740
plant at seedling stage (A) and flowering stage (B) 741
Supplemental Figure 4 B napus with higher NUE showed increased NO3- concentration in 742
the xylem sap at seedling and flowering stages 743
Supplemental Figure 5 NO3- concentration in the xylem sap as affected by inhibitor 744
treatments in B napus and in the energy pumpsrsquo mutants of A thaliana (col-0 vha-a2 vha-a3 745
avp1) 746
Supplemental Figure 6 Reduced VSC for NO3- in roots drives long-distance transport of 747
NO3- from roots to shoots in the A thaliana wild type (Ws) and mutant (clca-2) 748
Supplemental Figure 7 Differences of NR and GS activities between the two B napus (H 749
and L genotypes) at seedling and flowering stages 750
Supplemental Figure 8 Amino acid sequences of BnNRT15 and BnNRT18 751
Supplemental Figure 9 Functions of AtNRT15 and AtNRT18 genes in root tissues of A 752
thaliana in controlling NO3- long-distance transport from root to shoots 753
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
Supplemental Figure 10 B napus BnNRT15 and BnNRT18 genes in roots were 754
coordinately modulated to facilitate NO3- long distance transport from roots to shoots 755
Supplemental Table 1 Differences in stomatal conductance and transpiration rate between 756
the two B napus genotypes 757
Supplemental Table 2 Sequences of primera used for qRT-PCR 758
759
Acknowledgements 760
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 761
Shanghai Institutes for Biological Sciences) for providing nrt15-3 and nrt18-2 seeds 762
This study was supported by the National Natural Science Foundation of China (Grant 763
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 764
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 765
The Ministry of Education in China Open Novel Science Foundation of Hunan province 766
(13K062) the National Key Laboratory of Plant Molecular Genetics and the Twelfth 767
Five-Year National Science and technology support program (2012BAD15BO4) 768
769
Figure 1 B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 770
NO3- in roots at the seedling stage 771
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 772
genotype 814 Specific activities of the tonoplast proton pumps are expressed as μmol Pi 773
released mg-1 protein h-1 NO3- fluxes are expressed as pmol NO3
- cm-2 S-1 Mature vacuoles 774
were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 775
measuring solution between the two positions 1 μm and 11 μm from the vacuole surface 776
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles recording the 777
stable reading data The background was recorded by vibrating the electrode in measuring 778
solution without vacuoles 779
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 780
for NO3- content and NO3
- accumulation normalized against the specific activity of the 781
vacuole acid phosphatase (ACP) as described in Materials and Methods and plotted as μmol 782
NO3- per μmol p-nitrophenol the end product of ACP assay 783
Proton pump activities in root tissues between H and L genotypes are shown at the seedling 784
stage (A) Different letters at the top of the histogram bars denote significant differences of 785
V-ATPase in root tissues between H and L genotypes (Plt005) an asterisk () at the top of the 786
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
histogram bars indicates significant difference in V-PPase activity in root tissues between H 787
and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788
Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789
genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790
bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791
bars on the figures indicate SD (n=6) 792
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793
seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794
to the total NO3- in protoplasts 795
NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796
total NO3-in the cytosol and was calculated as total NO3
- in protoplasts ndash total NO3- in 797
vacuole Different letters at the top of histogram bars denote significant differences of total 798
NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799
(n=6) 800
801
Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802
roots to shoots at the seedling stage 803
H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804
Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805
the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806
as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807
the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808
the histogram bars denote significant differences at Plt005 809
Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810
in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811
genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812
of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813
SD (n=3) different letters at the top of the histogram bars denote significant differences at 814
Plt005 level 815
The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816
at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817
genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818
different letters at the top of the histogram bars denote significant differences at Plt005 819
820
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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Xu GH Fan XR Miller AJ (2012) Plant nitrogen assimilation and use efficiency The Annual Review of Plant Biology 63153-182Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yandeau-Nelson MD Laurens L Shi Z Xia H Smith AM Guiltinan MJ (2011) Starch-Branching Enzyme IIa Is Required for ProperDiurnal Cycling of Starch in Leaves of Maize [OA] Plant Physiology 156479-490
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Zhang GB Yi HY Gong JM (2014) The Arabidopsis EthyleneJasmonic acid-NRT signaling module coordinates nitrate reallocationand the trade-off between growth and environmental adaption The Plant Cell 26(10)3984-3998
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Zhang ZH Song HX Liu Qing Rong XM Xie GX Peng JW Zhang YP (2009) Study on differences of nitrogen efficiency andnitrogen response in different oilseed rape (Brassica napus L) varieties Asian Journal of Crop Science 1(2)105-112
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Zhang ZH Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2010) Nitrogen redistribution characteristics ofoilseed rape varieties with different nitrogen use efficiencies during later growth period Communications in Soil Science andPlant Analysis 41(14) 1693-1706
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Zhang ZH Huang HT Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2012) Research advances on nitratenitrogen reutilization by proton pump of tonoplast and its relation to nitrogen use efficiency Australian Journal of Crop Science6(9) 1377-1382
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- Parsed Citations
- Reviewer PDF
- ONE
- TWO
- THREE
- FOUR
- FIVE
- SIX
- SEVEN
- T-ONE
- T-TWO
- Parsed Citations
-
16
our hypothesis that concentration of NO3- in the xylem sap and shoot would be significantly 440
higher in the H genotype than in the L genotype (Fig 2EFFig S2EF Fig S4) The NO3- 441
long-distance transport from root to shoot was affected by cytosolic NO3- in roots of both A 442
thaliana and B napus genotypes (Fig 4-5 Fig S6) The activities of the tonoplast 443
proton-pumps in roots were significantly lower in the H genotype than in the L genotype (Fig 444
1AB) Consequently less NO3- was sequestered into the vacuole and more NO3
- was retained 445
in cytosol (Fig 1CD) BnNRT15 expression was then up-regulated resulting in greater 446
loading into the xylem while BnNRT18 was down-regulated (Fig 2AB) probably to 447
suppress NO3- unloading from xylem sap Consequently a higher proportion of NO3
- was 448
transported from root to shoots in the H genotype(Fig 2C-D Fig S4) 449
Moreover photosynthetic carbon fixation (Table 2) and activities of both NR and GS 450
were higher in shoots of the H genotype (Fig S7) both of which promote higher N 451
assimilation These results agreed with previous reports (Smirnoff et al 1985 Andrews et al 452
1986 Tang et al 2012 Tang et al 2013) where higher NO3- transport from root to shoot 453
higher photosynthetic rate and N assimilation efficiency in shoots were suggested as essential 454
mechanisms for high-NUE in crop plants These qualities endowed the B napus H genotype 455
Xiangyou15 with higher NUE as compared with the L genotype 814 456
NO3- long-distance transport in B napus is not only regulated by BnNRT15 and 457
BnNRT18 in root tissues (Fig 4 Fig 5 Fig S6) but also by stomatal conductance and 458
transpiration by indirectly controlling xylem sap flow and long-distance transport 459
(Dechorgnat et al 2011 Krapp et al 2014) Stomatal conductance and transpiration rates of 460
the H genotype were significantly higher than those of the L genotype (Table S1) a result 461
consistent with other studies describing characteristics of high-NUE plants (Daniel-Vedele et 462
al 1998 Wilkinson et al 2007 Dechorgnat et al 2011 Krapp et al 2014) 463
464
Response of NUE to NO3- short-distance transport 465
NUE is also strongly controlled by NO3- short-distance distribution between the vacuole 466
and cytosol (Han et al 2015a) A lower proportion of NO3- accumulating in vacuoles and a 467
higher proportion retained in the cytosol will contribute to better NUE in crop plants (Han et 468
al 2015b) The energy required for vacuolar NO3- sequestration is provided by the tonoplast 469
proton-pumps (Gaxiola et al 2001 Brux et al 2008 Krebs et al 2010) while the 470
distribution channel CLCa enables transport of NO3- across the vacuolar membrane (Angeli et 471
al 2006 Wege et al 2014) The activities of the tonoplast proton-pumps (V-ATPase and 472
V-PPase) in leaves of the H genotype were significantly lower than those of the L genotype at 473
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17
flowering stages (Han et al 2015aHan et al 2015b) thus we expected similar differences in 474
root tissues Our results confirmed that the activities of tonoplast proton-pumps in root tissues 475
of B napus H genotype and A thaliana mutants were lower than those in the L genotype and 476
A thaliana wild type leading to less NO3- influx into vacuoles and more in the cytosol (Fig 1 477
Fig 5A-D) Moreover the enzymes catalyzing N assimilation are located in the cytoplasm 478
where NO3- could be assimilated (Martinoia et al 1981 Han et al 2015a) or immediately 479
loaded into xylem sap for long-distance transport from roots to shoots (Lin et al 2008) 480
Consequently the NO3- concentration in root tissues of the H genotype and A thaliana 481
mutants defective in tonoplast proton-pumps was significantly lower than that in the L 482
genotype (Fig 2E) and in A thaliana wild type (Fig 6C) Therefore the higher proportion of 483
NO3- transported to shoot and the lower concentrations retained in root vacuoles contributed 484
to the higher NUE in Xiangyou15 and A thaliana mutants (vha-a2 vha-a3 and avp1) (Table 485
1Fig 6A) Further studies are needed to assess the impacts of regulating vacuolar 486
sequestration of NO3- and rates of long-distance transport for enhancing NUE in other crop 487
species and genotypes 488
These data suggested that NO3- distribution between vacuoles and cytosol within cells 489
regulates NO3- long-distance transport from root to shoot and ultimately affects NUE NO3
- 490
loading into the xylem sap is an active transport process (Lin et al 2008) therefore not 491
regulated by absolute concentration of NO3- in the cytosol but rather by genes involved in 492
NO3- long-distance transport (NRT15 and NRT18) Apparently the expression of NRT15 the 493
gene responsible for xylem loading is regulated by NO3-concentration in the cytosol 494
Therefore both NO3- short-distance transport across the tonoplast and its long-distance 495
transport from root to shoot are important determinants of NUE in both B napus and A 496
thaliana (Table 1 and Fig 6) 497
498
Roles of NRT15 and NRT18 in NO3- long-distance transport in B napus and A 499
thaliana 500
Our results (Figs S9 S10) showed that the proportion of NO3- distributed from root to 501
shoot is strongly controlled by NRT15 in accord with previous studies (Lin et al 2008 Li et 502
al 2010) The data suggest that the high-NUE genotypes possess lower activities of tonoplast 503
proton-pumps (V-ATPase and V-PPase) resulting in less NO3- accumulation in vacuoles and 504
more NO3- retention in cytosol (Fig 7) The higher NO3
- in the cytosol then up-regulated 505
NRT15 which then down-regulates NRT18 As a result more NO3- is loaded into the xylem 506
system by NRT15 mechanism and less NO3- is unloaded via NRT18 (Fig 7) Differential 507
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18
regulation of these NRT genes in high-NUE plants increased transport of NO3- from root to 508
shoot through the xylem vascular tissues (Fig 7) This coordinated NO3- long- and 509
short-distance transport likely fine-tunes NO3- allocation and modulates the balance of NO3
- 510
distribution between roots and shoots (Fig 7) 511
Our research also revealed differences between A thaliana and B napus in the 512
expression of the wild-type NRT genes in response to NO3- in the protoplast Moreover using 513
A thaliananrt15-3 mutants we uncovered possible regulatory interactions between a 514
functioning (wild-type) NRT15 gene and NRT18 expression in response to NO3- 515
concentration Because mutants defective in nrt15 are not available for B napus we could 516
not confirm that the respective NRT15 and NRT18 systems behave in an analogous way in 517
these plant species Based on Harper et al (2012) research database our homology 518
comparisons and gene function annotations provided strong evidence for the roles of 519
BnNRT15 and BnNRT18 in B napus Finally our studies were conducted using two model 520
dicotyledonous plant species for which we had ample data on responses to specific inhibitors 521
(B napus) or specific mutants were available with results forming the bases for the model 522
presented in Figure 7 Further research using genetically diverse plant species (eg 523
monocotyledonous crops such as rice wheat and corn) will help validate this model and its 524
broader application across species The information generated in this study could have future 525
application for improving NUE in commercial crops with consequent reduction in nitrogen 526
use and benefits to the environment 527
528
Materials and Methods 529
Plant material 530
The two oilseed rape (Bnapus) cultivars used in this study have been characterized 531
before as high- (Xiangyou15 referred to as H genotype hereafter) and low- (814 referred to 532
as L genotype) nitrogen use efficiency (NUE) genotypes (Zhang et al 2009 Han et al 2015b) 533
Here we define NUE as the total biomass per unit of N uptake by the plant with total 534
biomass includes roots shoots and grains The two genotypes were provided by the Hunan 535
Sub-Center of Improvement Center of National Oil Crops Hunan China 536
The Arabidopsis thaliana wild-type Columbia-0 (col-0) was used as control for 537
V-ATPase (vha-a2 and vha-a3) V-PPase (avp1) nrt15-3 and nrt18-2 mutants whereas the A 538
thaliana wild-type Wassilewskija (Ws) was used as control for clca-2 mutants A thaliana 539
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19
mutants and transgenic lines (nrt15-3 and nrt18-2) have been described previously (Chen et 540
al 2012 Zhang et al 2014) The mutant lines vha-a2 (Salk_142642) and vha-a3 541
(Salk_122135) described by Krebs et al (2010) were obtained from the Arabidopsis 542
Biological Resources Center (ABRC) The mutant line avp1 (GK-005004) described in Li et 543
al (2005) was obtained from the European Arabidopsis Stock Centre while the mutant line 544
clca-2 (FST 171A06) described in Angeli et al (2006) was from the Institute National de la 545
Recherche Agronomique (INRA) collection in France together with the T-DNA mutants used 546
for genotyping to select pure mutant lines 547
548
Growth conditions 549
B napus plants were grown hydroponically in ceramic pots(20cmtimes15cm) filled with a 550
nutrient solution in the greenhouse under natural light as described in Han et al (2015b) The 551
pots were arranged in a completely randomized design with six biological replications The 552
nutrient solution was replaced every 3 days and its pH adjusted to 55 daily Experiments were 553
conducted at the field station of Hunan Agricultural University Southern China 554
A thaliana plants were grown in a nutrient solution in plastic pots as described in Arteca 555
et al (2000) and Gong et al (2003) The solution was changed every 3 days with pH adjusted 556
daily to 58 Pots were arranged in a completely randomized design with six biological 557
replications The nutrient solution used for both species consisted of 125mM KNO3 558
0625mM KH2PO4 05mM MgSO4 05mM Ca (NO3)2middot4H2O 0025mM Fe-EDTA 025 559
mlL-1 micronutrients (stock solution concentrations 70mM B 14mM Mn 1mM Zn 05mM 560
Cu and 02mM Mo) The experiments were conducted at Hunan Agricultural University in a 561
phytotron set at 70 relative humidity16h-light8h-dark cycle and constant temperature of 562
22degC 563
564
Experimental treatments and phenotyping 565
All measurements made on B napus plants were conducted either at the seedling stage 566
(2 months after transplanting) or at flowering (5 months after transplanting) The whole root 567
tissue was harvested and used for analyses at either stage For shoot measurements the fourth 568
leaf from the bottom was used for measurements made at seedling stage whereas the 12th leaf 569
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
from the bottom was harvested for measurements made at flowering stage For A thaliana 570
whole root and shoot of four-week-old plants were sampled and used for different assays 571
572
Vacuolar proton-pumps inhibitor treatments 573
Inhibition of vacuolar proton pumps of B napus (Xiangyou15) was conducted at the seedling 574
stage in hydroponic culture as described in Han et al (2015a) with minor modifications 575
Plants grown in hydroponic solution were used as control Inhibitor treatment was conducted 576
using several chemicals Bafi(25 nmol L-1 Bafilomycin A1) an inhibitor of V-ATPase 577
DCCD(10 μmol L-1DCCD + 50 mmolL-1 Na2SO3) specifically inhibits V-PPase and 578
Bafi+DCCD in a 11 ratio as inhibitor of both V-ATPase and V-PPase The inhibitors were 579
applied in the hydroponic solutions for 24 h Fresh plant tissue samples from all treatments 580
were collected for different assays 581
582
NO3- induced gene expression 583
Four-week old A thaliana mutants (nrt15-3nrt18-2) were grown in hydroponics with 584
225mM (NH4)2succinate for 3 days and shifted to hydroponics with 45mM NO3- for 12 h 585
then root tissues were collected to assess relative expression of AtNRT15 and AtNRT18 586
genes Seedlings of B napus (Xiangyou15) plants grown hydroponically were treated with 587
5mM (NH4)2succinate for 3 d and shifted to fresh hydroponic solution containing 15 mM 588
NO3- for 12 h Root tissue was then collected to assess the relative expression of BnNRT15 589
and BnNRT18 genes using quantitative RT-PCR 590
591
ACC treatment 592
Hydroponically grown seedlings of B napus were subjected to ACC treatment as described in 593
Zhang et al (2014) with minor modifications Seedlings of the two genotypes Xiangyou15 594
and 814 were transferred to a nutrient solution containing 002mM L-1 ACC for 6 h or grown 595
in normal hydroponic solution as control Root tissues were then collected to assay relative 596
expression of BnNRT15 and BnNRT18 using quantitative RT-PCR Another set of B napus 597
plants were grown in ACC-containing hydroponic solution but supplemented with 15NO3- (22 598
15N) replacing the unlabeled NO3- for 1 h Root and shoot tissues were collected separately 599
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
21
for 15N measurement using a continuous-flow isotope ratio mass spectrometer coupled with a 600
C-N elemental analyzer (ANCA-MS PDZ Europa) 601
602
Biomass and N concentration 603
Grain yield dry biomass and N concentration were determined in plant samples at 604
harvest Plants were harvested (including senescing leaves) then dried in an oven for 30 min 605
at 105degC then at 70degC to a constant weight and weighed N concentration was determined 606
using the Kjeldahl method (Shi et al 2010 Han et al 2015b)02g of dried samples were 607
digested with H2SO4-H2O2 in a 100-ml Kjeldahl digestion flask after which N concentration 608
was determined using a Foss Auto Analyser Unit (Kjeldahl 8400) 609
610
V-ATPase and V-PPase activities 611
Roots (10 g) of B napus were collected at seedling and flowering stages and that of A 612
thaliana (05 g) sampled at seedling stage then used for determining the activities of 613
V-ATPase and V-PPase (Han et al 2015b) Activities of these two energy pumps within 614
microsomal membranes were followed colorimetrically based on released Pi as described by 615
Zhu et al (2001) and Krebs et al (2010) 616
617
Assay of NO3- flux in vacuoles 618
Roots of both species were collected similarly as that used for V-ATPase and V-PPase 619
activities then used for vacuole isolation and measurement of NO3- flux in vacuoles as 620
described by Robert et al (2007) with minor modification (Han et al 2015b) Net fluxes of 621
NO3- in vacuoles were measured non-invasively using SIET (scanning ion-selective electrode 622
technique SIET system BIO-003A Younger USA Science and Technology Corporation 623
USA) An NO3--selective microelectrode used for the assay of NO3
- flux was vibrated in the 624
measuring solution between two positions 1 μm and 11 μm from the vacuole surface 625
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles Background 626
signal was recorded by vibrating the electrode in the measuring solution in the absence of 627
vacuoles Ion flux was calculated by Fickrsquos law of diffusion J = -D(dcdx) where J represents 628
the ion flux (picomolescm-2s-1) dcdx is the ion concentration gradient and D is the ion 629
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
diffusion constant in a particular medium The direction of the flux was derived from Fickrsquos 630
law of diffusion that relates the concentration gradient 631
632
Isolation of intact protoplasts and vacuoles for determining NO3- concentration 633
Similar to the above assays root tissues (10 g) were collected from B napus at seedling 634
and flowering stages and from A thaliana (05 g) seedlings and used to isolate intact 635
protoplasts and vacuoles as described by Robert et al (2007) with minor modifications as 636
outlined in Huang et al (2012) and Han et al(2015b) The purified protoplasts were divided 637
into two equal aliquots with one of them used for isolation of vacuoles The purified 638
protoplasts and vacuoles were then sub-sampled and used to determine NO3- concentrations 639
(Vogeli-Lange and Wagner 1990) and for enzyme activity assays (Ma et al 2005) 640
NO3-concentrations in protoplasts and vacuoles were measured by a continuous-flow 641
auto-analyzer (Auto Analyser 3 Bran and Luebbe Germany) as described previously (Han et 642
al 2015b)The activities of acid phosphatase (ACP) and cytochrome oxidase (COX) were 643
determined using plant ACP colorimetry- and COX-assay kits (GenMedSci Inc) following 644
the manufacturerrsquos instructions ACP activity specific to vacuoles was determined and used to 645
normalize NO3- accumulation We measured NO3
- in the protoplast outside the vacuole which 646
includes the cytosol and organelles eg mitochondria and Golgi Apparatus (Robert et 647
al2007) Since most of the NO3- in the protoplast outside the vacuole is located in the cytosol 648
(Krebs et al 2010) we refer to NO3- distribution between vacuoles and cytosol rather than 649
vacuole versus protoplast 650
651
Quantitative RT-PCR 652
Total RNA extracted from B napus roots was prepared using Trizol reagent (Invitrogen) 653
cDNAs were synthesized using M-MLV reverse transcriptase (Promega) following 654
manufacturerrsquos protocol The relative expression of BnNRT15 (EV220114) BnNRT18 655
(EV116423) and Bnactin (AF1118121) genes in plant roots were determined by quantitative 656
RT-PCR and run on a LightCycler instrument (Roche) with the SYBR Green Real-Time PCR 657
Master Mix Kit (TOYOBO Japan) under the following conditions 95degC for 2min then 45 658
cycles of 95degC for 10s 60degC for 10s and 72degC for 20s The primer sequences of BnNRT15 659
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
BnNRT18 and Bnactin genes were obtained from httpbrassicanbiacuk as described in 660
Harper et al (2012) and expression levels were normalized to Bnactin as control 661
Total RNA extracted from the 4-week-old A thaliana roots under the treatments 662
conditions described above were prepared using Trizol reagent (Invitrogen) The cDNAs were 663
synthesized using M-MLV reverse transcriptase (Promega) based on the manufacturerrsquos 664
protocol and as described in Li et al (2010)The primer sequences of quantitative RT-PCR 665
used in these assays are listed in Supplemental Table 2 and were described previously (Chen 666
et al 2012 Zhang et al 2014) and their expression was normalized to Atactin2 as control 667
668
Determination of nitrate distribution using 15NO3- 669
Hydroponically grown B napus (seedling and flowering stages) and A thaliana 670
(seedling) were transferred to 01mM CaSO4 solution for 1min then to their respective 671
hydroponic nutrient solutions with15NO3- (22 excess) replacing unlabeled NO3
- for 1h 672
Plants were then transferred to 01mM CaSO4 solution for 1min after which roots were 673
washed with deionized water Root and shoot samples were separated and dried at 105degC for 674
30min followed by 70degC for 3 d then grounded and used for assaying 15N content using a 675
continuous-flow isotope ratio mass spectrometer coupled with a carbon-nitrogen elemental 676
analyzer (ANCA-MS PDZ Europa) 677
678
Xylem sap collection and assay of nitrate concentration 679
We used the method of Tang et al (2012) for collecting xylem sap Plants were cut 680
leaving1cm segments with intact roots at seedling and flowering stages for B napus and at 681
seedling stage for A thaliana Roots were immediately immersed in their respective nutrition 682
solutions Weighed cotton was put on the cut surface to absorb extruding xylem sap for 1 h 683
The cotton was then wrapped in plastic film and the volume of xylem sap was calculated as 684
the weight gain of the cotton Xylem sap was then squeezed from the cotton using a syringe 685
and used for subsequent assay of nitrate concentration using a continuous-flow auto-analyzer 686
(Auto Analyser 3 Bran and Luebbe Germany) 687
To determine nitrate concentration samples (10 g fresh root and shoot at seedling and 688
flowering of B napus and 05 g fresh root and shoot of 4-week-old A thaliana) were frozen 689
in liquid N2 and ground with a mortar and pestle the powder was then transferred to a beaker 690
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
containing 70 ml deionized water and boiled for 30 min to extract nitrate then cooled and 691
made to 100 ml volume and 02 g activated carbon added to eliminate the effect of 692
chlorophyll The mixture was filtered and nitrate in the filtrate was determined using a 693
continuous-flow auto-analyser (Atuo-Analyser 3 Bran and Luebbe Germany) 694
695
Measurements of photosynthesis 696
All measurements of photosynthesis and related parameters were conducted at 1000 h 697
using intact leaves The 4th leaf from the bottom in B napus at seedling stage and the 12th leaf 698
from the bottom at flowering stage were used for determining chlorophyll concentration 699
photosynthetic rate intercellular CO2 concentration transpiration rate and stomatal 700
conductance Chlorophyll concentration was determined by SPAD-502 (Minolta Camera Co 701
Ltd Japan) (Luo et al 2006 Yandeau-Nelson et al 2011) Photosynthetic rate intercellular 702
CO2 concentration transpiration rate and stomatal conductance were measured using LI-6400 703
Portable Photosynthesis System (Li-Cor Biosciences Co Ltd USA) set at flow rate of 500 704
μmol s-1 photosynthetic photon flux density of 1000 μmol m-2 s-1 relative humidity of 65 705
CO2 concentration of 400ppm and ambient temperatures 706
707
NR and GS activities 708
Leaves with similar age as those used for photosynthesis in B napus were used for the 709
assay of nitrate reductase (NR) and glutamine synthetase (GS) activities NR activity was 710
measured by the modified method of Fan et al (2007) Fresh samples (05g) were frozen in 711
liquid N2 ground into powder in the presence of acid-washed sand and homogenized with 4 712
ml of extraction buffer (0025mol L-1phosphate buffer pH87 1211g L-1 cysteine 0372g L-1 713
EDTA) Homogenates were centrifuged at 30000timesg for 15 min at 4degC and the supernatants 714
were treated with sulfanilamide and α-naphthylamine reagents for colorimetric (540nm) 715
determination of nitrite as described by Fan et al (2007) 716
GS activity was determined using a modified reverseγ-glutamyltransferase method 717
(Wang et al2014) which measures the GS-catalyzed formation of glutamyl-γ-hydroxamate 718
from glutamine and hydroxylamine Fresh samples were frozen for 30 min at -20degC then 719
ground in 10ml Tris-HCl buffer and acid-washed sand The homogenates were filtered 720
through two layers of gauze and centrifuged at 8000timesg for 15 min at 4degC About 12ml of the 721
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
supernatant was added in a reaction mixture and treated as described (Wang et al2014) After 722
the GS-catalyzed reaction was stopped and the solution centrifuged glutamyl-γ723
-hydroxamate was quantified colorimetrically in the supernatant (at 485 nm) and the 724
concentration was determined using a standard curve 725
726
Statistical analyses 727
We used the SPSS software (Statistical Product and Service Solutions V130 USA) for 728
ANOVA and mean separation of main-effects and interactions using Duncanrsquos multiple range 729
test at Plt005Values are presented as means and SD of three or six replicates from three 730
independent experiments Different letters or an asterisk () associated with specific data (eg 731
at the top of histogram bars in figures or within tables) denote significant differences at 732
Plt005 733
734
Supplemental Data 735
Supplemental Figure 1 B napus with higher NUE showed lower vacuolar sequestration 736
capacity (VSC) for NO3-in roots at flowering stage 737
Supplemental Figure 2 B napus with higher NUE showed enhanced long-distance transport 738
of NO3- from roots to shoots at flowering stage 739
Supplemental Figure 3 The two B napus (H and L genotypes) showed the same total N per 740
plant at seedling stage (A) and flowering stage (B) 741
Supplemental Figure 4 B napus with higher NUE showed increased NO3- concentration in 742
the xylem sap at seedling and flowering stages 743
Supplemental Figure 5 NO3- concentration in the xylem sap as affected by inhibitor 744
treatments in B napus and in the energy pumpsrsquo mutants of A thaliana (col-0 vha-a2 vha-a3 745
avp1) 746
Supplemental Figure 6 Reduced VSC for NO3- in roots drives long-distance transport of 747
NO3- from roots to shoots in the A thaliana wild type (Ws) and mutant (clca-2) 748
Supplemental Figure 7 Differences of NR and GS activities between the two B napus (H 749
and L genotypes) at seedling and flowering stages 750
Supplemental Figure 8 Amino acid sequences of BnNRT15 and BnNRT18 751
Supplemental Figure 9 Functions of AtNRT15 and AtNRT18 genes in root tissues of A 752
thaliana in controlling NO3- long-distance transport from root to shoots 753
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
Supplemental Figure 10 B napus BnNRT15 and BnNRT18 genes in roots were 754
coordinately modulated to facilitate NO3- long distance transport from roots to shoots 755
Supplemental Table 1 Differences in stomatal conductance and transpiration rate between 756
the two B napus genotypes 757
Supplemental Table 2 Sequences of primera used for qRT-PCR 758
759
Acknowledgements 760
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 761
Shanghai Institutes for Biological Sciences) for providing nrt15-3 and nrt18-2 seeds 762
This study was supported by the National Natural Science Foundation of China (Grant 763
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 764
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 765
The Ministry of Education in China Open Novel Science Foundation of Hunan province 766
(13K062) the National Key Laboratory of Plant Molecular Genetics and the Twelfth 767
Five-Year National Science and technology support program (2012BAD15BO4) 768
769
Figure 1 B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 770
NO3- in roots at the seedling stage 771
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 772
genotype 814 Specific activities of the tonoplast proton pumps are expressed as μmol Pi 773
released mg-1 protein h-1 NO3- fluxes are expressed as pmol NO3
- cm-2 S-1 Mature vacuoles 774
were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 775
measuring solution between the two positions 1 μm and 11 μm from the vacuole surface 776
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles recording the 777
stable reading data The background was recorded by vibrating the electrode in measuring 778
solution without vacuoles 779
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 780
for NO3- content and NO3
- accumulation normalized against the specific activity of the 781
vacuole acid phosphatase (ACP) as described in Materials and Methods and plotted as μmol 782
NO3- per μmol p-nitrophenol the end product of ACP assay 783
Proton pump activities in root tissues between H and L genotypes are shown at the seedling 784
stage (A) Different letters at the top of the histogram bars denote significant differences of 785
V-ATPase in root tissues between H and L genotypes (Plt005) an asterisk () at the top of the 786
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
histogram bars indicates significant difference in V-PPase activity in root tissues between H 787
and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788
Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789
genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790
bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791
bars on the figures indicate SD (n=6) 792
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793
seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794
to the total NO3- in protoplasts 795
NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796
total NO3-in the cytosol and was calculated as total NO3
- in protoplasts ndash total NO3- in 797
vacuole Different letters at the top of histogram bars denote significant differences of total 798
NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799
(n=6) 800
801
Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802
roots to shoots at the seedling stage 803
H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804
Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805
the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806
as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807
the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808
the histogram bars denote significant differences at Plt005 809
Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810
in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811
genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812
of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813
SD (n=3) different letters at the top of the histogram bars denote significant differences at 814
Plt005 level 815
The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816
at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817
genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818
different letters at the top of the histogram bars denote significant differences at Plt005 819
820
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
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30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
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Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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17
flowering stages (Han et al 2015aHan et al 2015b) thus we expected similar differences in 474
root tissues Our results confirmed that the activities of tonoplast proton-pumps in root tissues 475
of B napus H genotype and A thaliana mutants were lower than those in the L genotype and 476
A thaliana wild type leading to less NO3- influx into vacuoles and more in the cytosol (Fig 1 477
Fig 5A-D) Moreover the enzymes catalyzing N assimilation are located in the cytoplasm 478
where NO3- could be assimilated (Martinoia et al 1981 Han et al 2015a) or immediately 479
loaded into xylem sap for long-distance transport from roots to shoots (Lin et al 2008) 480
Consequently the NO3- concentration in root tissues of the H genotype and A thaliana 481
mutants defective in tonoplast proton-pumps was significantly lower than that in the L 482
genotype (Fig 2E) and in A thaliana wild type (Fig 6C) Therefore the higher proportion of 483
NO3- transported to shoot and the lower concentrations retained in root vacuoles contributed 484
to the higher NUE in Xiangyou15 and A thaliana mutants (vha-a2 vha-a3 and avp1) (Table 485
1Fig 6A) Further studies are needed to assess the impacts of regulating vacuolar 486
sequestration of NO3- and rates of long-distance transport for enhancing NUE in other crop 487
species and genotypes 488
These data suggested that NO3- distribution between vacuoles and cytosol within cells 489
regulates NO3- long-distance transport from root to shoot and ultimately affects NUE NO3
- 490
loading into the xylem sap is an active transport process (Lin et al 2008) therefore not 491
regulated by absolute concentration of NO3- in the cytosol but rather by genes involved in 492
NO3- long-distance transport (NRT15 and NRT18) Apparently the expression of NRT15 the 493
gene responsible for xylem loading is regulated by NO3-concentration in the cytosol 494
Therefore both NO3- short-distance transport across the tonoplast and its long-distance 495
transport from root to shoot are important determinants of NUE in both B napus and A 496
thaliana (Table 1 and Fig 6) 497
498
Roles of NRT15 and NRT18 in NO3- long-distance transport in B napus and A 499
thaliana 500
Our results (Figs S9 S10) showed that the proportion of NO3- distributed from root to 501
shoot is strongly controlled by NRT15 in accord with previous studies (Lin et al 2008 Li et 502
al 2010) The data suggest that the high-NUE genotypes possess lower activities of tonoplast 503
proton-pumps (V-ATPase and V-PPase) resulting in less NO3- accumulation in vacuoles and 504
more NO3- retention in cytosol (Fig 7) The higher NO3
- in the cytosol then up-regulated 505
NRT15 which then down-regulates NRT18 As a result more NO3- is loaded into the xylem 506
system by NRT15 mechanism and less NO3- is unloaded via NRT18 (Fig 7) Differential 507
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18
regulation of these NRT genes in high-NUE plants increased transport of NO3- from root to 508
shoot through the xylem vascular tissues (Fig 7) This coordinated NO3- long- and 509
short-distance transport likely fine-tunes NO3- allocation and modulates the balance of NO3
- 510
distribution between roots and shoots (Fig 7) 511
Our research also revealed differences between A thaliana and B napus in the 512
expression of the wild-type NRT genes in response to NO3- in the protoplast Moreover using 513
A thaliananrt15-3 mutants we uncovered possible regulatory interactions between a 514
functioning (wild-type) NRT15 gene and NRT18 expression in response to NO3- 515
concentration Because mutants defective in nrt15 are not available for B napus we could 516
not confirm that the respective NRT15 and NRT18 systems behave in an analogous way in 517
these plant species Based on Harper et al (2012) research database our homology 518
comparisons and gene function annotations provided strong evidence for the roles of 519
BnNRT15 and BnNRT18 in B napus Finally our studies were conducted using two model 520
dicotyledonous plant species for which we had ample data on responses to specific inhibitors 521
(B napus) or specific mutants were available with results forming the bases for the model 522
presented in Figure 7 Further research using genetically diverse plant species (eg 523
monocotyledonous crops such as rice wheat and corn) will help validate this model and its 524
broader application across species The information generated in this study could have future 525
application for improving NUE in commercial crops with consequent reduction in nitrogen 526
use and benefits to the environment 527
528
Materials and Methods 529
Plant material 530
The two oilseed rape (Bnapus) cultivars used in this study have been characterized 531
before as high- (Xiangyou15 referred to as H genotype hereafter) and low- (814 referred to 532
as L genotype) nitrogen use efficiency (NUE) genotypes (Zhang et al 2009 Han et al 2015b) 533
Here we define NUE as the total biomass per unit of N uptake by the plant with total 534
biomass includes roots shoots and grains The two genotypes were provided by the Hunan 535
Sub-Center of Improvement Center of National Oil Crops Hunan China 536
The Arabidopsis thaliana wild-type Columbia-0 (col-0) was used as control for 537
V-ATPase (vha-a2 and vha-a3) V-PPase (avp1) nrt15-3 and nrt18-2 mutants whereas the A 538
thaliana wild-type Wassilewskija (Ws) was used as control for clca-2 mutants A thaliana 539
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19
mutants and transgenic lines (nrt15-3 and nrt18-2) have been described previously (Chen et 540
al 2012 Zhang et al 2014) The mutant lines vha-a2 (Salk_142642) and vha-a3 541
(Salk_122135) described by Krebs et al (2010) were obtained from the Arabidopsis 542
Biological Resources Center (ABRC) The mutant line avp1 (GK-005004) described in Li et 543
al (2005) was obtained from the European Arabidopsis Stock Centre while the mutant line 544
clca-2 (FST 171A06) described in Angeli et al (2006) was from the Institute National de la 545
Recherche Agronomique (INRA) collection in France together with the T-DNA mutants used 546
for genotyping to select pure mutant lines 547
548
Growth conditions 549
B napus plants were grown hydroponically in ceramic pots(20cmtimes15cm) filled with a 550
nutrient solution in the greenhouse under natural light as described in Han et al (2015b) The 551
pots were arranged in a completely randomized design with six biological replications The 552
nutrient solution was replaced every 3 days and its pH adjusted to 55 daily Experiments were 553
conducted at the field station of Hunan Agricultural University Southern China 554
A thaliana plants were grown in a nutrient solution in plastic pots as described in Arteca 555
et al (2000) and Gong et al (2003) The solution was changed every 3 days with pH adjusted 556
daily to 58 Pots were arranged in a completely randomized design with six biological 557
replications The nutrient solution used for both species consisted of 125mM KNO3 558
0625mM KH2PO4 05mM MgSO4 05mM Ca (NO3)2middot4H2O 0025mM Fe-EDTA 025 559
mlL-1 micronutrients (stock solution concentrations 70mM B 14mM Mn 1mM Zn 05mM 560
Cu and 02mM Mo) The experiments were conducted at Hunan Agricultural University in a 561
phytotron set at 70 relative humidity16h-light8h-dark cycle and constant temperature of 562
22degC 563
564
Experimental treatments and phenotyping 565
All measurements made on B napus plants were conducted either at the seedling stage 566
(2 months after transplanting) or at flowering (5 months after transplanting) The whole root 567
tissue was harvested and used for analyses at either stage For shoot measurements the fourth 568
leaf from the bottom was used for measurements made at seedling stage whereas the 12th leaf 569
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
from the bottom was harvested for measurements made at flowering stage For A thaliana 570
whole root and shoot of four-week-old plants were sampled and used for different assays 571
572
Vacuolar proton-pumps inhibitor treatments 573
Inhibition of vacuolar proton pumps of B napus (Xiangyou15) was conducted at the seedling 574
stage in hydroponic culture as described in Han et al (2015a) with minor modifications 575
Plants grown in hydroponic solution were used as control Inhibitor treatment was conducted 576
using several chemicals Bafi(25 nmol L-1 Bafilomycin A1) an inhibitor of V-ATPase 577
DCCD(10 μmol L-1DCCD + 50 mmolL-1 Na2SO3) specifically inhibits V-PPase and 578
Bafi+DCCD in a 11 ratio as inhibitor of both V-ATPase and V-PPase The inhibitors were 579
applied in the hydroponic solutions for 24 h Fresh plant tissue samples from all treatments 580
were collected for different assays 581
582
NO3- induced gene expression 583
Four-week old A thaliana mutants (nrt15-3nrt18-2) were grown in hydroponics with 584
225mM (NH4)2succinate for 3 days and shifted to hydroponics with 45mM NO3- for 12 h 585
then root tissues were collected to assess relative expression of AtNRT15 and AtNRT18 586
genes Seedlings of B napus (Xiangyou15) plants grown hydroponically were treated with 587
5mM (NH4)2succinate for 3 d and shifted to fresh hydroponic solution containing 15 mM 588
NO3- for 12 h Root tissue was then collected to assess the relative expression of BnNRT15 589
and BnNRT18 genes using quantitative RT-PCR 590
591
ACC treatment 592
Hydroponically grown seedlings of B napus were subjected to ACC treatment as described in 593
Zhang et al (2014) with minor modifications Seedlings of the two genotypes Xiangyou15 594
and 814 were transferred to a nutrient solution containing 002mM L-1 ACC for 6 h or grown 595
in normal hydroponic solution as control Root tissues were then collected to assay relative 596
expression of BnNRT15 and BnNRT18 using quantitative RT-PCR Another set of B napus 597
plants were grown in ACC-containing hydroponic solution but supplemented with 15NO3- (22 598
15N) replacing the unlabeled NO3- for 1 h Root and shoot tissues were collected separately 599
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21
for 15N measurement using a continuous-flow isotope ratio mass spectrometer coupled with a 600
C-N elemental analyzer (ANCA-MS PDZ Europa) 601
602
Biomass and N concentration 603
Grain yield dry biomass and N concentration were determined in plant samples at 604
harvest Plants were harvested (including senescing leaves) then dried in an oven for 30 min 605
at 105degC then at 70degC to a constant weight and weighed N concentration was determined 606
using the Kjeldahl method (Shi et al 2010 Han et al 2015b)02g of dried samples were 607
digested with H2SO4-H2O2 in a 100-ml Kjeldahl digestion flask after which N concentration 608
was determined using a Foss Auto Analyser Unit (Kjeldahl 8400) 609
610
V-ATPase and V-PPase activities 611
Roots (10 g) of B napus were collected at seedling and flowering stages and that of A 612
thaliana (05 g) sampled at seedling stage then used for determining the activities of 613
V-ATPase and V-PPase (Han et al 2015b) Activities of these two energy pumps within 614
microsomal membranes were followed colorimetrically based on released Pi as described by 615
Zhu et al (2001) and Krebs et al (2010) 616
617
Assay of NO3- flux in vacuoles 618
Roots of both species were collected similarly as that used for V-ATPase and V-PPase 619
activities then used for vacuole isolation and measurement of NO3- flux in vacuoles as 620
described by Robert et al (2007) with minor modification (Han et al 2015b) Net fluxes of 621
NO3- in vacuoles were measured non-invasively using SIET (scanning ion-selective electrode 622
technique SIET system BIO-003A Younger USA Science and Technology Corporation 623
USA) An NO3--selective microelectrode used for the assay of NO3
- flux was vibrated in the 624
measuring solution between two positions 1 μm and 11 μm from the vacuole surface 625
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles Background 626
signal was recorded by vibrating the electrode in the measuring solution in the absence of 627
vacuoles Ion flux was calculated by Fickrsquos law of diffusion J = -D(dcdx) where J represents 628
the ion flux (picomolescm-2s-1) dcdx is the ion concentration gradient and D is the ion 629
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
diffusion constant in a particular medium The direction of the flux was derived from Fickrsquos 630
law of diffusion that relates the concentration gradient 631
632
Isolation of intact protoplasts and vacuoles for determining NO3- concentration 633
Similar to the above assays root tissues (10 g) were collected from B napus at seedling 634
and flowering stages and from A thaliana (05 g) seedlings and used to isolate intact 635
protoplasts and vacuoles as described by Robert et al (2007) with minor modifications as 636
outlined in Huang et al (2012) and Han et al(2015b) The purified protoplasts were divided 637
into two equal aliquots with one of them used for isolation of vacuoles The purified 638
protoplasts and vacuoles were then sub-sampled and used to determine NO3- concentrations 639
(Vogeli-Lange and Wagner 1990) and for enzyme activity assays (Ma et al 2005) 640
NO3-concentrations in protoplasts and vacuoles were measured by a continuous-flow 641
auto-analyzer (Auto Analyser 3 Bran and Luebbe Germany) as described previously (Han et 642
al 2015b)The activities of acid phosphatase (ACP) and cytochrome oxidase (COX) were 643
determined using plant ACP colorimetry- and COX-assay kits (GenMedSci Inc) following 644
the manufacturerrsquos instructions ACP activity specific to vacuoles was determined and used to 645
normalize NO3- accumulation We measured NO3
- in the protoplast outside the vacuole which 646
includes the cytosol and organelles eg mitochondria and Golgi Apparatus (Robert et 647
al2007) Since most of the NO3- in the protoplast outside the vacuole is located in the cytosol 648
(Krebs et al 2010) we refer to NO3- distribution between vacuoles and cytosol rather than 649
vacuole versus protoplast 650
651
Quantitative RT-PCR 652
Total RNA extracted from B napus roots was prepared using Trizol reagent (Invitrogen) 653
cDNAs were synthesized using M-MLV reverse transcriptase (Promega) following 654
manufacturerrsquos protocol The relative expression of BnNRT15 (EV220114) BnNRT18 655
(EV116423) and Bnactin (AF1118121) genes in plant roots were determined by quantitative 656
RT-PCR and run on a LightCycler instrument (Roche) with the SYBR Green Real-Time PCR 657
Master Mix Kit (TOYOBO Japan) under the following conditions 95degC for 2min then 45 658
cycles of 95degC for 10s 60degC for 10s and 72degC for 20s The primer sequences of BnNRT15 659
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
BnNRT18 and Bnactin genes were obtained from httpbrassicanbiacuk as described in 660
Harper et al (2012) and expression levels were normalized to Bnactin as control 661
Total RNA extracted from the 4-week-old A thaliana roots under the treatments 662
conditions described above were prepared using Trizol reagent (Invitrogen) The cDNAs were 663
synthesized using M-MLV reverse transcriptase (Promega) based on the manufacturerrsquos 664
protocol and as described in Li et al (2010)The primer sequences of quantitative RT-PCR 665
used in these assays are listed in Supplemental Table 2 and were described previously (Chen 666
et al 2012 Zhang et al 2014) and their expression was normalized to Atactin2 as control 667
668
Determination of nitrate distribution using 15NO3- 669
Hydroponically grown B napus (seedling and flowering stages) and A thaliana 670
(seedling) were transferred to 01mM CaSO4 solution for 1min then to their respective 671
hydroponic nutrient solutions with15NO3- (22 excess) replacing unlabeled NO3
- for 1h 672
Plants were then transferred to 01mM CaSO4 solution for 1min after which roots were 673
washed with deionized water Root and shoot samples were separated and dried at 105degC for 674
30min followed by 70degC for 3 d then grounded and used for assaying 15N content using a 675
continuous-flow isotope ratio mass spectrometer coupled with a carbon-nitrogen elemental 676
analyzer (ANCA-MS PDZ Europa) 677
678
Xylem sap collection and assay of nitrate concentration 679
We used the method of Tang et al (2012) for collecting xylem sap Plants were cut 680
leaving1cm segments with intact roots at seedling and flowering stages for B napus and at 681
seedling stage for A thaliana Roots were immediately immersed in their respective nutrition 682
solutions Weighed cotton was put on the cut surface to absorb extruding xylem sap for 1 h 683
The cotton was then wrapped in plastic film and the volume of xylem sap was calculated as 684
the weight gain of the cotton Xylem sap was then squeezed from the cotton using a syringe 685
and used for subsequent assay of nitrate concentration using a continuous-flow auto-analyzer 686
(Auto Analyser 3 Bran and Luebbe Germany) 687
To determine nitrate concentration samples (10 g fresh root and shoot at seedling and 688
flowering of B napus and 05 g fresh root and shoot of 4-week-old A thaliana) were frozen 689
in liquid N2 and ground with a mortar and pestle the powder was then transferred to a beaker 690
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
containing 70 ml deionized water and boiled for 30 min to extract nitrate then cooled and 691
made to 100 ml volume and 02 g activated carbon added to eliminate the effect of 692
chlorophyll The mixture was filtered and nitrate in the filtrate was determined using a 693
continuous-flow auto-analyser (Atuo-Analyser 3 Bran and Luebbe Germany) 694
695
Measurements of photosynthesis 696
All measurements of photosynthesis and related parameters were conducted at 1000 h 697
using intact leaves The 4th leaf from the bottom in B napus at seedling stage and the 12th leaf 698
from the bottom at flowering stage were used for determining chlorophyll concentration 699
photosynthetic rate intercellular CO2 concentration transpiration rate and stomatal 700
conductance Chlorophyll concentration was determined by SPAD-502 (Minolta Camera Co 701
Ltd Japan) (Luo et al 2006 Yandeau-Nelson et al 2011) Photosynthetic rate intercellular 702
CO2 concentration transpiration rate and stomatal conductance were measured using LI-6400 703
Portable Photosynthesis System (Li-Cor Biosciences Co Ltd USA) set at flow rate of 500 704
μmol s-1 photosynthetic photon flux density of 1000 μmol m-2 s-1 relative humidity of 65 705
CO2 concentration of 400ppm and ambient temperatures 706
707
NR and GS activities 708
Leaves with similar age as those used for photosynthesis in B napus were used for the 709
assay of nitrate reductase (NR) and glutamine synthetase (GS) activities NR activity was 710
measured by the modified method of Fan et al (2007) Fresh samples (05g) were frozen in 711
liquid N2 ground into powder in the presence of acid-washed sand and homogenized with 4 712
ml of extraction buffer (0025mol L-1phosphate buffer pH87 1211g L-1 cysteine 0372g L-1 713
EDTA) Homogenates were centrifuged at 30000timesg for 15 min at 4degC and the supernatants 714
were treated with sulfanilamide and α-naphthylamine reagents for colorimetric (540nm) 715
determination of nitrite as described by Fan et al (2007) 716
GS activity was determined using a modified reverseγ-glutamyltransferase method 717
(Wang et al2014) which measures the GS-catalyzed formation of glutamyl-γ-hydroxamate 718
from glutamine and hydroxylamine Fresh samples were frozen for 30 min at -20degC then 719
ground in 10ml Tris-HCl buffer and acid-washed sand The homogenates were filtered 720
through two layers of gauze and centrifuged at 8000timesg for 15 min at 4degC About 12ml of the 721
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
supernatant was added in a reaction mixture and treated as described (Wang et al2014) After 722
the GS-catalyzed reaction was stopped and the solution centrifuged glutamyl-γ723
-hydroxamate was quantified colorimetrically in the supernatant (at 485 nm) and the 724
concentration was determined using a standard curve 725
726
Statistical analyses 727
We used the SPSS software (Statistical Product and Service Solutions V130 USA) for 728
ANOVA and mean separation of main-effects and interactions using Duncanrsquos multiple range 729
test at Plt005Values are presented as means and SD of three or six replicates from three 730
independent experiments Different letters or an asterisk () associated with specific data (eg 731
at the top of histogram bars in figures or within tables) denote significant differences at 732
Plt005 733
734
Supplemental Data 735
Supplemental Figure 1 B napus with higher NUE showed lower vacuolar sequestration 736
capacity (VSC) for NO3-in roots at flowering stage 737
Supplemental Figure 2 B napus with higher NUE showed enhanced long-distance transport 738
of NO3- from roots to shoots at flowering stage 739
Supplemental Figure 3 The two B napus (H and L genotypes) showed the same total N per 740
plant at seedling stage (A) and flowering stage (B) 741
Supplemental Figure 4 B napus with higher NUE showed increased NO3- concentration in 742
the xylem sap at seedling and flowering stages 743
Supplemental Figure 5 NO3- concentration in the xylem sap as affected by inhibitor 744
treatments in B napus and in the energy pumpsrsquo mutants of A thaliana (col-0 vha-a2 vha-a3 745
avp1) 746
Supplemental Figure 6 Reduced VSC for NO3- in roots drives long-distance transport of 747
NO3- from roots to shoots in the A thaliana wild type (Ws) and mutant (clca-2) 748
Supplemental Figure 7 Differences of NR and GS activities between the two B napus (H 749
and L genotypes) at seedling and flowering stages 750
Supplemental Figure 8 Amino acid sequences of BnNRT15 and BnNRT18 751
Supplemental Figure 9 Functions of AtNRT15 and AtNRT18 genes in root tissues of A 752
thaliana in controlling NO3- long-distance transport from root to shoots 753
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
Supplemental Figure 10 B napus BnNRT15 and BnNRT18 genes in roots were 754
coordinately modulated to facilitate NO3- long distance transport from roots to shoots 755
Supplemental Table 1 Differences in stomatal conductance and transpiration rate between 756
the two B napus genotypes 757
Supplemental Table 2 Sequences of primera used for qRT-PCR 758
759
Acknowledgements 760
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 761
Shanghai Institutes for Biological Sciences) for providing nrt15-3 and nrt18-2 seeds 762
This study was supported by the National Natural Science Foundation of China (Grant 763
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 764
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 765
The Ministry of Education in China Open Novel Science Foundation of Hunan province 766
(13K062) the National Key Laboratory of Plant Molecular Genetics and the Twelfth 767
Five-Year National Science and technology support program (2012BAD15BO4) 768
769
Figure 1 B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 770
NO3- in roots at the seedling stage 771
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 772
genotype 814 Specific activities of the tonoplast proton pumps are expressed as μmol Pi 773
released mg-1 protein h-1 NO3- fluxes are expressed as pmol NO3
- cm-2 S-1 Mature vacuoles 774
were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 775
measuring solution between the two positions 1 μm and 11 μm from the vacuole surface 776
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles recording the 777
stable reading data The background was recorded by vibrating the electrode in measuring 778
solution without vacuoles 779
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 780
for NO3- content and NO3
- accumulation normalized against the specific activity of the 781
vacuole acid phosphatase (ACP) as described in Materials and Methods and plotted as μmol 782
NO3- per μmol p-nitrophenol the end product of ACP assay 783
Proton pump activities in root tissues between H and L genotypes are shown at the seedling 784
stage (A) Different letters at the top of the histogram bars denote significant differences of 785
V-ATPase in root tissues between H and L genotypes (Plt005) an asterisk () at the top of the 786
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
histogram bars indicates significant difference in V-PPase activity in root tissues between H 787
and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788
Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789
genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790
bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791
bars on the figures indicate SD (n=6) 792
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793
seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794
to the total NO3- in protoplasts 795
NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796
total NO3-in the cytosol and was calculated as total NO3
- in protoplasts ndash total NO3- in 797
vacuole Different letters at the top of histogram bars denote significant differences of total 798
NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799
(n=6) 800
801
Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802
roots to shoots at the seedling stage 803
H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804
Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805
the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806
as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807
the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808
the histogram bars denote significant differences at Plt005 809
Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810
in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811
genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812
of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813
SD (n=3) different letters at the top of the histogram bars denote significant differences at 814
Plt005 level 815
The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816
at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817
genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818
different letters at the top of the histogram bars denote significant differences at Plt005 819
820
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
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31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
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Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
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b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
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Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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Tang Z Fan XR Li Qing Feng HM Miller AJ Shen QR Xu GH (2012) Knockdown of a rice stellar nitrate transporter alters long-distance translocation but not root influx Plant Physiology 1602052-2063
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Wang YY Tsay YF (2011) Arabidopsis nitrate transporter NRT19 is important in phloem nitrate transport The Plant Cell 231945-1957
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Xu GH Fan XR Miller AJ (2012) Plant nitrogen assimilation and use efficiency The Annual Review of Plant Biology 63153-182Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yandeau-Nelson MD Laurens L Shi Z Xia H Smith AM Guiltinan MJ (2011) Starch-Branching Enzyme IIa Is Required for ProperDiurnal Cycling of Starch in Leaves of Maize [OA] Plant Physiology 156479-490
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Zhang GB Yi HY Gong JM (2014) The Arabidopsis EthyleneJasmonic acid-NRT signaling module coordinates nitrate reallocationand the trade-off between growth and environmental adaption The Plant Cell 26(10)3984-3998
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Zhang ZH Song HX Liu Qing Rong XM Xie GX Peng JW Zhang YP (2009) Study on differences of nitrogen efficiency andnitrogen response in different oilseed rape (Brassica napus L) varieties Asian Journal of Crop Science 1(2)105-112
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Zhang ZH Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2010) Nitrogen redistribution characteristics ofoilseed rape varieties with different nitrogen use efficiencies during later growth period Communications in Soil Science andPlant Analysis 41(14) 1693-1706
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Zhang ZH Huang HT Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2012) Research advances on nitratenitrogen reutilization by proton pump of tonoplast and its relation to nitrogen use efficiency Australian Journal of Crop Science6(9) 1377-1382
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- Parsed Citations
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- ONE
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18
regulation of these NRT genes in high-NUE plants increased transport of NO3- from root to 508
shoot through the xylem vascular tissues (Fig 7) This coordinated NO3- long- and 509
short-distance transport likely fine-tunes NO3- allocation and modulates the balance of NO3
- 510
distribution between roots and shoots (Fig 7) 511
Our research also revealed differences between A thaliana and B napus in the 512
expression of the wild-type NRT genes in response to NO3- in the protoplast Moreover using 513
A thaliananrt15-3 mutants we uncovered possible regulatory interactions between a 514
functioning (wild-type) NRT15 gene and NRT18 expression in response to NO3- 515
concentration Because mutants defective in nrt15 are not available for B napus we could 516
not confirm that the respective NRT15 and NRT18 systems behave in an analogous way in 517
these plant species Based on Harper et al (2012) research database our homology 518
comparisons and gene function annotations provided strong evidence for the roles of 519
BnNRT15 and BnNRT18 in B napus Finally our studies were conducted using two model 520
dicotyledonous plant species for which we had ample data on responses to specific inhibitors 521
(B napus) or specific mutants were available with results forming the bases for the model 522
presented in Figure 7 Further research using genetically diverse plant species (eg 523
monocotyledonous crops such as rice wheat and corn) will help validate this model and its 524
broader application across species The information generated in this study could have future 525
application for improving NUE in commercial crops with consequent reduction in nitrogen 526
use and benefits to the environment 527
528
Materials and Methods 529
Plant material 530
The two oilseed rape (Bnapus) cultivars used in this study have been characterized 531
before as high- (Xiangyou15 referred to as H genotype hereafter) and low- (814 referred to 532
as L genotype) nitrogen use efficiency (NUE) genotypes (Zhang et al 2009 Han et al 2015b) 533
Here we define NUE as the total biomass per unit of N uptake by the plant with total 534
biomass includes roots shoots and grains The two genotypes were provided by the Hunan 535
Sub-Center of Improvement Center of National Oil Crops Hunan China 536
The Arabidopsis thaliana wild-type Columbia-0 (col-0) was used as control for 537
V-ATPase (vha-a2 and vha-a3) V-PPase (avp1) nrt15-3 and nrt18-2 mutants whereas the A 538
thaliana wild-type Wassilewskija (Ws) was used as control for clca-2 mutants A thaliana 539
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
19
mutants and transgenic lines (nrt15-3 and nrt18-2) have been described previously (Chen et 540
al 2012 Zhang et al 2014) The mutant lines vha-a2 (Salk_142642) and vha-a3 541
(Salk_122135) described by Krebs et al (2010) were obtained from the Arabidopsis 542
Biological Resources Center (ABRC) The mutant line avp1 (GK-005004) described in Li et 543
al (2005) was obtained from the European Arabidopsis Stock Centre while the mutant line 544
clca-2 (FST 171A06) described in Angeli et al (2006) was from the Institute National de la 545
Recherche Agronomique (INRA) collection in France together with the T-DNA mutants used 546
for genotyping to select pure mutant lines 547
548
Growth conditions 549
B napus plants were grown hydroponically in ceramic pots(20cmtimes15cm) filled with a 550
nutrient solution in the greenhouse under natural light as described in Han et al (2015b) The 551
pots were arranged in a completely randomized design with six biological replications The 552
nutrient solution was replaced every 3 days and its pH adjusted to 55 daily Experiments were 553
conducted at the field station of Hunan Agricultural University Southern China 554
A thaliana plants were grown in a nutrient solution in plastic pots as described in Arteca 555
et al (2000) and Gong et al (2003) The solution was changed every 3 days with pH adjusted 556
daily to 58 Pots were arranged in a completely randomized design with six biological 557
replications The nutrient solution used for both species consisted of 125mM KNO3 558
0625mM KH2PO4 05mM MgSO4 05mM Ca (NO3)2middot4H2O 0025mM Fe-EDTA 025 559
mlL-1 micronutrients (stock solution concentrations 70mM B 14mM Mn 1mM Zn 05mM 560
Cu and 02mM Mo) The experiments were conducted at Hunan Agricultural University in a 561
phytotron set at 70 relative humidity16h-light8h-dark cycle and constant temperature of 562
22degC 563
564
Experimental treatments and phenotyping 565
All measurements made on B napus plants were conducted either at the seedling stage 566
(2 months after transplanting) or at flowering (5 months after transplanting) The whole root 567
tissue was harvested and used for analyses at either stage For shoot measurements the fourth 568
leaf from the bottom was used for measurements made at seedling stage whereas the 12th leaf 569
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
from the bottom was harvested for measurements made at flowering stage For A thaliana 570
whole root and shoot of four-week-old plants were sampled and used for different assays 571
572
Vacuolar proton-pumps inhibitor treatments 573
Inhibition of vacuolar proton pumps of B napus (Xiangyou15) was conducted at the seedling 574
stage in hydroponic culture as described in Han et al (2015a) with minor modifications 575
Plants grown in hydroponic solution were used as control Inhibitor treatment was conducted 576
using several chemicals Bafi(25 nmol L-1 Bafilomycin A1) an inhibitor of V-ATPase 577
DCCD(10 μmol L-1DCCD + 50 mmolL-1 Na2SO3) specifically inhibits V-PPase and 578
Bafi+DCCD in a 11 ratio as inhibitor of both V-ATPase and V-PPase The inhibitors were 579
applied in the hydroponic solutions for 24 h Fresh plant tissue samples from all treatments 580
were collected for different assays 581
582
NO3- induced gene expression 583
Four-week old A thaliana mutants (nrt15-3nrt18-2) were grown in hydroponics with 584
225mM (NH4)2succinate for 3 days and shifted to hydroponics with 45mM NO3- for 12 h 585
then root tissues were collected to assess relative expression of AtNRT15 and AtNRT18 586
genes Seedlings of B napus (Xiangyou15) plants grown hydroponically were treated with 587
5mM (NH4)2succinate for 3 d and shifted to fresh hydroponic solution containing 15 mM 588
NO3- for 12 h Root tissue was then collected to assess the relative expression of BnNRT15 589
and BnNRT18 genes using quantitative RT-PCR 590
591
ACC treatment 592
Hydroponically grown seedlings of B napus were subjected to ACC treatment as described in 593
Zhang et al (2014) with minor modifications Seedlings of the two genotypes Xiangyou15 594
and 814 were transferred to a nutrient solution containing 002mM L-1 ACC for 6 h or grown 595
in normal hydroponic solution as control Root tissues were then collected to assay relative 596
expression of BnNRT15 and BnNRT18 using quantitative RT-PCR Another set of B napus 597
plants were grown in ACC-containing hydroponic solution but supplemented with 15NO3- (22 598
15N) replacing the unlabeled NO3- for 1 h Root and shoot tissues were collected separately 599
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
21
for 15N measurement using a continuous-flow isotope ratio mass spectrometer coupled with a 600
C-N elemental analyzer (ANCA-MS PDZ Europa) 601
602
Biomass and N concentration 603
Grain yield dry biomass and N concentration were determined in plant samples at 604
harvest Plants were harvested (including senescing leaves) then dried in an oven for 30 min 605
at 105degC then at 70degC to a constant weight and weighed N concentration was determined 606
using the Kjeldahl method (Shi et al 2010 Han et al 2015b)02g of dried samples were 607
digested with H2SO4-H2O2 in a 100-ml Kjeldahl digestion flask after which N concentration 608
was determined using a Foss Auto Analyser Unit (Kjeldahl 8400) 609
610
V-ATPase and V-PPase activities 611
Roots (10 g) of B napus were collected at seedling and flowering stages and that of A 612
thaliana (05 g) sampled at seedling stage then used for determining the activities of 613
V-ATPase and V-PPase (Han et al 2015b) Activities of these two energy pumps within 614
microsomal membranes were followed colorimetrically based on released Pi as described by 615
Zhu et al (2001) and Krebs et al (2010) 616
617
Assay of NO3- flux in vacuoles 618
Roots of both species were collected similarly as that used for V-ATPase and V-PPase 619
activities then used for vacuole isolation and measurement of NO3- flux in vacuoles as 620
described by Robert et al (2007) with minor modification (Han et al 2015b) Net fluxes of 621
NO3- in vacuoles were measured non-invasively using SIET (scanning ion-selective electrode 622
technique SIET system BIO-003A Younger USA Science and Technology Corporation 623
USA) An NO3--selective microelectrode used for the assay of NO3
- flux was vibrated in the 624
measuring solution between two positions 1 μm and 11 μm from the vacuole surface 625
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles Background 626
signal was recorded by vibrating the electrode in the measuring solution in the absence of 627
vacuoles Ion flux was calculated by Fickrsquos law of diffusion J = -D(dcdx) where J represents 628
the ion flux (picomolescm-2s-1) dcdx is the ion concentration gradient and D is the ion 629
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
diffusion constant in a particular medium The direction of the flux was derived from Fickrsquos 630
law of diffusion that relates the concentration gradient 631
632
Isolation of intact protoplasts and vacuoles for determining NO3- concentration 633
Similar to the above assays root tissues (10 g) were collected from B napus at seedling 634
and flowering stages and from A thaliana (05 g) seedlings and used to isolate intact 635
protoplasts and vacuoles as described by Robert et al (2007) with minor modifications as 636
outlined in Huang et al (2012) and Han et al(2015b) The purified protoplasts were divided 637
into two equal aliquots with one of them used for isolation of vacuoles The purified 638
protoplasts and vacuoles were then sub-sampled and used to determine NO3- concentrations 639
(Vogeli-Lange and Wagner 1990) and for enzyme activity assays (Ma et al 2005) 640
NO3-concentrations in protoplasts and vacuoles were measured by a continuous-flow 641
auto-analyzer (Auto Analyser 3 Bran and Luebbe Germany) as described previously (Han et 642
al 2015b)The activities of acid phosphatase (ACP) and cytochrome oxidase (COX) were 643
determined using plant ACP colorimetry- and COX-assay kits (GenMedSci Inc) following 644
the manufacturerrsquos instructions ACP activity specific to vacuoles was determined and used to 645
normalize NO3- accumulation We measured NO3
- in the protoplast outside the vacuole which 646
includes the cytosol and organelles eg mitochondria and Golgi Apparatus (Robert et 647
al2007) Since most of the NO3- in the protoplast outside the vacuole is located in the cytosol 648
(Krebs et al 2010) we refer to NO3- distribution between vacuoles and cytosol rather than 649
vacuole versus protoplast 650
651
Quantitative RT-PCR 652
Total RNA extracted from B napus roots was prepared using Trizol reagent (Invitrogen) 653
cDNAs were synthesized using M-MLV reverse transcriptase (Promega) following 654
manufacturerrsquos protocol The relative expression of BnNRT15 (EV220114) BnNRT18 655
(EV116423) and Bnactin (AF1118121) genes in plant roots were determined by quantitative 656
RT-PCR and run on a LightCycler instrument (Roche) with the SYBR Green Real-Time PCR 657
Master Mix Kit (TOYOBO Japan) under the following conditions 95degC for 2min then 45 658
cycles of 95degC for 10s 60degC for 10s and 72degC for 20s The primer sequences of BnNRT15 659
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
BnNRT18 and Bnactin genes were obtained from httpbrassicanbiacuk as described in 660
Harper et al (2012) and expression levels were normalized to Bnactin as control 661
Total RNA extracted from the 4-week-old A thaliana roots under the treatments 662
conditions described above were prepared using Trizol reagent (Invitrogen) The cDNAs were 663
synthesized using M-MLV reverse transcriptase (Promega) based on the manufacturerrsquos 664
protocol and as described in Li et al (2010)The primer sequences of quantitative RT-PCR 665
used in these assays are listed in Supplemental Table 2 and were described previously (Chen 666
et al 2012 Zhang et al 2014) and their expression was normalized to Atactin2 as control 667
668
Determination of nitrate distribution using 15NO3- 669
Hydroponically grown B napus (seedling and flowering stages) and A thaliana 670
(seedling) were transferred to 01mM CaSO4 solution for 1min then to their respective 671
hydroponic nutrient solutions with15NO3- (22 excess) replacing unlabeled NO3
- for 1h 672
Plants were then transferred to 01mM CaSO4 solution for 1min after which roots were 673
washed with deionized water Root and shoot samples were separated and dried at 105degC for 674
30min followed by 70degC for 3 d then grounded and used for assaying 15N content using a 675
continuous-flow isotope ratio mass spectrometer coupled with a carbon-nitrogen elemental 676
analyzer (ANCA-MS PDZ Europa) 677
678
Xylem sap collection and assay of nitrate concentration 679
We used the method of Tang et al (2012) for collecting xylem sap Plants were cut 680
leaving1cm segments with intact roots at seedling and flowering stages for B napus and at 681
seedling stage for A thaliana Roots were immediately immersed in their respective nutrition 682
solutions Weighed cotton was put on the cut surface to absorb extruding xylem sap for 1 h 683
The cotton was then wrapped in plastic film and the volume of xylem sap was calculated as 684
the weight gain of the cotton Xylem sap was then squeezed from the cotton using a syringe 685
and used for subsequent assay of nitrate concentration using a continuous-flow auto-analyzer 686
(Auto Analyser 3 Bran and Luebbe Germany) 687
To determine nitrate concentration samples (10 g fresh root and shoot at seedling and 688
flowering of B napus and 05 g fresh root and shoot of 4-week-old A thaliana) were frozen 689
in liquid N2 and ground with a mortar and pestle the powder was then transferred to a beaker 690
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
containing 70 ml deionized water and boiled for 30 min to extract nitrate then cooled and 691
made to 100 ml volume and 02 g activated carbon added to eliminate the effect of 692
chlorophyll The mixture was filtered and nitrate in the filtrate was determined using a 693
continuous-flow auto-analyser (Atuo-Analyser 3 Bran and Luebbe Germany) 694
695
Measurements of photosynthesis 696
All measurements of photosynthesis and related parameters were conducted at 1000 h 697
using intact leaves The 4th leaf from the bottom in B napus at seedling stage and the 12th leaf 698
from the bottom at flowering stage were used for determining chlorophyll concentration 699
photosynthetic rate intercellular CO2 concentration transpiration rate and stomatal 700
conductance Chlorophyll concentration was determined by SPAD-502 (Minolta Camera Co 701
Ltd Japan) (Luo et al 2006 Yandeau-Nelson et al 2011) Photosynthetic rate intercellular 702
CO2 concentration transpiration rate and stomatal conductance were measured using LI-6400 703
Portable Photosynthesis System (Li-Cor Biosciences Co Ltd USA) set at flow rate of 500 704
μmol s-1 photosynthetic photon flux density of 1000 μmol m-2 s-1 relative humidity of 65 705
CO2 concentration of 400ppm and ambient temperatures 706
707
NR and GS activities 708
Leaves with similar age as those used for photosynthesis in B napus were used for the 709
assay of nitrate reductase (NR) and glutamine synthetase (GS) activities NR activity was 710
measured by the modified method of Fan et al (2007) Fresh samples (05g) were frozen in 711
liquid N2 ground into powder in the presence of acid-washed sand and homogenized with 4 712
ml of extraction buffer (0025mol L-1phosphate buffer pH87 1211g L-1 cysteine 0372g L-1 713
EDTA) Homogenates were centrifuged at 30000timesg for 15 min at 4degC and the supernatants 714
were treated with sulfanilamide and α-naphthylamine reagents for colorimetric (540nm) 715
determination of nitrite as described by Fan et al (2007) 716
GS activity was determined using a modified reverseγ-glutamyltransferase method 717
(Wang et al2014) which measures the GS-catalyzed formation of glutamyl-γ-hydroxamate 718
from glutamine and hydroxylamine Fresh samples were frozen for 30 min at -20degC then 719
ground in 10ml Tris-HCl buffer and acid-washed sand The homogenates were filtered 720
through two layers of gauze and centrifuged at 8000timesg for 15 min at 4degC About 12ml of the 721
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
supernatant was added in a reaction mixture and treated as described (Wang et al2014) After 722
the GS-catalyzed reaction was stopped and the solution centrifuged glutamyl-γ723
-hydroxamate was quantified colorimetrically in the supernatant (at 485 nm) and the 724
concentration was determined using a standard curve 725
726
Statistical analyses 727
We used the SPSS software (Statistical Product and Service Solutions V130 USA) for 728
ANOVA and mean separation of main-effects and interactions using Duncanrsquos multiple range 729
test at Plt005Values are presented as means and SD of three or six replicates from three 730
independent experiments Different letters or an asterisk () associated with specific data (eg 731
at the top of histogram bars in figures or within tables) denote significant differences at 732
Plt005 733
734
Supplemental Data 735
Supplemental Figure 1 B napus with higher NUE showed lower vacuolar sequestration 736
capacity (VSC) for NO3-in roots at flowering stage 737
Supplemental Figure 2 B napus with higher NUE showed enhanced long-distance transport 738
of NO3- from roots to shoots at flowering stage 739
Supplemental Figure 3 The two B napus (H and L genotypes) showed the same total N per 740
plant at seedling stage (A) and flowering stage (B) 741
Supplemental Figure 4 B napus with higher NUE showed increased NO3- concentration in 742
the xylem sap at seedling and flowering stages 743
Supplemental Figure 5 NO3- concentration in the xylem sap as affected by inhibitor 744
treatments in B napus and in the energy pumpsrsquo mutants of A thaliana (col-0 vha-a2 vha-a3 745
avp1) 746
Supplemental Figure 6 Reduced VSC for NO3- in roots drives long-distance transport of 747
NO3- from roots to shoots in the A thaliana wild type (Ws) and mutant (clca-2) 748
Supplemental Figure 7 Differences of NR and GS activities between the two B napus (H 749
and L genotypes) at seedling and flowering stages 750
Supplemental Figure 8 Amino acid sequences of BnNRT15 and BnNRT18 751
Supplemental Figure 9 Functions of AtNRT15 and AtNRT18 genes in root tissues of A 752
thaliana in controlling NO3- long-distance transport from root to shoots 753
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
Supplemental Figure 10 B napus BnNRT15 and BnNRT18 genes in roots were 754
coordinately modulated to facilitate NO3- long distance transport from roots to shoots 755
Supplemental Table 1 Differences in stomatal conductance and transpiration rate between 756
the two B napus genotypes 757
Supplemental Table 2 Sequences of primera used for qRT-PCR 758
759
Acknowledgements 760
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 761
Shanghai Institutes for Biological Sciences) for providing nrt15-3 and nrt18-2 seeds 762
This study was supported by the National Natural Science Foundation of China (Grant 763
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 764
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 765
The Ministry of Education in China Open Novel Science Foundation of Hunan province 766
(13K062) the National Key Laboratory of Plant Molecular Genetics and the Twelfth 767
Five-Year National Science and technology support program (2012BAD15BO4) 768
769
Figure 1 B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 770
NO3- in roots at the seedling stage 771
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 772
genotype 814 Specific activities of the tonoplast proton pumps are expressed as μmol Pi 773
released mg-1 protein h-1 NO3- fluxes are expressed as pmol NO3
- cm-2 S-1 Mature vacuoles 774
were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 775
measuring solution between the two positions 1 μm and 11 μm from the vacuole surface 776
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles recording the 777
stable reading data The background was recorded by vibrating the electrode in measuring 778
solution without vacuoles 779
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 780
for NO3- content and NO3
- accumulation normalized against the specific activity of the 781
vacuole acid phosphatase (ACP) as described in Materials and Methods and plotted as μmol 782
NO3- per μmol p-nitrophenol the end product of ACP assay 783
Proton pump activities in root tissues between H and L genotypes are shown at the seedling 784
stage (A) Different letters at the top of the histogram bars denote significant differences of 785
V-ATPase in root tissues between H and L genotypes (Plt005) an asterisk () at the top of the 786
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
histogram bars indicates significant difference in V-PPase activity in root tissues between H 787
and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788
Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789
genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790
bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791
bars on the figures indicate SD (n=6) 792
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793
seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794
to the total NO3- in protoplasts 795
NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796
total NO3-in the cytosol and was calculated as total NO3
- in protoplasts ndash total NO3- in 797
vacuole Different letters at the top of histogram bars denote significant differences of total 798
NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799
(n=6) 800
801
Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802
roots to shoots at the seedling stage 803
H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804
Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805
the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806
as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807
the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808
the histogram bars denote significant differences at Plt005 809
Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810
in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811
genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812
of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813
SD (n=3) different letters at the top of the histogram bars denote significant differences at 814
Plt005 level 815
The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816
at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817
genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818
different letters at the top of the histogram bars denote significant differences at Plt005 819
820
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
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b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
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Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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- Parsed Citations
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19
mutants and transgenic lines (nrt15-3 and nrt18-2) have been described previously (Chen et 540
al 2012 Zhang et al 2014) The mutant lines vha-a2 (Salk_142642) and vha-a3 541
(Salk_122135) described by Krebs et al (2010) were obtained from the Arabidopsis 542
Biological Resources Center (ABRC) The mutant line avp1 (GK-005004) described in Li et 543
al (2005) was obtained from the European Arabidopsis Stock Centre while the mutant line 544
clca-2 (FST 171A06) described in Angeli et al (2006) was from the Institute National de la 545
Recherche Agronomique (INRA) collection in France together with the T-DNA mutants used 546
for genotyping to select pure mutant lines 547
548
Growth conditions 549
B napus plants were grown hydroponically in ceramic pots(20cmtimes15cm) filled with a 550
nutrient solution in the greenhouse under natural light as described in Han et al (2015b) The 551
pots were arranged in a completely randomized design with six biological replications The 552
nutrient solution was replaced every 3 days and its pH adjusted to 55 daily Experiments were 553
conducted at the field station of Hunan Agricultural University Southern China 554
A thaliana plants were grown in a nutrient solution in plastic pots as described in Arteca 555
et al (2000) and Gong et al (2003) The solution was changed every 3 days with pH adjusted 556
daily to 58 Pots were arranged in a completely randomized design with six biological 557
replications The nutrient solution used for both species consisted of 125mM KNO3 558
0625mM KH2PO4 05mM MgSO4 05mM Ca (NO3)2middot4H2O 0025mM Fe-EDTA 025 559
mlL-1 micronutrients (stock solution concentrations 70mM B 14mM Mn 1mM Zn 05mM 560
Cu and 02mM Mo) The experiments were conducted at Hunan Agricultural University in a 561
phytotron set at 70 relative humidity16h-light8h-dark cycle and constant temperature of 562
22degC 563
564
Experimental treatments and phenotyping 565
All measurements made on B napus plants were conducted either at the seedling stage 566
(2 months after transplanting) or at flowering (5 months after transplanting) The whole root 567
tissue was harvested and used for analyses at either stage For shoot measurements the fourth 568
leaf from the bottom was used for measurements made at seedling stage whereas the 12th leaf 569
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20
from the bottom was harvested for measurements made at flowering stage For A thaliana 570
whole root and shoot of four-week-old plants were sampled and used for different assays 571
572
Vacuolar proton-pumps inhibitor treatments 573
Inhibition of vacuolar proton pumps of B napus (Xiangyou15) was conducted at the seedling 574
stage in hydroponic culture as described in Han et al (2015a) with minor modifications 575
Plants grown in hydroponic solution were used as control Inhibitor treatment was conducted 576
using several chemicals Bafi(25 nmol L-1 Bafilomycin A1) an inhibitor of V-ATPase 577
DCCD(10 μmol L-1DCCD + 50 mmolL-1 Na2SO3) specifically inhibits V-PPase and 578
Bafi+DCCD in a 11 ratio as inhibitor of both V-ATPase and V-PPase The inhibitors were 579
applied in the hydroponic solutions for 24 h Fresh plant tissue samples from all treatments 580
were collected for different assays 581
582
NO3- induced gene expression 583
Four-week old A thaliana mutants (nrt15-3nrt18-2) were grown in hydroponics with 584
225mM (NH4)2succinate for 3 days and shifted to hydroponics with 45mM NO3- for 12 h 585
then root tissues were collected to assess relative expression of AtNRT15 and AtNRT18 586
genes Seedlings of B napus (Xiangyou15) plants grown hydroponically were treated with 587
5mM (NH4)2succinate for 3 d and shifted to fresh hydroponic solution containing 15 mM 588
NO3- for 12 h Root tissue was then collected to assess the relative expression of BnNRT15 589
and BnNRT18 genes using quantitative RT-PCR 590
591
ACC treatment 592
Hydroponically grown seedlings of B napus were subjected to ACC treatment as described in 593
Zhang et al (2014) with minor modifications Seedlings of the two genotypes Xiangyou15 594
and 814 were transferred to a nutrient solution containing 002mM L-1 ACC for 6 h or grown 595
in normal hydroponic solution as control Root tissues were then collected to assay relative 596
expression of BnNRT15 and BnNRT18 using quantitative RT-PCR Another set of B napus 597
plants were grown in ACC-containing hydroponic solution but supplemented with 15NO3- (22 598
15N) replacing the unlabeled NO3- for 1 h Root and shoot tissues were collected separately 599
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21
for 15N measurement using a continuous-flow isotope ratio mass spectrometer coupled with a 600
C-N elemental analyzer (ANCA-MS PDZ Europa) 601
602
Biomass and N concentration 603
Grain yield dry biomass and N concentration were determined in plant samples at 604
harvest Plants were harvested (including senescing leaves) then dried in an oven for 30 min 605
at 105degC then at 70degC to a constant weight and weighed N concentration was determined 606
using the Kjeldahl method (Shi et al 2010 Han et al 2015b)02g of dried samples were 607
digested with H2SO4-H2O2 in a 100-ml Kjeldahl digestion flask after which N concentration 608
was determined using a Foss Auto Analyser Unit (Kjeldahl 8400) 609
610
V-ATPase and V-PPase activities 611
Roots (10 g) of B napus were collected at seedling and flowering stages and that of A 612
thaliana (05 g) sampled at seedling stage then used for determining the activities of 613
V-ATPase and V-PPase (Han et al 2015b) Activities of these two energy pumps within 614
microsomal membranes were followed colorimetrically based on released Pi as described by 615
Zhu et al (2001) and Krebs et al (2010) 616
617
Assay of NO3- flux in vacuoles 618
Roots of both species were collected similarly as that used for V-ATPase and V-PPase 619
activities then used for vacuole isolation and measurement of NO3- flux in vacuoles as 620
described by Robert et al (2007) with minor modification (Han et al 2015b) Net fluxes of 621
NO3- in vacuoles were measured non-invasively using SIET (scanning ion-selective electrode 622
technique SIET system BIO-003A Younger USA Science and Technology Corporation 623
USA) An NO3--selective microelectrode used for the assay of NO3
- flux was vibrated in the 624
measuring solution between two positions 1 μm and 11 μm from the vacuole surface 625
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles Background 626
signal was recorded by vibrating the electrode in the measuring solution in the absence of 627
vacuoles Ion flux was calculated by Fickrsquos law of diffusion J = -D(dcdx) where J represents 628
the ion flux (picomolescm-2s-1) dcdx is the ion concentration gradient and D is the ion 629
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
diffusion constant in a particular medium The direction of the flux was derived from Fickrsquos 630
law of diffusion that relates the concentration gradient 631
632
Isolation of intact protoplasts and vacuoles for determining NO3- concentration 633
Similar to the above assays root tissues (10 g) were collected from B napus at seedling 634
and flowering stages and from A thaliana (05 g) seedlings and used to isolate intact 635
protoplasts and vacuoles as described by Robert et al (2007) with minor modifications as 636
outlined in Huang et al (2012) and Han et al(2015b) The purified protoplasts were divided 637
into two equal aliquots with one of them used for isolation of vacuoles The purified 638
protoplasts and vacuoles were then sub-sampled and used to determine NO3- concentrations 639
(Vogeli-Lange and Wagner 1990) and for enzyme activity assays (Ma et al 2005) 640
NO3-concentrations in protoplasts and vacuoles were measured by a continuous-flow 641
auto-analyzer (Auto Analyser 3 Bran and Luebbe Germany) as described previously (Han et 642
al 2015b)The activities of acid phosphatase (ACP) and cytochrome oxidase (COX) were 643
determined using plant ACP colorimetry- and COX-assay kits (GenMedSci Inc) following 644
the manufacturerrsquos instructions ACP activity specific to vacuoles was determined and used to 645
normalize NO3- accumulation We measured NO3
- in the protoplast outside the vacuole which 646
includes the cytosol and organelles eg mitochondria and Golgi Apparatus (Robert et 647
al2007) Since most of the NO3- in the protoplast outside the vacuole is located in the cytosol 648
(Krebs et al 2010) we refer to NO3- distribution between vacuoles and cytosol rather than 649
vacuole versus protoplast 650
651
Quantitative RT-PCR 652
Total RNA extracted from B napus roots was prepared using Trizol reagent (Invitrogen) 653
cDNAs were synthesized using M-MLV reverse transcriptase (Promega) following 654
manufacturerrsquos protocol The relative expression of BnNRT15 (EV220114) BnNRT18 655
(EV116423) and Bnactin (AF1118121) genes in plant roots were determined by quantitative 656
RT-PCR and run on a LightCycler instrument (Roche) with the SYBR Green Real-Time PCR 657
Master Mix Kit (TOYOBO Japan) under the following conditions 95degC for 2min then 45 658
cycles of 95degC for 10s 60degC for 10s and 72degC for 20s The primer sequences of BnNRT15 659
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
BnNRT18 and Bnactin genes were obtained from httpbrassicanbiacuk as described in 660
Harper et al (2012) and expression levels were normalized to Bnactin as control 661
Total RNA extracted from the 4-week-old A thaliana roots under the treatments 662
conditions described above were prepared using Trizol reagent (Invitrogen) The cDNAs were 663
synthesized using M-MLV reverse transcriptase (Promega) based on the manufacturerrsquos 664
protocol and as described in Li et al (2010)The primer sequences of quantitative RT-PCR 665
used in these assays are listed in Supplemental Table 2 and were described previously (Chen 666
et al 2012 Zhang et al 2014) and their expression was normalized to Atactin2 as control 667
668
Determination of nitrate distribution using 15NO3- 669
Hydroponically grown B napus (seedling and flowering stages) and A thaliana 670
(seedling) were transferred to 01mM CaSO4 solution for 1min then to their respective 671
hydroponic nutrient solutions with15NO3- (22 excess) replacing unlabeled NO3
- for 1h 672
Plants were then transferred to 01mM CaSO4 solution for 1min after which roots were 673
washed with deionized water Root and shoot samples were separated and dried at 105degC for 674
30min followed by 70degC for 3 d then grounded and used for assaying 15N content using a 675
continuous-flow isotope ratio mass spectrometer coupled with a carbon-nitrogen elemental 676
analyzer (ANCA-MS PDZ Europa) 677
678
Xylem sap collection and assay of nitrate concentration 679
We used the method of Tang et al (2012) for collecting xylem sap Plants were cut 680
leaving1cm segments with intact roots at seedling and flowering stages for B napus and at 681
seedling stage for A thaliana Roots were immediately immersed in their respective nutrition 682
solutions Weighed cotton was put on the cut surface to absorb extruding xylem sap for 1 h 683
The cotton was then wrapped in plastic film and the volume of xylem sap was calculated as 684
the weight gain of the cotton Xylem sap was then squeezed from the cotton using a syringe 685
and used for subsequent assay of nitrate concentration using a continuous-flow auto-analyzer 686
(Auto Analyser 3 Bran and Luebbe Germany) 687
To determine nitrate concentration samples (10 g fresh root and shoot at seedling and 688
flowering of B napus and 05 g fresh root and shoot of 4-week-old A thaliana) were frozen 689
in liquid N2 and ground with a mortar and pestle the powder was then transferred to a beaker 690
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
containing 70 ml deionized water and boiled for 30 min to extract nitrate then cooled and 691
made to 100 ml volume and 02 g activated carbon added to eliminate the effect of 692
chlorophyll The mixture was filtered and nitrate in the filtrate was determined using a 693
continuous-flow auto-analyser (Atuo-Analyser 3 Bran and Luebbe Germany) 694
695
Measurements of photosynthesis 696
All measurements of photosynthesis and related parameters were conducted at 1000 h 697
using intact leaves The 4th leaf from the bottom in B napus at seedling stage and the 12th leaf 698
from the bottom at flowering stage were used for determining chlorophyll concentration 699
photosynthetic rate intercellular CO2 concentration transpiration rate and stomatal 700
conductance Chlorophyll concentration was determined by SPAD-502 (Minolta Camera Co 701
Ltd Japan) (Luo et al 2006 Yandeau-Nelson et al 2011) Photosynthetic rate intercellular 702
CO2 concentration transpiration rate and stomatal conductance were measured using LI-6400 703
Portable Photosynthesis System (Li-Cor Biosciences Co Ltd USA) set at flow rate of 500 704
μmol s-1 photosynthetic photon flux density of 1000 μmol m-2 s-1 relative humidity of 65 705
CO2 concentration of 400ppm and ambient temperatures 706
707
NR and GS activities 708
Leaves with similar age as those used for photosynthesis in B napus were used for the 709
assay of nitrate reductase (NR) and glutamine synthetase (GS) activities NR activity was 710
measured by the modified method of Fan et al (2007) Fresh samples (05g) were frozen in 711
liquid N2 ground into powder in the presence of acid-washed sand and homogenized with 4 712
ml of extraction buffer (0025mol L-1phosphate buffer pH87 1211g L-1 cysteine 0372g L-1 713
EDTA) Homogenates were centrifuged at 30000timesg for 15 min at 4degC and the supernatants 714
were treated with sulfanilamide and α-naphthylamine reagents for colorimetric (540nm) 715
determination of nitrite as described by Fan et al (2007) 716
GS activity was determined using a modified reverseγ-glutamyltransferase method 717
(Wang et al2014) which measures the GS-catalyzed formation of glutamyl-γ-hydroxamate 718
from glutamine and hydroxylamine Fresh samples were frozen for 30 min at -20degC then 719
ground in 10ml Tris-HCl buffer and acid-washed sand The homogenates were filtered 720
through two layers of gauze and centrifuged at 8000timesg for 15 min at 4degC About 12ml of the 721
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
supernatant was added in a reaction mixture and treated as described (Wang et al2014) After 722
the GS-catalyzed reaction was stopped and the solution centrifuged glutamyl-γ723
-hydroxamate was quantified colorimetrically in the supernatant (at 485 nm) and the 724
concentration was determined using a standard curve 725
726
Statistical analyses 727
We used the SPSS software (Statistical Product and Service Solutions V130 USA) for 728
ANOVA and mean separation of main-effects and interactions using Duncanrsquos multiple range 729
test at Plt005Values are presented as means and SD of three or six replicates from three 730
independent experiments Different letters or an asterisk () associated with specific data (eg 731
at the top of histogram bars in figures or within tables) denote significant differences at 732
Plt005 733
734
Supplemental Data 735
Supplemental Figure 1 B napus with higher NUE showed lower vacuolar sequestration 736
capacity (VSC) for NO3-in roots at flowering stage 737
Supplemental Figure 2 B napus with higher NUE showed enhanced long-distance transport 738
of NO3- from roots to shoots at flowering stage 739
Supplemental Figure 3 The two B napus (H and L genotypes) showed the same total N per 740
plant at seedling stage (A) and flowering stage (B) 741
Supplemental Figure 4 B napus with higher NUE showed increased NO3- concentration in 742
the xylem sap at seedling and flowering stages 743
Supplemental Figure 5 NO3- concentration in the xylem sap as affected by inhibitor 744
treatments in B napus and in the energy pumpsrsquo mutants of A thaliana (col-0 vha-a2 vha-a3 745
avp1) 746
Supplemental Figure 6 Reduced VSC for NO3- in roots drives long-distance transport of 747
NO3- from roots to shoots in the A thaliana wild type (Ws) and mutant (clca-2) 748
Supplemental Figure 7 Differences of NR and GS activities between the two B napus (H 749
and L genotypes) at seedling and flowering stages 750
Supplemental Figure 8 Amino acid sequences of BnNRT15 and BnNRT18 751
Supplemental Figure 9 Functions of AtNRT15 and AtNRT18 genes in root tissues of A 752
thaliana in controlling NO3- long-distance transport from root to shoots 753
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
Supplemental Figure 10 B napus BnNRT15 and BnNRT18 genes in roots were 754
coordinately modulated to facilitate NO3- long distance transport from roots to shoots 755
Supplemental Table 1 Differences in stomatal conductance and transpiration rate between 756
the two B napus genotypes 757
Supplemental Table 2 Sequences of primera used for qRT-PCR 758
759
Acknowledgements 760
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 761
Shanghai Institutes for Biological Sciences) for providing nrt15-3 and nrt18-2 seeds 762
This study was supported by the National Natural Science Foundation of China (Grant 763
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 764
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 765
The Ministry of Education in China Open Novel Science Foundation of Hunan province 766
(13K062) the National Key Laboratory of Plant Molecular Genetics and the Twelfth 767
Five-Year National Science and technology support program (2012BAD15BO4) 768
769
Figure 1 B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 770
NO3- in roots at the seedling stage 771
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 772
genotype 814 Specific activities of the tonoplast proton pumps are expressed as μmol Pi 773
released mg-1 protein h-1 NO3- fluxes are expressed as pmol NO3
- cm-2 S-1 Mature vacuoles 774
were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 775
measuring solution between the two positions 1 μm and 11 μm from the vacuole surface 776
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles recording the 777
stable reading data The background was recorded by vibrating the electrode in measuring 778
solution without vacuoles 779
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 780
for NO3- content and NO3
- accumulation normalized against the specific activity of the 781
vacuole acid phosphatase (ACP) as described in Materials and Methods and plotted as μmol 782
NO3- per μmol p-nitrophenol the end product of ACP assay 783
Proton pump activities in root tissues between H and L genotypes are shown at the seedling 784
stage (A) Different letters at the top of the histogram bars denote significant differences of 785
V-ATPase in root tissues between H and L genotypes (Plt005) an asterisk () at the top of the 786
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
histogram bars indicates significant difference in V-PPase activity in root tissues between H 787
and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788
Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789
genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790
bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791
bars on the figures indicate SD (n=6) 792
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793
seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794
to the total NO3- in protoplasts 795
NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796
total NO3-in the cytosol and was calculated as total NO3
- in protoplasts ndash total NO3- in 797
vacuole Different letters at the top of histogram bars denote significant differences of total 798
NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799
(n=6) 800
801
Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802
roots to shoots at the seedling stage 803
H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804
Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805
the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806
as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807
the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808
the histogram bars denote significant differences at Plt005 809
Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810
in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811
genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812
of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813
SD (n=3) different letters at the top of the histogram bars denote significant differences at 814
Plt005 level 815
The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816
at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817
genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818
different letters at the top of the histogram bars denote significant differences at Plt005 819
820
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
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b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
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Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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Harper AL Trick M Higgins J Fraser F Clissold L Wells R Hattori C Werner P Bancroft I (2012) Associative transcriptomics oftraits in the polyploidy crop species Brassica napus Nature Biotechnology 30 798-802
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Huang J Zhang Y Peng JS Zhong C Yi HY Ow DW Gong JM (2012) Fission yeast HMT1 lowers seed Cadmium throughphytochelatin-dependent vacuolar sequestration in Arabidopsis Plant Physiology 158(4) 1779-1788
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Krapp A David LC Chardin C Cirin T Marmagne A Leprince AC Chaillou S Ferrario-Mery S Meyer C Daniel-VedeleF (2014)Nitrate transport and signaling in Arabidopsis Journal of Experiment Botany 65789-798
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Krebs M Beyhl D Gorlich E Al-Rasheid KA Marten I Stierhof YD Hedrich R Schumacher K (2010) Arabidopsis V-ATPase activityat the tonoplast is required for efficient nutrient storage but not for sodium accumulation PNAS1073251-3256
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Leran S Varala K Boyer JC Chiurazzi M Crawford N Daniel-Vedele J David L Rebecca D (2014) A unified nomenclature ofNITRATE TRANSPORTER 1PEPTIDE TRANSPORTER family members in plants Trends in Plant Science 19(1) 5-9
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Lin SH Kou HF Canivenc G Lin CS Lepetit M Hsu PK Tillard P Lin HL Wang YY Tsai CB Gojon A Tsay YF (2008) Mutation ofthe Arabidopsis NRT15 nitrate transporter causes defective root-to-shoot nitrate transport The Plant Cell 202514-2528
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Li JY Fu YL Pike SM Bao J Tian W Zhang Y Chen CZ Zhang Y Li HM Huang J Li LG Schroeder J Gassmann W Gong JM(2010) The arabidopsis nitrate transporter NRT18 functions in nitrate removal from the xylem sap and mediates cadmiumtolerance The Plant Cell 22 1633-1646
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Li JS Yang HB Peer WA Richter G Blakeslee J Bandyopadhyay A Titapiwantakun B Undurraga S Khodakovskaya M RichardsE Krizek B Murphy AS Gilroy S Gaxiola R (2005) Arabidopsis H+-PPase AVP1 regulates auxin-mediated organ developmentScience 310 (121) 121-125
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Luo JK Sun SB Jia LJ Chen W Shen QR (2006) The mechanism of nitrate accumulation in Pakchoi [BrassicacampestrisLsspChinensis (L)] Plant and Soil 282291-300
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Ma JF Ueno D Zhao FJ McGrath SP (2005) Subcellular localization of Cd and Zn in the leaves of a Cd-hyperaccumulating ecotypeof ThlaspicaerulescensPlanta 220 731-736
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Martinoia E Massonneau A Frangne N (2000) Transport processes of solutes across the vacuolar membrane of higher plantsPlant Cell Physiology 41(11)1175-1186
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Martinoia E Heck U Wiemken A (1981) Vacuoles as storage compartments for nitrate in barley leaves Nature 289 292-294Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Miller AJ Smith SJ (2008) Cytosolic nitrate ion homeostasis could it have a role in sensing nitrogen status Annals ofBotany101485-489
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Robert S Zouhar J Carter C Raikhel N (2007) Isolation of intact vacuoles from Arabidopsis rosette leaf-derived protoplastsNature Protocols 2(2)259-262
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Schroeder JL Delhaize E Frommer WB Guerinot ML Harrison MJ Herrera-Estrella L Horie T Kochian LV Munns R NishizawaNK Tsay YF Sanders D (2013) Using membrane transporters to improve crops for sustainable food production Nature 497 60-66
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Shi WM Xu WF Li SM Zhao XQ Dong GQ (2010) Responses of two rice cultivars differing in seedling-stage nitrogen useefficiency to growth under low-nitrogen conditions Plant and Soil 326291-302
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Shen QR Tang L Xu YC (2003) A review on the behavior of nitrate in vacuoles of plants ActaPedologicaSinica40(3)465-470Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Smirnoff N Stewart G (1985) Nitrate assimilation and translocation by higher plants Comparative physiology and ecologicalconsequences Physiologia Plantarum 64 133-140
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Tang TF Sun XC Hu CX Tan QL Zhao XH (2013) Genotypic differences in nitrate uptake translocation and assimilation of twoChinese cabbage cultivars [Brassica campestris L ssp Chinesnsis (L)] Plant Physiology and Biochemistry7014-20
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Tang Z Fan XR Li Qing Feng HM Miller AJ Shen QR Xu GH (2012) Knockdown of a rice stellar nitrate transporter alters long-distance translocation but not root influx Plant Physiology 1602052-2063
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Vogeli-Lange R Wagner GJ (1990) Subcellular localization of cadmium and cadmium-binding peptides in tobacco leavesimplication of a transport function for cadmium-binding peptides Plant Physiology 921086-1093
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Wang GL Ding GD Li L Cai HM Ye XS Zou J Xu FS (2014) Identification and characterization of improved nitrogen efficiency ininterspecific hybridized new-type Brassica napus Annals of Botany 114(3) 549-559
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Wang YY Tsay YF (2011) Arabidopsis nitrate transporter NRT19 is important in phloem nitrate transport The Plant Cell 231945-1957
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Wege S Angeli AD Droillard MJ Kroniewicz L Merlot S Cornu D Gambale F Martinoia E Barbier-Brygoo H Thomine SLeonhardt N Filleur S (2014) Phosphorylation of the vacuolar anion exchanger AtCLCa is required for the stomatal response toabscisic acid Science Signalling 7(333) ra65
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Wilkinson S Bacon MA Davies WJ (2007) Nitrate signaling to stomatal and growing leaves interactions with soil drying ABA andxylem sap pH in maize Journal of Experimental Botany 58(7) 1705-1716
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Xu GH Fan XR Miller AJ (2012) Plant nitrogen assimilation and use efficiency The Annual Review of Plant Biology 63153-182Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yandeau-Nelson MD Laurens L Shi Z Xia H Smith AM Guiltinan MJ (2011) Starch-Branching Enzyme IIa Is Required for ProperDiurnal Cycling of Starch in Leaves of Maize [OA] Plant Physiology 156479-490
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Zhang GB Yi HY Gong JM (2014) The Arabidopsis EthyleneJasmonic acid-NRT signaling module coordinates nitrate reallocationand the trade-off between growth and environmental adaption The Plant Cell 26(10)3984-3998
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Zhang ZH Song HX Liu Qing Rong XM Xie GX Peng JW Zhang YP (2009) Study on differences of nitrogen efficiency andnitrogen response in different oilseed rape (Brassica napus L) varieties Asian Journal of Crop Science 1(2)105-112
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Zhang ZH Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2010) Nitrogen redistribution characteristics ofoilseed rape varieties with different nitrogen use efficiencies during later growth period Communications in Soil Science andPlant Analysis 41(14) 1693-1706
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Zhang ZH Huang HT Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2012) Research advances on nitratenitrogen reutilization by proton pump of tonoplast and its relation to nitrogen use efficiency Australian Journal of Crop Science6(9) 1377-1382
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- Parsed Citations
- Reviewer PDF
- ONE
- TWO
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- T-TWO
- Parsed Citations
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20
from the bottom was harvested for measurements made at flowering stage For A thaliana 570
whole root and shoot of four-week-old plants were sampled and used for different assays 571
572
Vacuolar proton-pumps inhibitor treatments 573
Inhibition of vacuolar proton pumps of B napus (Xiangyou15) was conducted at the seedling 574
stage in hydroponic culture as described in Han et al (2015a) with minor modifications 575
Plants grown in hydroponic solution were used as control Inhibitor treatment was conducted 576
using several chemicals Bafi(25 nmol L-1 Bafilomycin A1) an inhibitor of V-ATPase 577
DCCD(10 μmol L-1DCCD + 50 mmolL-1 Na2SO3) specifically inhibits V-PPase and 578
Bafi+DCCD in a 11 ratio as inhibitor of both V-ATPase and V-PPase The inhibitors were 579
applied in the hydroponic solutions for 24 h Fresh plant tissue samples from all treatments 580
were collected for different assays 581
582
NO3- induced gene expression 583
Four-week old A thaliana mutants (nrt15-3nrt18-2) were grown in hydroponics with 584
225mM (NH4)2succinate for 3 days and shifted to hydroponics with 45mM NO3- for 12 h 585
then root tissues were collected to assess relative expression of AtNRT15 and AtNRT18 586
genes Seedlings of B napus (Xiangyou15) plants grown hydroponically were treated with 587
5mM (NH4)2succinate for 3 d and shifted to fresh hydroponic solution containing 15 mM 588
NO3- for 12 h Root tissue was then collected to assess the relative expression of BnNRT15 589
and BnNRT18 genes using quantitative RT-PCR 590
591
ACC treatment 592
Hydroponically grown seedlings of B napus were subjected to ACC treatment as described in 593
Zhang et al (2014) with minor modifications Seedlings of the two genotypes Xiangyou15 594
and 814 were transferred to a nutrient solution containing 002mM L-1 ACC for 6 h or grown 595
in normal hydroponic solution as control Root tissues were then collected to assay relative 596
expression of BnNRT15 and BnNRT18 using quantitative RT-PCR Another set of B napus 597
plants were grown in ACC-containing hydroponic solution but supplemented with 15NO3- (22 598
15N) replacing the unlabeled NO3- for 1 h Root and shoot tissues were collected separately 599
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
21
for 15N measurement using a continuous-flow isotope ratio mass spectrometer coupled with a 600
C-N elemental analyzer (ANCA-MS PDZ Europa) 601
602
Biomass and N concentration 603
Grain yield dry biomass and N concentration were determined in plant samples at 604
harvest Plants were harvested (including senescing leaves) then dried in an oven for 30 min 605
at 105degC then at 70degC to a constant weight and weighed N concentration was determined 606
using the Kjeldahl method (Shi et al 2010 Han et al 2015b)02g of dried samples were 607
digested with H2SO4-H2O2 in a 100-ml Kjeldahl digestion flask after which N concentration 608
was determined using a Foss Auto Analyser Unit (Kjeldahl 8400) 609
610
V-ATPase and V-PPase activities 611
Roots (10 g) of B napus were collected at seedling and flowering stages and that of A 612
thaliana (05 g) sampled at seedling stage then used for determining the activities of 613
V-ATPase and V-PPase (Han et al 2015b) Activities of these two energy pumps within 614
microsomal membranes were followed colorimetrically based on released Pi as described by 615
Zhu et al (2001) and Krebs et al (2010) 616
617
Assay of NO3- flux in vacuoles 618
Roots of both species were collected similarly as that used for V-ATPase and V-PPase 619
activities then used for vacuole isolation and measurement of NO3- flux in vacuoles as 620
described by Robert et al (2007) with minor modification (Han et al 2015b) Net fluxes of 621
NO3- in vacuoles were measured non-invasively using SIET (scanning ion-selective electrode 622
technique SIET system BIO-003A Younger USA Science and Technology Corporation 623
USA) An NO3--selective microelectrode used for the assay of NO3
- flux was vibrated in the 624
measuring solution between two positions 1 μm and 11 μm from the vacuole surface 625
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles Background 626
signal was recorded by vibrating the electrode in the measuring solution in the absence of 627
vacuoles Ion flux was calculated by Fickrsquos law of diffusion J = -D(dcdx) where J represents 628
the ion flux (picomolescm-2s-1) dcdx is the ion concentration gradient and D is the ion 629
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
diffusion constant in a particular medium The direction of the flux was derived from Fickrsquos 630
law of diffusion that relates the concentration gradient 631
632
Isolation of intact protoplasts and vacuoles for determining NO3- concentration 633
Similar to the above assays root tissues (10 g) were collected from B napus at seedling 634
and flowering stages and from A thaliana (05 g) seedlings and used to isolate intact 635
protoplasts and vacuoles as described by Robert et al (2007) with minor modifications as 636
outlined in Huang et al (2012) and Han et al(2015b) The purified protoplasts were divided 637
into two equal aliquots with one of them used for isolation of vacuoles The purified 638
protoplasts and vacuoles were then sub-sampled and used to determine NO3- concentrations 639
(Vogeli-Lange and Wagner 1990) and for enzyme activity assays (Ma et al 2005) 640
NO3-concentrations in protoplasts and vacuoles were measured by a continuous-flow 641
auto-analyzer (Auto Analyser 3 Bran and Luebbe Germany) as described previously (Han et 642
al 2015b)The activities of acid phosphatase (ACP) and cytochrome oxidase (COX) were 643
determined using plant ACP colorimetry- and COX-assay kits (GenMedSci Inc) following 644
the manufacturerrsquos instructions ACP activity specific to vacuoles was determined and used to 645
normalize NO3- accumulation We measured NO3
- in the protoplast outside the vacuole which 646
includes the cytosol and organelles eg mitochondria and Golgi Apparatus (Robert et 647
al2007) Since most of the NO3- in the protoplast outside the vacuole is located in the cytosol 648
(Krebs et al 2010) we refer to NO3- distribution between vacuoles and cytosol rather than 649
vacuole versus protoplast 650
651
Quantitative RT-PCR 652
Total RNA extracted from B napus roots was prepared using Trizol reagent (Invitrogen) 653
cDNAs were synthesized using M-MLV reverse transcriptase (Promega) following 654
manufacturerrsquos protocol The relative expression of BnNRT15 (EV220114) BnNRT18 655
(EV116423) and Bnactin (AF1118121) genes in plant roots were determined by quantitative 656
RT-PCR and run on a LightCycler instrument (Roche) with the SYBR Green Real-Time PCR 657
Master Mix Kit (TOYOBO Japan) under the following conditions 95degC for 2min then 45 658
cycles of 95degC for 10s 60degC for 10s and 72degC for 20s The primer sequences of BnNRT15 659
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
BnNRT18 and Bnactin genes were obtained from httpbrassicanbiacuk as described in 660
Harper et al (2012) and expression levels were normalized to Bnactin as control 661
Total RNA extracted from the 4-week-old A thaliana roots under the treatments 662
conditions described above were prepared using Trizol reagent (Invitrogen) The cDNAs were 663
synthesized using M-MLV reverse transcriptase (Promega) based on the manufacturerrsquos 664
protocol and as described in Li et al (2010)The primer sequences of quantitative RT-PCR 665
used in these assays are listed in Supplemental Table 2 and were described previously (Chen 666
et al 2012 Zhang et al 2014) and their expression was normalized to Atactin2 as control 667
668
Determination of nitrate distribution using 15NO3- 669
Hydroponically grown B napus (seedling and flowering stages) and A thaliana 670
(seedling) were transferred to 01mM CaSO4 solution for 1min then to their respective 671
hydroponic nutrient solutions with15NO3- (22 excess) replacing unlabeled NO3
- for 1h 672
Plants were then transferred to 01mM CaSO4 solution for 1min after which roots were 673
washed with deionized water Root and shoot samples were separated and dried at 105degC for 674
30min followed by 70degC for 3 d then grounded and used for assaying 15N content using a 675
continuous-flow isotope ratio mass spectrometer coupled with a carbon-nitrogen elemental 676
analyzer (ANCA-MS PDZ Europa) 677
678
Xylem sap collection and assay of nitrate concentration 679
We used the method of Tang et al (2012) for collecting xylem sap Plants were cut 680
leaving1cm segments with intact roots at seedling and flowering stages for B napus and at 681
seedling stage for A thaliana Roots were immediately immersed in their respective nutrition 682
solutions Weighed cotton was put on the cut surface to absorb extruding xylem sap for 1 h 683
The cotton was then wrapped in plastic film and the volume of xylem sap was calculated as 684
the weight gain of the cotton Xylem sap was then squeezed from the cotton using a syringe 685
and used for subsequent assay of nitrate concentration using a continuous-flow auto-analyzer 686
(Auto Analyser 3 Bran and Luebbe Germany) 687
To determine nitrate concentration samples (10 g fresh root and shoot at seedling and 688
flowering of B napus and 05 g fresh root and shoot of 4-week-old A thaliana) were frozen 689
in liquid N2 and ground with a mortar and pestle the powder was then transferred to a beaker 690
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
containing 70 ml deionized water and boiled for 30 min to extract nitrate then cooled and 691
made to 100 ml volume and 02 g activated carbon added to eliminate the effect of 692
chlorophyll The mixture was filtered and nitrate in the filtrate was determined using a 693
continuous-flow auto-analyser (Atuo-Analyser 3 Bran and Luebbe Germany) 694
695
Measurements of photosynthesis 696
All measurements of photosynthesis and related parameters were conducted at 1000 h 697
using intact leaves The 4th leaf from the bottom in B napus at seedling stage and the 12th leaf 698
from the bottom at flowering stage were used for determining chlorophyll concentration 699
photosynthetic rate intercellular CO2 concentration transpiration rate and stomatal 700
conductance Chlorophyll concentration was determined by SPAD-502 (Minolta Camera Co 701
Ltd Japan) (Luo et al 2006 Yandeau-Nelson et al 2011) Photosynthetic rate intercellular 702
CO2 concentration transpiration rate and stomatal conductance were measured using LI-6400 703
Portable Photosynthesis System (Li-Cor Biosciences Co Ltd USA) set at flow rate of 500 704
μmol s-1 photosynthetic photon flux density of 1000 μmol m-2 s-1 relative humidity of 65 705
CO2 concentration of 400ppm and ambient temperatures 706
707
NR and GS activities 708
Leaves with similar age as those used for photosynthesis in B napus were used for the 709
assay of nitrate reductase (NR) and glutamine synthetase (GS) activities NR activity was 710
measured by the modified method of Fan et al (2007) Fresh samples (05g) were frozen in 711
liquid N2 ground into powder in the presence of acid-washed sand and homogenized with 4 712
ml of extraction buffer (0025mol L-1phosphate buffer pH87 1211g L-1 cysteine 0372g L-1 713
EDTA) Homogenates were centrifuged at 30000timesg for 15 min at 4degC and the supernatants 714
were treated with sulfanilamide and α-naphthylamine reagents for colorimetric (540nm) 715
determination of nitrite as described by Fan et al (2007) 716
GS activity was determined using a modified reverseγ-glutamyltransferase method 717
(Wang et al2014) which measures the GS-catalyzed formation of glutamyl-γ-hydroxamate 718
from glutamine and hydroxylamine Fresh samples were frozen for 30 min at -20degC then 719
ground in 10ml Tris-HCl buffer and acid-washed sand The homogenates were filtered 720
through two layers of gauze and centrifuged at 8000timesg for 15 min at 4degC About 12ml of the 721
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
supernatant was added in a reaction mixture and treated as described (Wang et al2014) After 722
the GS-catalyzed reaction was stopped and the solution centrifuged glutamyl-γ723
-hydroxamate was quantified colorimetrically in the supernatant (at 485 nm) and the 724
concentration was determined using a standard curve 725
726
Statistical analyses 727
We used the SPSS software (Statistical Product and Service Solutions V130 USA) for 728
ANOVA and mean separation of main-effects and interactions using Duncanrsquos multiple range 729
test at Plt005Values are presented as means and SD of three or six replicates from three 730
independent experiments Different letters or an asterisk () associated with specific data (eg 731
at the top of histogram bars in figures or within tables) denote significant differences at 732
Plt005 733
734
Supplemental Data 735
Supplemental Figure 1 B napus with higher NUE showed lower vacuolar sequestration 736
capacity (VSC) for NO3-in roots at flowering stage 737
Supplemental Figure 2 B napus with higher NUE showed enhanced long-distance transport 738
of NO3- from roots to shoots at flowering stage 739
Supplemental Figure 3 The two B napus (H and L genotypes) showed the same total N per 740
plant at seedling stage (A) and flowering stage (B) 741
Supplemental Figure 4 B napus with higher NUE showed increased NO3- concentration in 742
the xylem sap at seedling and flowering stages 743
Supplemental Figure 5 NO3- concentration in the xylem sap as affected by inhibitor 744
treatments in B napus and in the energy pumpsrsquo mutants of A thaliana (col-0 vha-a2 vha-a3 745
avp1) 746
Supplemental Figure 6 Reduced VSC for NO3- in roots drives long-distance transport of 747
NO3- from roots to shoots in the A thaliana wild type (Ws) and mutant (clca-2) 748
Supplemental Figure 7 Differences of NR and GS activities between the two B napus (H 749
and L genotypes) at seedling and flowering stages 750
Supplemental Figure 8 Amino acid sequences of BnNRT15 and BnNRT18 751
Supplemental Figure 9 Functions of AtNRT15 and AtNRT18 genes in root tissues of A 752
thaliana in controlling NO3- long-distance transport from root to shoots 753
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
Supplemental Figure 10 B napus BnNRT15 and BnNRT18 genes in roots were 754
coordinately modulated to facilitate NO3- long distance transport from roots to shoots 755
Supplemental Table 1 Differences in stomatal conductance and transpiration rate between 756
the two B napus genotypes 757
Supplemental Table 2 Sequences of primera used for qRT-PCR 758
759
Acknowledgements 760
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 761
Shanghai Institutes for Biological Sciences) for providing nrt15-3 and nrt18-2 seeds 762
This study was supported by the National Natural Science Foundation of China (Grant 763
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 764
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 765
The Ministry of Education in China Open Novel Science Foundation of Hunan province 766
(13K062) the National Key Laboratory of Plant Molecular Genetics and the Twelfth 767
Five-Year National Science and technology support program (2012BAD15BO4) 768
769
Figure 1 B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 770
NO3- in roots at the seedling stage 771
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 772
genotype 814 Specific activities of the tonoplast proton pumps are expressed as μmol Pi 773
released mg-1 protein h-1 NO3- fluxes are expressed as pmol NO3
- cm-2 S-1 Mature vacuoles 774
were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 775
measuring solution between the two positions 1 μm and 11 μm from the vacuole surface 776
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles recording the 777
stable reading data The background was recorded by vibrating the electrode in measuring 778
solution without vacuoles 779
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 780
for NO3- content and NO3
- accumulation normalized against the specific activity of the 781
vacuole acid phosphatase (ACP) as described in Materials and Methods and plotted as μmol 782
NO3- per μmol p-nitrophenol the end product of ACP assay 783
Proton pump activities in root tissues between H and L genotypes are shown at the seedling 784
stage (A) Different letters at the top of the histogram bars denote significant differences of 785
V-ATPase in root tissues between H and L genotypes (Plt005) an asterisk () at the top of the 786
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
histogram bars indicates significant difference in V-PPase activity in root tissues between H 787
and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788
Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789
genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790
bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791
bars on the figures indicate SD (n=6) 792
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793
seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794
to the total NO3- in protoplasts 795
NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796
total NO3-in the cytosol and was calculated as total NO3
- in protoplasts ndash total NO3- in 797
vacuole Different letters at the top of histogram bars denote significant differences of total 798
NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799
(n=6) 800
801
Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802
roots to shoots at the seedling stage 803
H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804
Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805
the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806
as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807
the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808
the histogram bars denote significant differences at Plt005 809
Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810
in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811
genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812
of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813
SD (n=3) different letters at the top of the histogram bars denote significant differences at 814
Plt005 level 815
The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816
at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817
genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818
different letters at the top of the histogram bars denote significant differences at Plt005 819
820
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
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- Parsed Citations
- Reviewer PDF
- ONE
- TWO
- THREE
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- SIX
- SEVEN
- T-ONE
- T-TWO
- Parsed Citations
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21
for 15N measurement using a continuous-flow isotope ratio mass spectrometer coupled with a 600
C-N elemental analyzer (ANCA-MS PDZ Europa) 601
602
Biomass and N concentration 603
Grain yield dry biomass and N concentration were determined in plant samples at 604
harvest Plants were harvested (including senescing leaves) then dried in an oven for 30 min 605
at 105degC then at 70degC to a constant weight and weighed N concentration was determined 606
using the Kjeldahl method (Shi et al 2010 Han et al 2015b)02g of dried samples were 607
digested with H2SO4-H2O2 in a 100-ml Kjeldahl digestion flask after which N concentration 608
was determined using a Foss Auto Analyser Unit (Kjeldahl 8400) 609
610
V-ATPase and V-PPase activities 611
Roots (10 g) of B napus were collected at seedling and flowering stages and that of A 612
thaliana (05 g) sampled at seedling stage then used for determining the activities of 613
V-ATPase and V-PPase (Han et al 2015b) Activities of these two energy pumps within 614
microsomal membranes were followed colorimetrically based on released Pi as described by 615
Zhu et al (2001) and Krebs et al (2010) 616
617
Assay of NO3- flux in vacuoles 618
Roots of both species were collected similarly as that used for V-ATPase and V-PPase 619
activities then used for vacuole isolation and measurement of NO3- flux in vacuoles as 620
described by Robert et al (2007) with minor modification (Han et al 2015b) Net fluxes of 621
NO3- in vacuoles were measured non-invasively using SIET (scanning ion-selective electrode 622
technique SIET system BIO-003A Younger USA Science and Technology Corporation 623
USA) An NO3--selective microelectrode used for the assay of NO3
- flux was vibrated in the 624
measuring solution between two positions 1 μm and 11 μm from the vacuole surface 625
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles Background 626
signal was recorded by vibrating the electrode in the measuring solution in the absence of 627
vacuoles Ion flux was calculated by Fickrsquos law of diffusion J = -D(dcdx) where J represents 628
the ion flux (picomolescm-2s-1) dcdx is the ion concentration gradient and D is the ion 629
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22
diffusion constant in a particular medium The direction of the flux was derived from Fickrsquos 630
law of diffusion that relates the concentration gradient 631
632
Isolation of intact protoplasts and vacuoles for determining NO3- concentration 633
Similar to the above assays root tissues (10 g) were collected from B napus at seedling 634
and flowering stages and from A thaliana (05 g) seedlings and used to isolate intact 635
protoplasts and vacuoles as described by Robert et al (2007) with minor modifications as 636
outlined in Huang et al (2012) and Han et al(2015b) The purified protoplasts were divided 637
into two equal aliquots with one of them used for isolation of vacuoles The purified 638
protoplasts and vacuoles were then sub-sampled and used to determine NO3- concentrations 639
(Vogeli-Lange and Wagner 1990) and for enzyme activity assays (Ma et al 2005) 640
NO3-concentrations in protoplasts and vacuoles were measured by a continuous-flow 641
auto-analyzer (Auto Analyser 3 Bran and Luebbe Germany) as described previously (Han et 642
al 2015b)The activities of acid phosphatase (ACP) and cytochrome oxidase (COX) were 643
determined using plant ACP colorimetry- and COX-assay kits (GenMedSci Inc) following 644
the manufacturerrsquos instructions ACP activity specific to vacuoles was determined and used to 645
normalize NO3- accumulation We measured NO3
- in the protoplast outside the vacuole which 646
includes the cytosol and organelles eg mitochondria and Golgi Apparatus (Robert et 647
al2007) Since most of the NO3- in the protoplast outside the vacuole is located in the cytosol 648
(Krebs et al 2010) we refer to NO3- distribution between vacuoles and cytosol rather than 649
vacuole versus protoplast 650
651
Quantitative RT-PCR 652
Total RNA extracted from B napus roots was prepared using Trizol reagent (Invitrogen) 653
cDNAs were synthesized using M-MLV reverse transcriptase (Promega) following 654
manufacturerrsquos protocol The relative expression of BnNRT15 (EV220114) BnNRT18 655
(EV116423) and Bnactin (AF1118121) genes in plant roots were determined by quantitative 656
RT-PCR and run on a LightCycler instrument (Roche) with the SYBR Green Real-Time PCR 657
Master Mix Kit (TOYOBO Japan) under the following conditions 95degC for 2min then 45 658
cycles of 95degC for 10s 60degC for 10s and 72degC for 20s The primer sequences of BnNRT15 659
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
BnNRT18 and Bnactin genes were obtained from httpbrassicanbiacuk as described in 660
Harper et al (2012) and expression levels were normalized to Bnactin as control 661
Total RNA extracted from the 4-week-old A thaliana roots under the treatments 662
conditions described above were prepared using Trizol reagent (Invitrogen) The cDNAs were 663
synthesized using M-MLV reverse transcriptase (Promega) based on the manufacturerrsquos 664
protocol and as described in Li et al (2010)The primer sequences of quantitative RT-PCR 665
used in these assays are listed in Supplemental Table 2 and were described previously (Chen 666
et al 2012 Zhang et al 2014) and their expression was normalized to Atactin2 as control 667
668
Determination of nitrate distribution using 15NO3- 669
Hydroponically grown B napus (seedling and flowering stages) and A thaliana 670
(seedling) were transferred to 01mM CaSO4 solution for 1min then to their respective 671
hydroponic nutrient solutions with15NO3- (22 excess) replacing unlabeled NO3
- for 1h 672
Plants were then transferred to 01mM CaSO4 solution for 1min after which roots were 673
washed with deionized water Root and shoot samples were separated and dried at 105degC for 674
30min followed by 70degC for 3 d then grounded and used for assaying 15N content using a 675
continuous-flow isotope ratio mass spectrometer coupled with a carbon-nitrogen elemental 676
analyzer (ANCA-MS PDZ Europa) 677
678
Xylem sap collection and assay of nitrate concentration 679
We used the method of Tang et al (2012) for collecting xylem sap Plants were cut 680
leaving1cm segments with intact roots at seedling and flowering stages for B napus and at 681
seedling stage for A thaliana Roots were immediately immersed in their respective nutrition 682
solutions Weighed cotton was put on the cut surface to absorb extruding xylem sap for 1 h 683
The cotton was then wrapped in plastic film and the volume of xylem sap was calculated as 684
the weight gain of the cotton Xylem sap was then squeezed from the cotton using a syringe 685
and used for subsequent assay of nitrate concentration using a continuous-flow auto-analyzer 686
(Auto Analyser 3 Bran and Luebbe Germany) 687
To determine nitrate concentration samples (10 g fresh root and shoot at seedling and 688
flowering of B napus and 05 g fresh root and shoot of 4-week-old A thaliana) were frozen 689
in liquid N2 and ground with a mortar and pestle the powder was then transferred to a beaker 690
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
containing 70 ml deionized water and boiled for 30 min to extract nitrate then cooled and 691
made to 100 ml volume and 02 g activated carbon added to eliminate the effect of 692
chlorophyll The mixture was filtered and nitrate in the filtrate was determined using a 693
continuous-flow auto-analyser (Atuo-Analyser 3 Bran and Luebbe Germany) 694
695
Measurements of photosynthesis 696
All measurements of photosynthesis and related parameters were conducted at 1000 h 697
using intact leaves The 4th leaf from the bottom in B napus at seedling stage and the 12th leaf 698
from the bottom at flowering stage were used for determining chlorophyll concentration 699
photosynthetic rate intercellular CO2 concentration transpiration rate and stomatal 700
conductance Chlorophyll concentration was determined by SPAD-502 (Minolta Camera Co 701
Ltd Japan) (Luo et al 2006 Yandeau-Nelson et al 2011) Photosynthetic rate intercellular 702
CO2 concentration transpiration rate and stomatal conductance were measured using LI-6400 703
Portable Photosynthesis System (Li-Cor Biosciences Co Ltd USA) set at flow rate of 500 704
μmol s-1 photosynthetic photon flux density of 1000 μmol m-2 s-1 relative humidity of 65 705
CO2 concentration of 400ppm and ambient temperatures 706
707
NR and GS activities 708
Leaves with similar age as those used for photosynthesis in B napus were used for the 709
assay of nitrate reductase (NR) and glutamine synthetase (GS) activities NR activity was 710
measured by the modified method of Fan et al (2007) Fresh samples (05g) were frozen in 711
liquid N2 ground into powder in the presence of acid-washed sand and homogenized with 4 712
ml of extraction buffer (0025mol L-1phosphate buffer pH87 1211g L-1 cysteine 0372g L-1 713
EDTA) Homogenates were centrifuged at 30000timesg for 15 min at 4degC and the supernatants 714
were treated with sulfanilamide and α-naphthylamine reagents for colorimetric (540nm) 715
determination of nitrite as described by Fan et al (2007) 716
GS activity was determined using a modified reverseγ-glutamyltransferase method 717
(Wang et al2014) which measures the GS-catalyzed formation of glutamyl-γ-hydroxamate 718
from glutamine and hydroxylamine Fresh samples were frozen for 30 min at -20degC then 719
ground in 10ml Tris-HCl buffer and acid-washed sand The homogenates were filtered 720
through two layers of gauze and centrifuged at 8000timesg for 15 min at 4degC About 12ml of the 721
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
supernatant was added in a reaction mixture and treated as described (Wang et al2014) After 722
the GS-catalyzed reaction was stopped and the solution centrifuged glutamyl-γ723
-hydroxamate was quantified colorimetrically in the supernatant (at 485 nm) and the 724
concentration was determined using a standard curve 725
726
Statistical analyses 727
We used the SPSS software (Statistical Product and Service Solutions V130 USA) for 728
ANOVA and mean separation of main-effects and interactions using Duncanrsquos multiple range 729
test at Plt005Values are presented as means and SD of three or six replicates from three 730
independent experiments Different letters or an asterisk () associated with specific data (eg 731
at the top of histogram bars in figures or within tables) denote significant differences at 732
Plt005 733
734
Supplemental Data 735
Supplemental Figure 1 B napus with higher NUE showed lower vacuolar sequestration 736
capacity (VSC) for NO3-in roots at flowering stage 737
Supplemental Figure 2 B napus with higher NUE showed enhanced long-distance transport 738
of NO3- from roots to shoots at flowering stage 739
Supplemental Figure 3 The two B napus (H and L genotypes) showed the same total N per 740
plant at seedling stage (A) and flowering stage (B) 741
Supplemental Figure 4 B napus with higher NUE showed increased NO3- concentration in 742
the xylem sap at seedling and flowering stages 743
Supplemental Figure 5 NO3- concentration in the xylem sap as affected by inhibitor 744
treatments in B napus and in the energy pumpsrsquo mutants of A thaliana (col-0 vha-a2 vha-a3 745
avp1) 746
Supplemental Figure 6 Reduced VSC for NO3- in roots drives long-distance transport of 747
NO3- from roots to shoots in the A thaliana wild type (Ws) and mutant (clca-2) 748
Supplemental Figure 7 Differences of NR and GS activities between the two B napus (H 749
and L genotypes) at seedling and flowering stages 750
Supplemental Figure 8 Amino acid sequences of BnNRT15 and BnNRT18 751
Supplemental Figure 9 Functions of AtNRT15 and AtNRT18 genes in root tissues of A 752
thaliana in controlling NO3- long-distance transport from root to shoots 753
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
Supplemental Figure 10 B napus BnNRT15 and BnNRT18 genes in roots were 754
coordinately modulated to facilitate NO3- long distance transport from roots to shoots 755
Supplemental Table 1 Differences in stomatal conductance and transpiration rate between 756
the two B napus genotypes 757
Supplemental Table 2 Sequences of primera used for qRT-PCR 758
759
Acknowledgements 760
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 761
Shanghai Institutes for Biological Sciences) for providing nrt15-3 and nrt18-2 seeds 762
This study was supported by the National Natural Science Foundation of China (Grant 763
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 764
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 765
The Ministry of Education in China Open Novel Science Foundation of Hunan province 766
(13K062) the National Key Laboratory of Plant Molecular Genetics and the Twelfth 767
Five-Year National Science and technology support program (2012BAD15BO4) 768
769
Figure 1 B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 770
NO3- in roots at the seedling stage 771
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 772
genotype 814 Specific activities of the tonoplast proton pumps are expressed as μmol Pi 773
released mg-1 protein h-1 NO3- fluxes are expressed as pmol NO3
- cm-2 S-1 Mature vacuoles 774
were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 775
measuring solution between the two positions 1 μm and 11 μm from the vacuole surface 776
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles recording the 777
stable reading data The background was recorded by vibrating the electrode in measuring 778
solution without vacuoles 779
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 780
for NO3- content and NO3
- accumulation normalized against the specific activity of the 781
vacuole acid phosphatase (ACP) as described in Materials and Methods and plotted as μmol 782
NO3- per μmol p-nitrophenol the end product of ACP assay 783
Proton pump activities in root tissues between H and L genotypes are shown at the seedling 784
stage (A) Different letters at the top of the histogram bars denote significant differences of 785
V-ATPase in root tissues between H and L genotypes (Plt005) an asterisk () at the top of the 786
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
histogram bars indicates significant difference in V-PPase activity in root tissues between H 787
and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788
Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789
genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790
bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791
bars on the figures indicate SD (n=6) 792
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793
seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794
to the total NO3- in protoplasts 795
NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796
total NO3-in the cytosol and was calculated as total NO3
- in protoplasts ndash total NO3- in 797
vacuole Different letters at the top of histogram bars denote significant differences of total 798
NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799
(n=6) 800
801
Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802
roots to shoots at the seedling stage 803
H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804
Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805
the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806
as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807
the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808
the histogram bars denote significant differences at Plt005 809
Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810
in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811
genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812
of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813
SD (n=3) different letters at the top of the histogram bars denote significant differences at 814
Plt005 level 815
The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816
at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817
genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818
different letters at the top of the histogram bars denote significant differences at Plt005 819
820
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
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Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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22
diffusion constant in a particular medium The direction of the flux was derived from Fickrsquos 630
law of diffusion that relates the concentration gradient 631
632
Isolation of intact protoplasts and vacuoles for determining NO3- concentration 633
Similar to the above assays root tissues (10 g) were collected from B napus at seedling 634
and flowering stages and from A thaliana (05 g) seedlings and used to isolate intact 635
protoplasts and vacuoles as described by Robert et al (2007) with minor modifications as 636
outlined in Huang et al (2012) and Han et al(2015b) The purified protoplasts were divided 637
into two equal aliquots with one of them used for isolation of vacuoles The purified 638
protoplasts and vacuoles were then sub-sampled and used to determine NO3- concentrations 639
(Vogeli-Lange and Wagner 1990) and for enzyme activity assays (Ma et al 2005) 640
NO3-concentrations in protoplasts and vacuoles were measured by a continuous-flow 641
auto-analyzer (Auto Analyser 3 Bran and Luebbe Germany) as described previously (Han et 642
al 2015b)The activities of acid phosphatase (ACP) and cytochrome oxidase (COX) were 643
determined using plant ACP colorimetry- and COX-assay kits (GenMedSci Inc) following 644
the manufacturerrsquos instructions ACP activity specific to vacuoles was determined and used to 645
normalize NO3- accumulation We measured NO3
- in the protoplast outside the vacuole which 646
includes the cytosol and organelles eg mitochondria and Golgi Apparatus (Robert et 647
al2007) Since most of the NO3- in the protoplast outside the vacuole is located in the cytosol 648
(Krebs et al 2010) we refer to NO3- distribution between vacuoles and cytosol rather than 649
vacuole versus protoplast 650
651
Quantitative RT-PCR 652
Total RNA extracted from B napus roots was prepared using Trizol reagent (Invitrogen) 653
cDNAs were synthesized using M-MLV reverse transcriptase (Promega) following 654
manufacturerrsquos protocol The relative expression of BnNRT15 (EV220114) BnNRT18 655
(EV116423) and Bnactin (AF1118121) genes in plant roots were determined by quantitative 656
RT-PCR and run on a LightCycler instrument (Roche) with the SYBR Green Real-Time PCR 657
Master Mix Kit (TOYOBO Japan) under the following conditions 95degC for 2min then 45 658
cycles of 95degC for 10s 60degC for 10s and 72degC for 20s The primer sequences of BnNRT15 659
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
BnNRT18 and Bnactin genes were obtained from httpbrassicanbiacuk as described in 660
Harper et al (2012) and expression levels were normalized to Bnactin as control 661
Total RNA extracted from the 4-week-old A thaliana roots under the treatments 662
conditions described above were prepared using Trizol reagent (Invitrogen) The cDNAs were 663
synthesized using M-MLV reverse transcriptase (Promega) based on the manufacturerrsquos 664
protocol and as described in Li et al (2010)The primer sequences of quantitative RT-PCR 665
used in these assays are listed in Supplemental Table 2 and were described previously (Chen 666
et al 2012 Zhang et al 2014) and their expression was normalized to Atactin2 as control 667
668
Determination of nitrate distribution using 15NO3- 669
Hydroponically grown B napus (seedling and flowering stages) and A thaliana 670
(seedling) were transferred to 01mM CaSO4 solution for 1min then to their respective 671
hydroponic nutrient solutions with15NO3- (22 excess) replacing unlabeled NO3
- for 1h 672
Plants were then transferred to 01mM CaSO4 solution for 1min after which roots were 673
washed with deionized water Root and shoot samples were separated and dried at 105degC for 674
30min followed by 70degC for 3 d then grounded and used for assaying 15N content using a 675
continuous-flow isotope ratio mass spectrometer coupled with a carbon-nitrogen elemental 676
analyzer (ANCA-MS PDZ Europa) 677
678
Xylem sap collection and assay of nitrate concentration 679
We used the method of Tang et al (2012) for collecting xylem sap Plants were cut 680
leaving1cm segments with intact roots at seedling and flowering stages for B napus and at 681
seedling stage for A thaliana Roots were immediately immersed in their respective nutrition 682
solutions Weighed cotton was put on the cut surface to absorb extruding xylem sap for 1 h 683
The cotton was then wrapped in plastic film and the volume of xylem sap was calculated as 684
the weight gain of the cotton Xylem sap was then squeezed from the cotton using a syringe 685
and used for subsequent assay of nitrate concentration using a continuous-flow auto-analyzer 686
(Auto Analyser 3 Bran and Luebbe Germany) 687
To determine nitrate concentration samples (10 g fresh root and shoot at seedling and 688
flowering of B napus and 05 g fresh root and shoot of 4-week-old A thaliana) were frozen 689
in liquid N2 and ground with a mortar and pestle the powder was then transferred to a beaker 690
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
containing 70 ml deionized water and boiled for 30 min to extract nitrate then cooled and 691
made to 100 ml volume and 02 g activated carbon added to eliminate the effect of 692
chlorophyll The mixture was filtered and nitrate in the filtrate was determined using a 693
continuous-flow auto-analyser (Atuo-Analyser 3 Bran and Luebbe Germany) 694
695
Measurements of photosynthesis 696
All measurements of photosynthesis and related parameters were conducted at 1000 h 697
using intact leaves The 4th leaf from the bottom in B napus at seedling stage and the 12th leaf 698
from the bottom at flowering stage were used for determining chlorophyll concentration 699
photosynthetic rate intercellular CO2 concentration transpiration rate and stomatal 700
conductance Chlorophyll concentration was determined by SPAD-502 (Minolta Camera Co 701
Ltd Japan) (Luo et al 2006 Yandeau-Nelson et al 2011) Photosynthetic rate intercellular 702
CO2 concentration transpiration rate and stomatal conductance were measured using LI-6400 703
Portable Photosynthesis System (Li-Cor Biosciences Co Ltd USA) set at flow rate of 500 704
μmol s-1 photosynthetic photon flux density of 1000 μmol m-2 s-1 relative humidity of 65 705
CO2 concentration of 400ppm and ambient temperatures 706
707
NR and GS activities 708
Leaves with similar age as those used for photosynthesis in B napus were used for the 709
assay of nitrate reductase (NR) and glutamine synthetase (GS) activities NR activity was 710
measured by the modified method of Fan et al (2007) Fresh samples (05g) were frozen in 711
liquid N2 ground into powder in the presence of acid-washed sand and homogenized with 4 712
ml of extraction buffer (0025mol L-1phosphate buffer pH87 1211g L-1 cysteine 0372g L-1 713
EDTA) Homogenates were centrifuged at 30000timesg for 15 min at 4degC and the supernatants 714
were treated with sulfanilamide and α-naphthylamine reagents for colorimetric (540nm) 715
determination of nitrite as described by Fan et al (2007) 716
GS activity was determined using a modified reverseγ-glutamyltransferase method 717
(Wang et al2014) which measures the GS-catalyzed formation of glutamyl-γ-hydroxamate 718
from glutamine and hydroxylamine Fresh samples were frozen for 30 min at -20degC then 719
ground in 10ml Tris-HCl buffer and acid-washed sand The homogenates were filtered 720
through two layers of gauze and centrifuged at 8000timesg for 15 min at 4degC About 12ml of the 721
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
supernatant was added in a reaction mixture and treated as described (Wang et al2014) After 722
the GS-catalyzed reaction was stopped and the solution centrifuged glutamyl-γ723
-hydroxamate was quantified colorimetrically in the supernatant (at 485 nm) and the 724
concentration was determined using a standard curve 725
726
Statistical analyses 727
We used the SPSS software (Statistical Product and Service Solutions V130 USA) for 728
ANOVA and mean separation of main-effects and interactions using Duncanrsquos multiple range 729
test at Plt005Values are presented as means and SD of three or six replicates from three 730
independent experiments Different letters or an asterisk () associated with specific data (eg 731
at the top of histogram bars in figures or within tables) denote significant differences at 732
Plt005 733
734
Supplemental Data 735
Supplemental Figure 1 B napus with higher NUE showed lower vacuolar sequestration 736
capacity (VSC) for NO3-in roots at flowering stage 737
Supplemental Figure 2 B napus with higher NUE showed enhanced long-distance transport 738
of NO3- from roots to shoots at flowering stage 739
Supplemental Figure 3 The two B napus (H and L genotypes) showed the same total N per 740
plant at seedling stage (A) and flowering stage (B) 741
Supplemental Figure 4 B napus with higher NUE showed increased NO3- concentration in 742
the xylem sap at seedling and flowering stages 743
Supplemental Figure 5 NO3- concentration in the xylem sap as affected by inhibitor 744
treatments in B napus and in the energy pumpsrsquo mutants of A thaliana (col-0 vha-a2 vha-a3 745
avp1) 746
Supplemental Figure 6 Reduced VSC for NO3- in roots drives long-distance transport of 747
NO3- from roots to shoots in the A thaliana wild type (Ws) and mutant (clca-2) 748
Supplemental Figure 7 Differences of NR and GS activities between the two B napus (H 749
and L genotypes) at seedling and flowering stages 750
Supplemental Figure 8 Amino acid sequences of BnNRT15 and BnNRT18 751
Supplemental Figure 9 Functions of AtNRT15 and AtNRT18 genes in root tissues of A 752
thaliana in controlling NO3- long-distance transport from root to shoots 753
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
Supplemental Figure 10 B napus BnNRT15 and BnNRT18 genes in roots were 754
coordinately modulated to facilitate NO3- long distance transport from roots to shoots 755
Supplemental Table 1 Differences in stomatal conductance and transpiration rate between 756
the two B napus genotypes 757
Supplemental Table 2 Sequences of primera used for qRT-PCR 758
759
Acknowledgements 760
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 761
Shanghai Institutes for Biological Sciences) for providing nrt15-3 and nrt18-2 seeds 762
This study was supported by the National Natural Science Foundation of China (Grant 763
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 764
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 765
The Ministry of Education in China Open Novel Science Foundation of Hunan province 766
(13K062) the National Key Laboratory of Plant Molecular Genetics and the Twelfth 767
Five-Year National Science and technology support program (2012BAD15BO4) 768
769
Figure 1 B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 770
NO3- in roots at the seedling stage 771
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 772
genotype 814 Specific activities of the tonoplast proton pumps are expressed as μmol Pi 773
released mg-1 protein h-1 NO3- fluxes are expressed as pmol NO3
- cm-2 S-1 Mature vacuoles 774
were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 775
measuring solution between the two positions 1 μm and 11 μm from the vacuole surface 776
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles recording the 777
stable reading data The background was recorded by vibrating the electrode in measuring 778
solution without vacuoles 779
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 780
for NO3- content and NO3
- accumulation normalized against the specific activity of the 781
vacuole acid phosphatase (ACP) as described in Materials and Methods and plotted as μmol 782
NO3- per μmol p-nitrophenol the end product of ACP assay 783
Proton pump activities in root tissues between H and L genotypes are shown at the seedling 784
stage (A) Different letters at the top of the histogram bars denote significant differences of 785
V-ATPase in root tissues between H and L genotypes (Plt005) an asterisk () at the top of the 786
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
histogram bars indicates significant difference in V-PPase activity in root tissues between H 787
and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788
Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789
genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790
bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791
bars on the figures indicate SD (n=6) 792
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793
seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794
to the total NO3- in protoplasts 795
NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796
total NO3-in the cytosol and was calculated as total NO3
- in protoplasts ndash total NO3- in 797
vacuole Different letters at the top of histogram bars denote significant differences of total 798
NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799
(n=6) 800
801
Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802
roots to shoots at the seedling stage 803
H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804
Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805
the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806
as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807
the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808
the histogram bars denote significant differences at Plt005 809
Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810
in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811
genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812
of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813
SD (n=3) different letters at the top of the histogram bars denote significant differences at 814
Plt005 level 815
The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816
at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817
genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818
different letters at the top of the histogram bars denote significant differences at Plt005 819
820
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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Wang GL Ding GD Li L Cai HM Ye XS Zou J Xu FS (2014) Identification and characterization of improved nitrogen efficiency ininterspecific hybridized new-type Brassica napus Annals of Botany 114(3) 549-559
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Wang YY Tsay YF (2011) Arabidopsis nitrate transporter NRT19 is important in phloem nitrate transport The Plant Cell 231945-1957
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wege S Angeli AD Droillard MJ Kroniewicz L Merlot S Cornu D Gambale F Martinoia E Barbier-Brygoo H Thomine SLeonhardt N Filleur S (2014) Phosphorylation of the vacuolar anion exchanger AtCLCa is required for the stomatal response toabscisic acid Science Signalling 7(333) ra65
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wilkinson S Bacon MA Davies WJ (2007) Nitrate signaling to stomatal and growing leaves interactions with soil drying ABA andxylem sap pH in maize Journal of Experimental Botany 58(7) 1705-1716
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xu GH Fan XR Miller AJ (2012) Plant nitrogen assimilation and use efficiency The Annual Review of Plant Biology 63153-182Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yandeau-Nelson MD Laurens L Shi Z Xia H Smith AM Guiltinan MJ (2011) Starch-Branching Enzyme IIa Is Required for ProperDiurnal Cycling of Starch in Leaves of Maize [OA] Plant Physiology 156479-490
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang GB Yi HY Gong JM (2014) The Arabidopsis EthyleneJasmonic acid-NRT signaling module coordinates nitrate reallocationand the trade-off between growth and environmental adaption The Plant Cell 26(10)3984-3998
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Song HX Liu Qing Rong XM Xie GX Peng JW Zhang YP (2009) Study on differences of nitrogen efficiency andnitrogen response in different oilseed rape (Brassica napus L) varieties Asian Journal of Crop Science 1(2)105-112
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2010) Nitrogen redistribution characteristics ofoilseed rape varieties with different nitrogen use efficiencies during later growth period Communications in Soil Science andPlant Analysis 41(14) 1693-1706
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Zhang ZH Huang HT Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2012) Research advances on nitratenitrogen reutilization by proton pump of tonoplast and its relation to nitrogen use efficiency Australian Journal of Crop Science6(9) 1377-1382
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Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
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- Parsed Citations
- Reviewer PDF
- ONE
- TWO
- THREE
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- SIX
- SEVEN
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- T-TWO
- Parsed Citations
-
23
BnNRT18 and Bnactin genes were obtained from httpbrassicanbiacuk as described in 660
Harper et al (2012) and expression levels were normalized to Bnactin as control 661
Total RNA extracted from the 4-week-old A thaliana roots under the treatments 662
conditions described above were prepared using Trizol reagent (Invitrogen) The cDNAs were 663
synthesized using M-MLV reverse transcriptase (Promega) based on the manufacturerrsquos 664
protocol and as described in Li et al (2010)The primer sequences of quantitative RT-PCR 665
used in these assays are listed in Supplemental Table 2 and were described previously (Chen 666
et al 2012 Zhang et al 2014) and their expression was normalized to Atactin2 as control 667
668
Determination of nitrate distribution using 15NO3- 669
Hydroponically grown B napus (seedling and flowering stages) and A thaliana 670
(seedling) were transferred to 01mM CaSO4 solution for 1min then to their respective 671
hydroponic nutrient solutions with15NO3- (22 excess) replacing unlabeled NO3
- for 1h 672
Plants were then transferred to 01mM CaSO4 solution for 1min after which roots were 673
washed with deionized water Root and shoot samples were separated and dried at 105degC for 674
30min followed by 70degC for 3 d then grounded and used for assaying 15N content using a 675
continuous-flow isotope ratio mass spectrometer coupled with a carbon-nitrogen elemental 676
analyzer (ANCA-MS PDZ Europa) 677
678
Xylem sap collection and assay of nitrate concentration 679
We used the method of Tang et al (2012) for collecting xylem sap Plants were cut 680
leaving1cm segments with intact roots at seedling and flowering stages for B napus and at 681
seedling stage for A thaliana Roots were immediately immersed in their respective nutrition 682
solutions Weighed cotton was put on the cut surface to absorb extruding xylem sap for 1 h 683
The cotton was then wrapped in plastic film and the volume of xylem sap was calculated as 684
the weight gain of the cotton Xylem sap was then squeezed from the cotton using a syringe 685
and used for subsequent assay of nitrate concentration using a continuous-flow auto-analyzer 686
(Auto Analyser 3 Bran and Luebbe Germany) 687
To determine nitrate concentration samples (10 g fresh root and shoot at seedling and 688
flowering of B napus and 05 g fresh root and shoot of 4-week-old A thaliana) were frozen 689
in liquid N2 and ground with a mortar and pestle the powder was then transferred to a beaker 690
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
containing 70 ml deionized water and boiled for 30 min to extract nitrate then cooled and 691
made to 100 ml volume and 02 g activated carbon added to eliminate the effect of 692
chlorophyll The mixture was filtered and nitrate in the filtrate was determined using a 693
continuous-flow auto-analyser (Atuo-Analyser 3 Bran and Luebbe Germany) 694
695
Measurements of photosynthesis 696
All measurements of photosynthesis and related parameters were conducted at 1000 h 697
using intact leaves The 4th leaf from the bottom in B napus at seedling stage and the 12th leaf 698
from the bottom at flowering stage were used for determining chlorophyll concentration 699
photosynthetic rate intercellular CO2 concentration transpiration rate and stomatal 700
conductance Chlorophyll concentration was determined by SPAD-502 (Minolta Camera Co 701
Ltd Japan) (Luo et al 2006 Yandeau-Nelson et al 2011) Photosynthetic rate intercellular 702
CO2 concentration transpiration rate and stomatal conductance were measured using LI-6400 703
Portable Photosynthesis System (Li-Cor Biosciences Co Ltd USA) set at flow rate of 500 704
μmol s-1 photosynthetic photon flux density of 1000 μmol m-2 s-1 relative humidity of 65 705
CO2 concentration of 400ppm and ambient temperatures 706
707
NR and GS activities 708
Leaves with similar age as those used for photosynthesis in B napus were used for the 709
assay of nitrate reductase (NR) and glutamine synthetase (GS) activities NR activity was 710
measured by the modified method of Fan et al (2007) Fresh samples (05g) were frozen in 711
liquid N2 ground into powder in the presence of acid-washed sand and homogenized with 4 712
ml of extraction buffer (0025mol L-1phosphate buffer pH87 1211g L-1 cysteine 0372g L-1 713
EDTA) Homogenates were centrifuged at 30000timesg for 15 min at 4degC and the supernatants 714
were treated with sulfanilamide and α-naphthylamine reagents for colorimetric (540nm) 715
determination of nitrite as described by Fan et al (2007) 716
GS activity was determined using a modified reverseγ-glutamyltransferase method 717
(Wang et al2014) which measures the GS-catalyzed formation of glutamyl-γ-hydroxamate 718
from glutamine and hydroxylamine Fresh samples were frozen for 30 min at -20degC then 719
ground in 10ml Tris-HCl buffer and acid-washed sand The homogenates were filtered 720
through two layers of gauze and centrifuged at 8000timesg for 15 min at 4degC About 12ml of the 721
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
supernatant was added in a reaction mixture and treated as described (Wang et al2014) After 722
the GS-catalyzed reaction was stopped and the solution centrifuged glutamyl-γ723
-hydroxamate was quantified colorimetrically in the supernatant (at 485 nm) and the 724
concentration was determined using a standard curve 725
726
Statistical analyses 727
We used the SPSS software (Statistical Product and Service Solutions V130 USA) for 728
ANOVA and mean separation of main-effects and interactions using Duncanrsquos multiple range 729
test at Plt005Values are presented as means and SD of three or six replicates from three 730
independent experiments Different letters or an asterisk () associated with specific data (eg 731
at the top of histogram bars in figures or within tables) denote significant differences at 732
Plt005 733
734
Supplemental Data 735
Supplemental Figure 1 B napus with higher NUE showed lower vacuolar sequestration 736
capacity (VSC) for NO3-in roots at flowering stage 737
Supplemental Figure 2 B napus with higher NUE showed enhanced long-distance transport 738
of NO3- from roots to shoots at flowering stage 739
Supplemental Figure 3 The two B napus (H and L genotypes) showed the same total N per 740
plant at seedling stage (A) and flowering stage (B) 741
Supplemental Figure 4 B napus with higher NUE showed increased NO3- concentration in 742
the xylem sap at seedling and flowering stages 743
Supplemental Figure 5 NO3- concentration in the xylem sap as affected by inhibitor 744
treatments in B napus and in the energy pumpsrsquo mutants of A thaliana (col-0 vha-a2 vha-a3 745
avp1) 746
Supplemental Figure 6 Reduced VSC for NO3- in roots drives long-distance transport of 747
NO3- from roots to shoots in the A thaliana wild type (Ws) and mutant (clca-2) 748
Supplemental Figure 7 Differences of NR and GS activities between the two B napus (H 749
and L genotypes) at seedling and flowering stages 750
Supplemental Figure 8 Amino acid sequences of BnNRT15 and BnNRT18 751
Supplemental Figure 9 Functions of AtNRT15 and AtNRT18 genes in root tissues of A 752
thaliana in controlling NO3- long-distance transport from root to shoots 753
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
Supplemental Figure 10 B napus BnNRT15 and BnNRT18 genes in roots were 754
coordinately modulated to facilitate NO3- long distance transport from roots to shoots 755
Supplemental Table 1 Differences in stomatal conductance and transpiration rate between 756
the two B napus genotypes 757
Supplemental Table 2 Sequences of primera used for qRT-PCR 758
759
Acknowledgements 760
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 761
Shanghai Institutes for Biological Sciences) for providing nrt15-3 and nrt18-2 seeds 762
This study was supported by the National Natural Science Foundation of China (Grant 763
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 764
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 765
The Ministry of Education in China Open Novel Science Foundation of Hunan province 766
(13K062) the National Key Laboratory of Plant Molecular Genetics and the Twelfth 767
Five-Year National Science and technology support program (2012BAD15BO4) 768
769
Figure 1 B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 770
NO3- in roots at the seedling stage 771
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 772
genotype 814 Specific activities of the tonoplast proton pumps are expressed as μmol Pi 773
released mg-1 protein h-1 NO3- fluxes are expressed as pmol NO3
- cm-2 S-1 Mature vacuoles 774
were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 775
measuring solution between the two positions 1 μm and 11 μm from the vacuole surface 776
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles recording the 777
stable reading data The background was recorded by vibrating the electrode in measuring 778
solution without vacuoles 779
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 780
for NO3- content and NO3
- accumulation normalized against the specific activity of the 781
vacuole acid phosphatase (ACP) as described in Materials and Methods and plotted as μmol 782
NO3- per μmol p-nitrophenol the end product of ACP assay 783
Proton pump activities in root tissues between H and L genotypes are shown at the seedling 784
stage (A) Different letters at the top of the histogram bars denote significant differences of 785
V-ATPase in root tissues between H and L genotypes (Plt005) an asterisk () at the top of the 786
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
histogram bars indicates significant difference in V-PPase activity in root tissues between H 787
and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788
Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789
genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790
bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791
bars on the figures indicate SD (n=6) 792
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793
seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794
to the total NO3- in protoplasts 795
NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796
total NO3-in the cytosol and was calculated as total NO3
- in protoplasts ndash total NO3- in 797
vacuole Different letters at the top of histogram bars denote significant differences of total 798
NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799
(n=6) 800
801
Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802
roots to shoots at the seedling stage 803
H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804
Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805
the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806
as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807
the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808
the histogram bars denote significant differences at Plt005 809
Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810
in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811
genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812
of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813
SD (n=3) different letters at the top of the histogram bars denote significant differences at 814
Plt005 level 815
The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816
at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817
genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818
different letters at the top of the histogram bars denote significant differences at Plt005 819
820
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
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b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
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Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
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Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ma JF Ueno D Zhao FJ McGrath SP (2005) Subcellular localization of Cd and Zn in the leaves of a Cd-hyperaccumulating ecotypeof ThlaspicaerulescensPlanta 220 731-736
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Martinoia E Massonneau A Frangne N (2000) Transport processes of solutes across the vacuolar membrane of higher plantsPlant Cell Physiology 41(11)1175-1186
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Martinoia E Heck U Wiemken A (1981) Vacuoles as storage compartments for nitrate in barley leaves Nature 289 292-294Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Miller AJ Smith SJ (2008) Cytosolic nitrate ion homeostasis could it have a role in sensing nitrogen status Annals ofBotany101485-489
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Robert S Zouhar J Carter C Raikhel N (2007) Isolation of intact vacuoles from Arabidopsis rosette leaf-derived protoplastsNature Protocols 2(2)259-262
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Schroeder JL Delhaize E Frommer WB Guerinot ML Harrison MJ Herrera-Estrella L Horie T Kochian LV Munns R NishizawaNK Tsay YF Sanders D (2013) Using membrane transporters to improve crops for sustainable food production Nature 497 60-66
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi WM Xu WF Li SM Zhao XQ Dong GQ (2010) Responses of two rice cultivars differing in seedling-stage nitrogen useefficiency to growth under low-nitrogen conditions Plant and Soil 326291-302
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shen QR Tang L Xu YC (2003) A review on the behavior of nitrate in vacuoles of plants ActaPedologicaSinica40(3)465-470Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Smirnoff N Stewart G (1985) Nitrate assimilation and translocation by higher plants Comparative physiology and ecologicalconsequences Physiologia Plantarum 64 133-140
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Tang TF Sun XC Hu CX Tan QL Zhao XH (2013) Genotypic differences in nitrate uptake translocation and assimilation of twoChinese cabbage cultivars [Brassica campestris L ssp Chinesnsis (L)] Plant Physiology and Biochemistry7014-20
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Tang Z Fan XR Li Qing Feng HM Miller AJ Shen QR Xu GH (2012) Knockdown of a rice stellar nitrate transporter alters long-distance translocation but not root influx Plant Physiology 1602052-2063
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Vogeli-Lange R Wagner GJ (1990) Subcellular localization of cadmium and cadmium-binding peptides in tobacco leavesimplication of a transport function for cadmium-binding peptides Plant Physiology 921086-1093
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Wang GL Ding GD Li L Cai HM Ye XS Zou J Xu FS (2014) Identification and characterization of improved nitrogen efficiency ininterspecific hybridized new-type Brassica napus Annals of Botany 114(3) 549-559
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
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Wang YY Tsay YF (2011) Arabidopsis nitrate transporter NRT19 is important in phloem nitrate transport The Plant Cell 231945-1957
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Wege S Angeli AD Droillard MJ Kroniewicz L Merlot S Cornu D Gambale F Martinoia E Barbier-Brygoo H Thomine SLeonhardt N Filleur S (2014) Phosphorylation of the vacuolar anion exchanger AtCLCa is required for the stomatal response toabscisic acid Science Signalling 7(333) ra65
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wilkinson S Bacon MA Davies WJ (2007) Nitrate signaling to stomatal and growing leaves interactions with soil drying ABA andxylem sap pH in maize Journal of Experimental Botany 58(7) 1705-1716
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Xu GH Fan XR Miller AJ (2012) Plant nitrogen assimilation and use efficiency The Annual Review of Plant Biology 63153-182Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yandeau-Nelson MD Laurens L Shi Z Xia H Smith AM Guiltinan MJ (2011) Starch-Branching Enzyme IIa Is Required for ProperDiurnal Cycling of Starch in Leaves of Maize [OA] Plant Physiology 156479-490
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Zhang GB Yi HY Gong JM (2014) The Arabidopsis EthyleneJasmonic acid-NRT signaling module coordinates nitrate reallocationand the trade-off between growth and environmental adaption The Plant Cell 26(10)3984-3998
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Zhang ZH Song HX Liu Qing Rong XM Xie GX Peng JW Zhang YP (2009) Study on differences of nitrogen efficiency andnitrogen response in different oilseed rape (Brassica napus L) varieties Asian Journal of Crop Science 1(2)105-112
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Zhang ZH Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2010) Nitrogen redistribution characteristics ofoilseed rape varieties with different nitrogen use efficiencies during later growth period Communications in Soil Science andPlant Analysis 41(14) 1693-1706
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Zhang ZH Huang HT Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2012) Research advances on nitratenitrogen reutilization by proton pump of tonoplast and its relation to nitrogen use efficiency Australian Journal of Crop Science6(9) 1377-1382
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- Parsed Citations
- Reviewer PDF
- ONE
- TWO
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- Parsed Citations
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24
containing 70 ml deionized water and boiled for 30 min to extract nitrate then cooled and 691
made to 100 ml volume and 02 g activated carbon added to eliminate the effect of 692
chlorophyll The mixture was filtered and nitrate in the filtrate was determined using a 693
continuous-flow auto-analyser (Atuo-Analyser 3 Bran and Luebbe Germany) 694
695
Measurements of photosynthesis 696
All measurements of photosynthesis and related parameters were conducted at 1000 h 697
using intact leaves The 4th leaf from the bottom in B napus at seedling stage and the 12th leaf 698
from the bottom at flowering stage were used for determining chlorophyll concentration 699
photosynthetic rate intercellular CO2 concentration transpiration rate and stomatal 700
conductance Chlorophyll concentration was determined by SPAD-502 (Minolta Camera Co 701
Ltd Japan) (Luo et al 2006 Yandeau-Nelson et al 2011) Photosynthetic rate intercellular 702
CO2 concentration transpiration rate and stomatal conductance were measured using LI-6400 703
Portable Photosynthesis System (Li-Cor Biosciences Co Ltd USA) set at flow rate of 500 704
μmol s-1 photosynthetic photon flux density of 1000 μmol m-2 s-1 relative humidity of 65 705
CO2 concentration of 400ppm and ambient temperatures 706
707
NR and GS activities 708
Leaves with similar age as those used for photosynthesis in B napus were used for the 709
assay of nitrate reductase (NR) and glutamine synthetase (GS) activities NR activity was 710
measured by the modified method of Fan et al (2007) Fresh samples (05g) were frozen in 711
liquid N2 ground into powder in the presence of acid-washed sand and homogenized with 4 712
ml of extraction buffer (0025mol L-1phosphate buffer pH87 1211g L-1 cysteine 0372g L-1 713
EDTA) Homogenates were centrifuged at 30000timesg for 15 min at 4degC and the supernatants 714
were treated with sulfanilamide and α-naphthylamine reagents for colorimetric (540nm) 715
determination of nitrite as described by Fan et al (2007) 716
GS activity was determined using a modified reverseγ-glutamyltransferase method 717
(Wang et al2014) which measures the GS-catalyzed formation of glutamyl-γ-hydroxamate 718
from glutamine and hydroxylamine Fresh samples were frozen for 30 min at -20degC then 719
ground in 10ml Tris-HCl buffer and acid-washed sand The homogenates were filtered 720
through two layers of gauze and centrifuged at 8000timesg for 15 min at 4degC About 12ml of the 721
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
supernatant was added in a reaction mixture and treated as described (Wang et al2014) After 722
the GS-catalyzed reaction was stopped and the solution centrifuged glutamyl-γ723
-hydroxamate was quantified colorimetrically in the supernatant (at 485 nm) and the 724
concentration was determined using a standard curve 725
726
Statistical analyses 727
We used the SPSS software (Statistical Product and Service Solutions V130 USA) for 728
ANOVA and mean separation of main-effects and interactions using Duncanrsquos multiple range 729
test at Plt005Values are presented as means and SD of three or six replicates from three 730
independent experiments Different letters or an asterisk () associated with specific data (eg 731
at the top of histogram bars in figures or within tables) denote significant differences at 732
Plt005 733
734
Supplemental Data 735
Supplemental Figure 1 B napus with higher NUE showed lower vacuolar sequestration 736
capacity (VSC) for NO3-in roots at flowering stage 737
Supplemental Figure 2 B napus with higher NUE showed enhanced long-distance transport 738
of NO3- from roots to shoots at flowering stage 739
Supplemental Figure 3 The two B napus (H and L genotypes) showed the same total N per 740
plant at seedling stage (A) and flowering stage (B) 741
Supplemental Figure 4 B napus with higher NUE showed increased NO3- concentration in 742
the xylem sap at seedling and flowering stages 743
Supplemental Figure 5 NO3- concentration in the xylem sap as affected by inhibitor 744
treatments in B napus and in the energy pumpsrsquo mutants of A thaliana (col-0 vha-a2 vha-a3 745
avp1) 746
Supplemental Figure 6 Reduced VSC for NO3- in roots drives long-distance transport of 747
NO3- from roots to shoots in the A thaliana wild type (Ws) and mutant (clca-2) 748
Supplemental Figure 7 Differences of NR and GS activities between the two B napus (H 749
and L genotypes) at seedling and flowering stages 750
Supplemental Figure 8 Amino acid sequences of BnNRT15 and BnNRT18 751
Supplemental Figure 9 Functions of AtNRT15 and AtNRT18 genes in root tissues of A 752
thaliana in controlling NO3- long-distance transport from root to shoots 753
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
Supplemental Figure 10 B napus BnNRT15 and BnNRT18 genes in roots were 754
coordinately modulated to facilitate NO3- long distance transport from roots to shoots 755
Supplemental Table 1 Differences in stomatal conductance and transpiration rate between 756
the two B napus genotypes 757
Supplemental Table 2 Sequences of primera used for qRT-PCR 758
759
Acknowledgements 760
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 761
Shanghai Institutes for Biological Sciences) for providing nrt15-3 and nrt18-2 seeds 762
This study was supported by the National Natural Science Foundation of China (Grant 763
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 764
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 765
The Ministry of Education in China Open Novel Science Foundation of Hunan province 766
(13K062) the National Key Laboratory of Plant Molecular Genetics and the Twelfth 767
Five-Year National Science and technology support program (2012BAD15BO4) 768
769
Figure 1 B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 770
NO3- in roots at the seedling stage 771
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 772
genotype 814 Specific activities of the tonoplast proton pumps are expressed as μmol Pi 773
released mg-1 protein h-1 NO3- fluxes are expressed as pmol NO3
- cm-2 S-1 Mature vacuoles 774
were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 775
measuring solution between the two positions 1 μm and 11 μm from the vacuole surface 776
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles recording the 777
stable reading data The background was recorded by vibrating the electrode in measuring 778
solution without vacuoles 779
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 780
for NO3- content and NO3
- accumulation normalized against the specific activity of the 781
vacuole acid phosphatase (ACP) as described in Materials and Methods and plotted as μmol 782
NO3- per μmol p-nitrophenol the end product of ACP assay 783
Proton pump activities in root tissues between H and L genotypes are shown at the seedling 784
stage (A) Different letters at the top of the histogram bars denote significant differences of 785
V-ATPase in root tissues between H and L genotypes (Plt005) an asterisk () at the top of the 786
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
histogram bars indicates significant difference in V-PPase activity in root tissues between H 787
and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788
Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789
genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790
bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791
bars on the figures indicate SD (n=6) 792
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793
seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794
to the total NO3- in protoplasts 795
NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796
total NO3-in the cytosol and was calculated as total NO3
- in protoplasts ndash total NO3- in 797
vacuole Different letters at the top of histogram bars denote significant differences of total 798
NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799
(n=6) 800
801
Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802
roots to shoots at the seedling stage 803
H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804
Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805
the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806
as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807
the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808
the histogram bars denote significant differences at Plt005 809
Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810
in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811
genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812
of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813
SD (n=3) different letters at the top of the histogram bars denote significant differences at 814
Plt005 level 815
The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816
at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817
genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818
different letters at the top of the histogram bars denote significant differences at Plt005 819
820
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
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NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
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b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
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Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
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Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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Li JS Yang HB Peer WA Richter G Blakeslee J Bandyopadhyay A Titapiwantakun B Undurraga S Khodakovskaya M RichardsE Krizek B Murphy AS Gilroy S Gaxiola R (2005) Arabidopsis H+-PPase AVP1 regulates auxin-mediated organ developmentScience 310 (121) 121-125
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Luo JK Sun SB Jia LJ Chen W Shen QR (2006) The mechanism of nitrate accumulation in Pakchoi [BrassicacampestrisLsspChinensis (L)] Plant and Soil 282291-300
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ma JF Ueno D Zhao FJ McGrath SP (2005) Subcellular localization of Cd and Zn in the leaves of a Cd-hyperaccumulating ecotypeof ThlaspicaerulescensPlanta 220 731-736
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Martinoia E Massonneau A Frangne N (2000) Transport processes of solutes across the vacuolar membrane of higher plantsPlant Cell Physiology 41(11)1175-1186
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Martinoia E Heck U Wiemken A (1981) Vacuoles as storage compartments for nitrate in barley leaves Nature 289 292-294Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Miller AJ Smith SJ (2008) Cytosolic nitrate ion homeostasis could it have a role in sensing nitrogen status Annals ofBotany101485-489
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Schroeder JL Delhaize E Frommer WB Guerinot ML Harrison MJ Herrera-Estrella L Horie T Kochian LV Munns R NishizawaNK Tsay YF Sanders D (2013) Using membrane transporters to improve crops for sustainable food production Nature 497 60-66
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Shi WM Xu WF Li SM Zhao XQ Dong GQ (2010) Responses of two rice cultivars differing in seedling-stage nitrogen useefficiency to growth under low-nitrogen conditions Plant and Soil 326291-302
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Shen QR Tang L Xu YC (2003) A review on the behavior of nitrate in vacuoles of plants ActaPedologicaSinica40(3)465-470Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Smirnoff N Stewart G (1985) Nitrate assimilation and translocation by higher plants Comparative physiology and ecologicalconsequences Physiologia Plantarum 64 133-140
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Tang Z Fan XR Li Qing Feng HM Miller AJ Shen QR Xu GH (2012) Knockdown of a rice stellar nitrate transporter alters long-distance translocation but not root influx Plant Physiology 1602052-2063
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Wang YY Tsay YF (2011) Arabidopsis nitrate transporter NRT19 is important in phloem nitrate transport The Plant Cell 231945-1957
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wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Reviewer PDF
- ONE
- TWO
- THREE
- FOUR
- FIVE
- SIX
- SEVEN
- T-ONE
- T-TWO
- Parsed Citations
-
25
supernatant was added in a reaction mixture and treated as described (Wang et al2014) After 722
the GS-catalyzed reaction was stopped and the solution centrifuged glutamyl-γ723
-hydroxamate was quantified colorimetrically in the supernatant (at 485 nm) and the 724
concentration was determined using a standard curve 725
726
Statistical analyses 727
We used the SPSS software (Statistical Product and Service Solutions V130 USA) for 728
ANOVA and mean separation of main-effects and interactions using Duncanrsquos multiple range 729
test at Plt005Values are presented as means and SD of three or six replicates from three 730
independent experiments Different letters or an asterisk () associated with specific data (eg 731
at the top of histogram bars in figures or within tables) denote significant differences at 732
Plt005 733
734
Supplemental Data 735
Supplemental Figure 1 B napus with higher NUE showed lower vacuolar sequestration 736
capacity (VSC) for NO3-in roots at flowering stage 737
Supplemental Figure 2 B napus with higher NUE showed enhanced long-distance transport 738
of NO3- from roots to shoots at flowering stage 739
Supplemental Figure 3 The two B napus (H and L genotypes) showed the same total N per 740
plant at seedling stage (A) and flowering stage (B) 741
Supplemental Figure 4 B napus with higher NUE showed increased NO3- concentration in 742
the xylem sap at seedling and flowering stages 743
Supplemental Figure 5 NO3- concentration in the xylem sap as affected by inhibitor 744
treatments in B napus and in the energy pumpsrsquo mutants of A thaliana (col-0 vha-a2 vha-a3 745
avp1) 746
Supplemental Figure 6 Reduced VSC for NO3- in roots drives long-distance transport of 747
NO3- from roots to shoots in the A thaliana wild type (Ws) and mutant (clca-2) 748
Supplemental Figure 7 Differences of NR and GS activities between the two B napus (H 749
and L genotypes) at seedling and flowering stages 750
Supplemental Figure 8 Amino acid sequences of BnNRT15 and BnNRT18 751
Supplemental Figure 9 Functions of AtNRT15 and AtNRT18 genes in root tissues of A 752
thaliana in controlling NO3- long-distance transport from root to shoots 753
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
Supplemental Figure 10 B napus BnNRT15 and BnNRT18 genes in roots were 754
coordinately modulated to facilitate NO3- long distance transport from roots to shoots 755
Supplemental Table 1 Differences in stomatal conductance and transpiration rate between 756
the two B napus genotypes 757
Supplemental Table 2 Sequences of primera used for qRT-PCR 758
759
Acknowledgements 760
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 761
Shanghai Institutes for Biological Sciences) for providing nrt15-3 and nrt18-2 seeds 762
This study was supported by the National Natural Science Foundation of China (Grant 763
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 764
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 765
The Ministry of Education in China Open Novel Science Foundation of Hunan province 766
(13K062) the National Key Laboratory of Plant Molecular Genetics and the Twelfth 767
Five-Year National Science and technology support program (2012BAD15BO4) 768
769
Figure 1 B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 770
NO3- in roots at the seedling stage 771
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 772
genotype 814 Specific activities of the tonoplast proton pumps are expressed as μmol Pi 773
released mg-1 protein h-1 NO3- fluxes are expressed as pmol NO3
- cm-2 S-1 Mature vacuoles 774
were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 775
measuring solution between the two positions 1 μm and 11 μm from the vacuole surface 776
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles recording the 777
stable reading data The background was recorded by vibrating the electrode in measuring 778
solution without vacuoles 779
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 780
for NO3- content and NO3
- accumulation normalized against the specific activity of the 781
vacuole acid phosphatase (ACP) as described in Materials and Methods and plotted as μmol 782
NO3- per μmol p-nitrophenol the end product of ACP assay 783
Proton pump activities in root tissues between H and L genotypes are shown at the seedling 784
stage (A) Different letters at the top of the histogram bars denote significant differences of 785
V-ATPase in root tissues between H and L genotypes (Plt005) an asterisk () at the top of the 786
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
histogram bars indicates significant difference in V-PPase activity in root tissues between H 787
and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788
Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789
genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790
bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791
bars on the figures indicate SD (n=6) 792
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793
seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794
to the total NO3- in protoplasts 795
NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796
total NO3-in the cytosol and was calculated as total NO3
- in protoplasts ndash total NO3- in 797
vacuole Different letters at the top of histogram bars denote significant differences of total 798
NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799
(n=6) 800
801
Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802
roots to shoots at the seedling stage 803
H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804
Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805
the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806
as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807
the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808
the histogram bars denote significant differences at Plt005 809
Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810
in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811
genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812
of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813
SD (n=3) different letters at the top of the histogram bars denote significant differences at 814
Plt005 level 815
The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816
at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817
genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818
different letters at the top of the histogram bars denote significant differences at Plt005 819
820
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
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b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
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Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
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Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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Schroeder JL Delhaize E Frommer WB Guerinot ML Harrison MJ Herrera-Estrella L Horie T Kochian LV Munns R NishizawaNK Tsay YF Sanders D (2013) Using membrane transporters to improve crops for sustainable food production Nature 497 60-66
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Shi WM Xu WF Li SM Zhao XQ Dong GQ (2010) Responses of two rice cultivars differing in seedling-stage nitrogen useefficiency to growth under low-nitrogen conditions Plant and Soil 326291-302
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Shen QR Tang L Xu YC (2003) A review on the behavior of nitrate in vacuoles of plants ActaPedologicaSinica40(3)465-470Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Smirnoff N Stewart G (1985) Nitrate assimilation and translocation by higher plants Comparative physiology and ecologicalconsequences Physiologia Plantarum 64 133-140
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Tang TF Sun XC Hu CX Tan QL Zhao XH (2013) Genotypic differences in nitrate uptake translocation and assimilation of twoChinese cabbage cultivars [Brassica campestris L ssp Chinesnsis (L)] Plant Physiology and Biochemistry7014-20
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Tang Z Fan XR Li Qing Feng HM Miller AJ Shen QR Xu GH (2012) Knockdown of a rice stellar nitrate transporter alters long-distance translocation but not root influx Plant Physiology 1602052-2063
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Vogeli-Lange R Wagner GJ (1990) Subcellular localization of cadmium and cadmium-binding peptides in tobacco leavesimplication of a transport function for cadmium-binding peptides Plant Physiology 921086-1093
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Wang GL Ding GD Li L Cai HM Ye XS Zou J Xu FS (2014) Identification and characterization of improved nitrogen efficiency ininterspecific hybridized new-type Brassica napus Annals of Botany 114(3) 549-559
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Wang YY Tsay YF (2011) Arabidopsis nitrate transporter NRT19 is important in phloem nitrate transport The Plant Cell 231945-1957
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Wege S Angeli AD Droillard MJ Kroniewicz L Merlot S Cornu D Gambale F Martinoia E Barbier-Brygoo H Thomine SLeonhardt N Filleur S (2014) Phosphorylation of the vacuolar anion exchanger AtCLCa is required for the stomatal response toabscisic acid Science Signalling 7(333) ra65
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Wilkinson S Bacon MA Davies WJ (2007) Nitrate signaling to stomatal and growing leaves interactions with soil drying ABA andxylem sap pH in maize Journal of Experimental Botany 58(7) 1705-1716
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Xu GH Fan XR Miller AJ (2012) Plant nitrogen assimilation and use efficiency The Annual Review of Plant Biology 63153-182Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
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- Reviewer PDF
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26
Supplemental Figure 10 B napus BnNRT15 and BnNRT18 genes in roots were 754
coordinately modulated to facilitate NO3- long distance transport from roots to shoots 755
Supplemental Table 1 Differences in stomatal conductance and transpiration rate between 756
the two B napus genotypes 757
Supplemental Table 2 Sequences of primera used for qRT-PCR 758
759
Acknowledgements 760
We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 761
Shanghai Institutes for Biological Sciences) for providing nrt15-3 and nrt18-2 seeds 762
This study was supported by the National Natural Science Foundation of China (Grant 763
No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 764
National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 765
The Ministry of Education in China Open Novel Science Foundation of Hunan province 766
(13K062) the National Key Laboratory of Plant Molecular Genetics and the Twelfth 767
Five-Year National Science and technology support program (2012BAD15BO4) 768
769
Figure 1 B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 770
NO3- in roots at the seedling stage 771
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 772
genotype 814 Specific activities of the tonoplast proton pumps are expressed as μmol Pi 773
released mg-1 protein h-1 NO3- fluxes are expressed as pmol NO3
- cm-2 S-1 Mature vacuoles 774
were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 775
measuring solution between the two positions 1 μm and 11 μm from the vacuole surface 776
(tonoplast) along an axis perpendicular to the tangent of the target vacuoles recording the 777
stable reading data The background was recorded by vibrating the electrode in measuring 778
solution without vacuoles 779
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 780
for NO3- content and NO3
- accumulation normalized against the specific activity of the 781
vacuole acid phosphatase (ACP) as described in Materials and Methods and plotted as μmol 782
NO3- per μmol p-nitrophenol the end product of ACP assay 783
Proton pump activities in root tissues between H and L genotypes are shown at the seedling 784
stage (A) Different letters at the top of the histogram bars denote significant differences of 785
V-ATPase in root tissues between H and L genotypes (Plt005) an asterisk () at the top of the 786
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
histogram bars indicates significant difference in V-PPase activity in root tissues between H 787
and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788
Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789
genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790
bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791
bars on the figures indicate SD (n=6) 792
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793
seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794
to the total NO3- in protoplasts 795
NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796
total NO3-in the cytosol and was calculated as total NO3
- in protoplasts ndash total NO3- in 797
vacuole Different letters at the top of histogram bars denote significant differences of total 798
NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799
(n=6) 800
801
Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802
roots to shoots at the seedling stage 803
H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804
Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805
the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806
as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807
the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808
the histogram bars denote significant differences at Plt005 809
Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810
in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811
genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812
of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813
SD (n=3) different letters at the top of the histogram bars denote significant differences at 814
Plt005 level 815
The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816
at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817
genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818
different letters at the top of the histogram bars denote significant differences at Plt005 819
820
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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Zhang ZH Song HX Liu Qing Rong XM Xie GX Peng JW Zhang YP (2009) Study on differences of nitrogen efficiency andnitrogen response in different oilseed rape (Brassica napus L) varieties Asian Journal of Crop Science 1(2)105-112
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2010) Nitrogen redistribution characteristics ofoilseed rape varieties with different nitrogen use efficiencies during later growth period Communications in Soil Science andPlant Analysis 41(14) 1693-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Huang HT Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2012) Research advances on nitratenitrogen reutilization by proton pump of tonoplast and its relation to nitrogen use efficiency Australian Journal of Crop Science6(9) 1377-1382
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhu ZJ Qian YR Pfeiffer W (2001) Effect of nitrogen form on the activity of tonoplast pyrophosphatase in tomato roots ActaBotanica Sinica 43(11) 1146-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
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- Parsed Citations
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27
histogram bars indicates significant difference in V-PPase activity in root tissues between H 787
and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788
Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789
genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790
bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791
bars on the figures indicate SD (n=6) 792
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793
seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794
to the total NO3- in protoplasts 795
NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796
total NO3-in the cytosol and was calculated as total NO3
- in protoplasts ndash total NO3- in 797
vacuole Different letters at the top of histogram bars denote significant differences of total 798
NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799
(n=6) 800
801
Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802
roots to shoots at the seedling stage 803
H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804
Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805
the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806
as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807
the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808
the histogram bars denote significant differences at Plt005 809
Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810
in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811
genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812
of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813
SD (n=3) different letters at the top of the histogram bars denote significant differences at 814
Plt005 level 815
The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816
at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817
genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818
different letters at the top of the histogram bars denote significant differences at Plt005 819
820
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
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29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
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30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
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31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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- Parsed Citations
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28
Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821
regulation of the NRT18 gene 822
Culture conditions and plant materials are defined in the Materials and Methods Seedling 823
stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824
225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825
which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826
were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827
relative to that of the Atactin2 gene 828
B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829
shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830
analysis as described in the Materials and Methods 831
Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832
col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833
nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834
for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835
the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836
relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837
Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838
(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840
841
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 842
shoots in the B napus (Xiangyou15) 843
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844
the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845
an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846
specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847
both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848
solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849
at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851
NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as described in the legend to 852
Figure 1 and in Materials and Methods 853
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
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30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
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31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
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Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
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b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
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- Parsed Citations
- Reviewer PDF
- ONE
- TWO
- THREE
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- SIX
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- Parsed Citations
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29
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854
napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855
denote significant differences in V-ATPase activities in root tissues between inhibitor 856
treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857
in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858
the figures indicate SD (n=6) 859
NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860
treatments (B) Different letters at the top of the histogram bars denote significant differences 861
of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862
(n=6) 863
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864
napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865
total NO3- in protoplasts Vertical bars indicate SD (n=6) 866
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 868
letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869
outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871
of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872
value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873
top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874
bars indicate SD (n=3) 875
Growth conditions for hydroponically grown plants with 15N treatmentare described in 876
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877
for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878
napus (H) Different letters at the top of the histogram bars denote significant differences 879
(Plt005) Vertical bars on the figures indicate SD (n=3) 880
881
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3
- from roots to 882
shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884
V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885
were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886
measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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Xu GH Fan XR Miller AJ (2012) Plant nitrogen assimilation and use efficiency The Annual Review of Plant Biology 63153-182Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yandeau-Nelson MD Laurens L Shi Z Xia H Smith AM Guiltinan MJ (2011) Starch-Branching Enzyme IIa Is Required for ProperDiurnal Cycling of Starch in Leaves of Maize [OA] Plant Physiology 156479-490
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Zhang ZH Huang HT Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2012) Research advances on nitratenitrogen reutilization by proton pump of tonoplast and its relation to nitrogen use efficiency Australian Journal of Crop Science6(9) 1377-1382
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- Parsed Citations
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- T-TWO
- Parsed Citations
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30
roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888
NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889
Methods 890
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891
different A thaliana plant materials (A) Different letters at the top of the histogram bars 892
denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893
materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896
mutants (B) Different letters at the top of the histogram bars denote significant differences in 897
NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899
thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900
total NO3- in protoplasts Vertical bars indicate SD (n=6) 901
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902
NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3- in vacuoles Different 903
letters at the top of the histogram bars denote significant differences in total NO3-in the 904
cytosol (Plt005) Vertical bars indicate SD (n=6) 905
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906
actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907
expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908
significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909
(n=3) 910
Growth conditions for hydroponically grown plants with 15N treatmentare described in 911
Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 912
for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913
are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914
bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=3) 915
916
Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917
thaliana 918
Plants grown hydroponically were sampled for further analysis at seedling stage and at 919
harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920
genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921
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31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
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Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
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Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
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Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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- Parsed Citations
- Reviewer PDF
- ONE
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31
were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922
nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923
used as control for clca-2 mutants 924
Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925
wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926
Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927
thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928
denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929
the figures indicate SD (n=6) 930
931
Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932
Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933
in root tissues Red lines display the route for regulation pathway 934
935
936
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Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
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b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
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Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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- Parsed Citations
- Reviewer PDF
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Figure 1 B napus with higher NUE showed lower vacuolar
sequestration capacity (VSC) for NO3- in roots at the seedling stage
H refers to the high-NUE oilseed rape genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Specific activities of the tonoplast
proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-
fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were
collected from the root tissues at seedling stage and a microelectrode
was vibrated in the measuring solution between the two positions 1
μm and 11 μm from the vacuole surface (tonoplast) along an axis
perpendicular to the tangent of the target vacuoles recording the stable
reading data The background was recorded by vibrating the electrode
in measuring solution without vacuoles
Protoplasts and vacuoles isolated from roots of hydroponically grown
plants were measured for NO3- content and NO3
- accumulation
normalized against the specific activity of the vacuole acid phosphatase
(ACP) as described in Materials and Methods and plotted as μmol
NO3- per μmol p-nitrophenol the end product of ACP assay
Proton pump activities in root tissues between H and L genotypes are
shown at the seedling stage (A) Different letters at the top of the
histogram bars denote significant differences of V-ATPase in root
tissues between H and L genotypes (Plt005) an asterisk () at the top
of the histogram bars indicates significant difference in V-PPase
activity in root tissues between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Mean rates of NO3- flux during 160 s within vacuoles of root tissues
between H and L genotypes are shown at the seedling stage (B)
Different letters at the top of the histogram bars denote significant
differences of NO3- flux between H and L genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown at the seedling stage (C) Values above the bars
represent the percentage of vacuolar NO3- relative to the total NO3
- in
protoplasts
NO3- accumulation in the cytosol of root tissues is shown at seedling
stage (D) P-V is the total NO3-in the cytosol and was calculated as
total NO3- in protoplasts ndash total NO3
- in vacuole Different letters at the
top of histogram bars denote significant differences of total NO3- in
cytosol between H and L genotypes (Plt005) Vertical bars on the
figures indicate SD (n=6)
A B
C D
μm
ol
Pi
mg
-1
pro
tein
h-1
Seedling stage
Influx
H L
Root
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
μm
ol
NO
3- μ
mol
p-n
itro
phen
ol
Seedling stage
NO
3- f
lux
(pm
ol
cm-2
s-1
)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
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D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
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b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
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Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
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Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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- Parsed Citations
- Reviewer PDF
- ONE
- TWO
- THREE
- FOUR
- FIVE
- SIX
- SEVEN
- T-ONE
- T-TWO
- Parsed Citations
-
NO
3
- concentr
ati
on
(μg g
-1
FW
)
Seedling stage
[NO
3
- ] S
R r
atio
s
Seedling stage
C
mm
ol1
5
N g
-1
DW
Seedling stage
E
[15
N]
SR
rati
os
Seedling stage
BnN
RT
15
rela
tiv
e
expre
ssio
n
Seedling stage
BnN
RT
18
rela
tiv
e
expre
ssio
n
Seedling stage
A B
D
F
Figure 2 B napus with higher NUE showed enhanced
long-distance transport of NO3- from roots to shoots at
the seedling stage
H refers to the high-NUE genotype Xiangyou15 and L
refers to the low-NUE genotype 814 Expression of the
BnNRT15 (A) and BnNRT18 (B) genes relative to that
of the actin gene in the root tissues of the two
genotypes at seedling stage was assessed by
quantitative RT-PCR as described in Materials and
Methods a value of 10 is equivalent to levels of
expression of the Bnactin gene Vertical bars on the
figures indicate SD (n=3) different letters at the top of
the histogram bars denote significant differences at
Plt005
Hydroponically grown B napus plants were subjected
to 15N-labeling treatment as described in Materials and
Methods The 15N concentration in the root and shoot
tissues of the two genotypes is shown at the seedling
stage (C) The [15N] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (D)Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 level
The NO3- concentration (μg g-1 FW) in root and shoot
tissues of the two genotypes are shown at the seedling
stage (E) The [NO3-] SR ratios in the root and shoot
tissues of the two genotypes are shown at the seedling
stage (F) Vertical bars on the figures indicate SD
(n=3) different letters at the top of the histogram bars
denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
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Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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- Parsed Citations
- Reviewer PDF
- ONE
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- T-TWO
- Parsed Citations
-
aa
00
05
10
15
20 A
tNR
T1
5 r
elat
ive
exp
ress
ion
col-0 nrt18-2
Root
b
a
0
04
08
12
16
AtN
RT
15
rel
ativ
e ex
pre
ssio
n
(NH4)2succinate KNO3
nrt18-2 Root
AtNRT15 AtNRT15 A
tNR
T1
8 r
elat
ive
exp
ress
ion
b
a
0
4
8
12
a a
0
4
8
12AtNRT18 AtNRT18 A
tNR
T1
8 r
elat
ive
exp
ress
ion
col-0 nrt15-3 (NH4)2succinate KNO3
b
a
00
05
10
15
20
Bn
NR
T1
5 r
elat
ive
exp
ress
ion
(NH4)2succinate KNO3
Root nrt15-3 Root
a
b
0
002
004
006
008
(NH4)2succinate KNO3
Root Root
A B
C D
E F
Bn
NR
T1
8 r
elat
ive
exp
ress
ion
BnNRT15 BnNRT18
Figure 3 NRT15 gene expression is up-regulated by NO3-
and might affect downstream regulation of the NRT18
gene
Culture conditions and plant materials are defined in the
Materials and Methods Seedling stage A thaliana mutant
plants (nrt18-2 and nrt15-3) were cultured hydroponically
with 225 mM (NH4)2succinate for 3 d and shifted to
hydroponics with 45 mM NO3- for 12 h after which root
tissues were collected for analysis Expression of AtNRT15
or AtNRT18 genes were assessed by quantitative RT-PCR
as described in Materials and Methods and presented
relative to that of the Atactin2 gene
B napus (Xiangyou15) was cultured in hydroponics with
75 mM (NH4)2succinate for 3 d and shifted to hydroponics
with 15 mM NO3- for 12 h after which root tissues were
collected for analysis as described in the Materials and
Methods
Relative expressions of the AtNRT15 genes are shown at
the seedling stage in root tissues of col-0 and nrt18-2 (A)
Relative expression of the AtNRT15 gene is shown in root
tissues of nrt18-2 plants treated with either
(NH4)2succinate for 3 d or shifted to hydroponics with NO3-
for 12 h at the seedling stage (B) Relative expressions of
the AtNRT18 genes are shown at the seedling stage in root
tissues of col-0 and nrt15-3 (C) Panel (D) show results for
the relative expression of the AtNRT18 genes under the
same conditions as in panel (B)
Relative expression of BnNRT15 (E) and BnNRT18 (F)
genes in root tissues of B napus (Xiangyou15) are shown
at the seedling stage Different letters at the top of the
histogram bars denote significant differences (Plt005)
Vertical bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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- Parsed Citations
- Reviewer PDF
- ONE
- TWO
- THREE
- FOUR
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- T-ONE
- T-TWO
- Parsed Citations
-
a
bb
c
0
001
002
003
004
Control Bafil DCCD B+D
Root
BnNRT18
c
bb
a
0
2
4
6
Control Bafil DCCD B+D
Root
BnNRT15
a
b
a b
0
5
10
15
20
25
Control Bafil DCCD B+D
Root
V-ATPase V-PPaseμ
mol
Pi
mg
-1 p
rote
in h
-1
A B
a
b
b
c
-350
-280
-210
-140
-70
0
control Bafi DCCD B+D
C D
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
932
852 821 745
0
2
4
6
8
Control Bafil DCCD B+D
Root
Vacuole Protoplast
c
b
b
a
0
04
08
12
Control Bafil DCCD B+D
Root
P-V
μm
ol
NO
3-
μm
ol
p-n
itro
phen
ol
BnN
RT
15
rel
ativ
e ex
pre
ssio
n
BnN
RT
18
rel
ativ
e ex
pre
ssio
n
Influx
[1
5N
] S
R r
atio
c
b b
a
000
025
050
075
100
Control Bafil DCCD B+D
G
[NO
3- ]
SR
rat
io
c
b b
a
0
2
4
6
8
10
Control Bafil DCCD B+D
H
Root
E F
NO
3- f
lux
(pm
ol
cm-2
s-1
)
Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3
-
from roots to shoots in the B napus (Xiangyou15)
Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were
conducted at the seedling stage Control normal hydroponics solution Bafi(25
nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD
+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a
11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase
The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h
Mature vacuoles were collected from the root tissues of the plant materials at the
seedling stage The NO3- flux measurement is described in the legend to Figure 1
Protoplasts and vacuoles isolated from roots of hydroponically grown plants were
assessed for NO3- accumulation plotted as μmol NO3
- per μmol p-nitrophenol as
described in the legend to Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root
tissues of B napus as affected by inhibitor treatments (A) Different letters at the
top of histogram bars denote significant differences in V-ATPase activities in
root tissues between inhibitor treatments (Plt005) asterisk () at the top of the
histogram bars denote significant differences in V-PPase activities in root tissues
between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for B napus between
inhibitor treatments (B) Different letters at the top of the histogram bars denote
significant differences of NO3- flux between inhibitor treatments (Plt005)
Vertical bars on the figures indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is
shown for B napus (C) Values above the bars represent the percentage of
vacuolar NO3- relative to the total NO3
- in protoplasts Vertical bars indicate SD
(n=6)
Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is
the total NO3- in the cytosol calculated as total NO3
- in protoplasts ndash total NO3-
in vacuoles Different letters at the top of the histogram bars denote significant
differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical
bars on the figures indicate SD (n=6)
Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR as described in
Materials and Methods a value of 10 is equivalent to levels of expression of the
Bnactin gene Different letters at the top of the histogram bars denote significant
differences in gene expression (Plt005) Vertical bars indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by inhibitor
treatments are depicted for B napus (G)The [NO3-] SRratioas affected by
inhibitor treatments are shown for B napus (H) Different letters at the top of the
histogram bars denote significant differences (Plt005) Vertical bars on the
figures indicate SD (n=3)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
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- Parsed Citations
- Reviewer PDF
- ONE
- TWO
- THREE
- FOUR
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- SIX
- SEVEN
- T-ONE
- T-TWO
- Parsed Citations
-
D
b
a
a
a
0
2
4
6
8
col-0 vha-a2 vha-a3 avp1
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
Root
P-V
AtN
RT
15
rela
tive
ex
pres
sion
b
aa a
00
05
10
15
20
25
col-0 vha-a2 vha-a3 avp1
Root
AtNRT15 E
Root
A
a
bb
a
0
01
02
03
04V-ATPase V-PPase
μm
ol P
i m
g-1 p
rote
in h
-1
col-0 vha-a2 vha-a3 avp1
Root
avp1 vha-a3 vha-a2 col-0
B
NO
3- flu
x
(pm
ol c
m-2
s-1
)
a
b
b
c
-2000
-1500
-1000
-500
0
C
940
826780
791
0
15
30
45Vacuole Protoplast
μm
ol N
O3- μ
mol
p-
nitr
ophe
nol
col-0 vha-a2 vha-a3 avp1
Root
H
[NO
3- ] S
R r
atio
cb
b
a
00
25
50
col-0 vha-a2 vha-a3 avp1
G
c
bb
a
0
02
04
06
08
[15
N]
SR
rat
io
col-0 vha-a2 vha-a3 avp1
a
b b
c
0000
0005
0010
0015
0020
0025
col-0 vha-a2 vha-a3 avp1
AtN
RT
18
rela
tive
ex
pres
sion
AtNRT18 F
Root
Influx
Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of
NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)
Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-
a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model
plant materials Mature vacuoles were collected from the root tissues of the
plant materials at the seedling stage The NO3- flux measurement is
described in the legend to Figure 1 Protoplasts and vacuoles isolated from
roots of hydroponically grown plants were assessed for NO3- accumulation
plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to
Figure 1 and in Materials and Methods
Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in
root tissues from different A thaliana plant materials (A) Different letters
at the top of the histogram bars denote significant differences in V-ATPase
activities in root tissues between A thaliana plant materials (Plt005)
asterisk () at the top of the histogram bars denote significant differences
of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD
(n=6)
NO3- fluxes within the vacuoles of root tissues are shown for A thaliana
between different mutants (B) Different letters at the top of the histogram
bars denote significant differences in NO3- flux between Arabidopsis plant
materials (Plt005) Vertical bars indicate SD (n=6)
Accumulation of NO3- inside the vacuole and in the protoplasts of root
tissues is shown for A thaliana (C) Values above the bars represent the
percentage of vacuolar NO3- relative to the total NO3
- in protoplasts
Vertical bars indicate SD (n=6)
Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)
P-V is the total NO3- in the cytosol calculated as total NO3
- in protoplasts
ndash total NO3- in vacuoles Different letters at the top of the histogram bars
denote significant differences in total NO3-in the cytosol (Plt005) Vertical
bars indicate SD (n=6)
Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown
relative to that of the actin gene assessed by quantitative RT-PCR a value
of 10 is equivalent to levels of expression of the Atactin2 gene Different
letters at the top of the histogram bars denote significant differences in
gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)
Growth conditions for hydroponically grown plants with 15N treatmentare
described in Materials and Methods The [15N] SR ratios as affected by
inhibitor treatments are depicted for various A thaliana mutants (G) The
[NO3-] SR ratio as affected by inhibitor treatments are depicted for various
A thaliana mutants (H) Different letters at the top of the histogram bars
denote significant differences (Plt005) Vertical bars on the figures
indicate SD (n=3)
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsAndrews M (1986) The partitioning of nitrate assimilation between root and shoot of higher plants Plant Cell Environment 9511-519
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Angeli AD Monachello D Ephritikhine G Frachisse JM Thomine S Gambale F Barbier-Brygoo H (2006) The nitrateprotonantiporter AtCLCa mediates nitrate accumulation in plant vacuoles Nature 442 939-942
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Arteca RN Arteca JM (2000) A novel method for growing Arabidopsis thaliana plants hydroponically Physiol Plantarum 108 188-193Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brux A Liu TY Krebs M Stierhof YD Lohmann JU Miersch O Wasternack C Sschumacher K (2008) Reduced V-ATPPase activityin the trans-Golgi network causes oxylipin-dependent hypocotyl growth inhibition in arabidopsis The Plant Cell 20 1088-1100
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen CZ Lv XF Li JY Yi HY Gong JM (2012) Arabidopsis NRT15 is another essential component in the regulation of nitratereallocation and stress tolerance Plant Physiology 159 1582-1590
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen XP Cui ZL Fan MS Vitousek P Zhao M Ma WQ Wang ZL Zhang WJ Yan XY Yang JC Deng XP Gao Q Zhang Q Guo SWRen J Li SQ Ye YL Wang ZH Huang JL Tang QY Sun YX Peng XL Zhang JW He MR Zhu YJ Xue JQ Wang GL Wu L An N WuLQ Ma L Zhang WF Zhang FS (2014) Producing more grain with lower environmental costs Nature 514 486-489
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiu CC Lin CS Hsia AP Su RC Lin HL Tsay YF (2004) Mutation of a nitrate transporter AtNRT14 results in a reduced petiolenitrate content and altered leaf development Plant Cell Physiology 45 1139-1148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chopin F Orsel M Dorbe MF Chardon F Truong HN Miller AJ Krapp A Daniel-VedeleF (2007) The Arabidopsis ATNRT27 nitratetransporter controls nitrate content in seeds Plant Cell 19 1590-1602
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Daniel-Vedele F Filleur S CabocheM (1998) Nitrate transport a key step in nitrate assimilation Current Opinion in Plant Biology 1235-239
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dechorgnat J Nguyen CT Armengaud P Jossier M Diatloff E Filleur S Daniel-Vedele F (2011) From the soil to the seedsthe longjourney of nitrate in plants Journal of Experimental Botany 62 1349-1359
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Fan SC Lin CS Hsu PK Lin SH TsayYF (2009) The Arabidopsis nitrate transporter NRT17 expressed in phloem is responsiblefor source-to-sink remobilization of nitrate Plant Cell 212750-2761
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Fan XR Jia L Li Y Smith SJ Miller AJ Shen QR (2007) Comparing nitrate storage and remobilization in two rice cultivars thatdiffer in their nitrogen use efficiency Journal of Experimental Botany 58(7)1729-1740
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gaxiola RA Li JS Undurraga S Dang LM Allen GJ Alper SL Flink GR (2001) Drought and salt tolerant plants result from overexpression of the AVP1 H+-pump PNAS 98(20) 11444-11449
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Glass ADM Britto DT Kaiser BN Kinghorn JR Kronzucker HJ Kumar A Okamoto M Rawat S Siddiqi MY Unkles SE Vidmar JJ(2002) The regulation of nitrate and ammonium transport systems in plants Journal of Experimental Botany 53855-864
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gong JM Lee DA Schroeder JI (2003) Long-distance root-to-shoot transport of phytochelatins and cadmium in Arabidopsis PNAS100 10118-10123
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Granstedt RC Huffaker RC (1982) Identification of the leaf vacuole as a major nitrate storage pool Plant Physiology70410-413Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han YL Liao Q Yu Y Song HX Liu Q Rong XM Gu JD Lepo JE Guan CY Zhang ZH (2015a) Nitrate reutilization mechanisms inthe tonoplast of two Brassica napus genotypes with different nitrogen use efficiency Acta Physiologia Planturum 3742
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han YL Liu Q Gu JD Gong JM Guan CY Lepo JE Rong XR Song HX Zhang ZH (2015b) V-ATPase and V-PPase at the Tonoplastaffect NO3- Content in Brassica napus by Controlling Distribution of NO3- between the Cytoplasm and Vacuole Journal of PlantGrowth Regulation 2015 34 22-34
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harper AL Trick M Higgins J Fraser F Clissold L Wells R Hattori C Werner P Bancroft I (2012) Associative transcriptomics oftraits in the polyploidy crop species Brassica napus Nature Biotechnology 30 798-802
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huang J Zhang Y Peng JS Zhong C Yi HY Ow DW Gong JM (2012) Fission yeast HMT1 lowers seed Cadmium throughphytochelatin-dependent vacuolar sequestration in Arabidopsis Plant Physiology 158(4) 1779-1788
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krapp A David LC Chardin C Cirin T Marmagne A Leprince AC Chaillou S Ferrario-Mery S Meyer C Daniel-VedeleF (2014)Nitrate transport and signaling in Arabidopsis Journal of Experiment Botany 65789-798
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Krebs M Beyhl D Gorlich E Al-Rasheid KA Marten I Stierhof YD Hedrich R Schumacher K (2010) Arabidopsis V-ATPase activityat the tonoplast is required for efficient nutrient storage but not for sodium accumulation PNAS1073251-3256
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Lin SH Kou HF Canivenc G Lin CS Lepetit M Hsu PK Tillard P Lin HL Wang YY Tsai CB Gojon A Tsay YF (2008) Mutation ofthe Arabidopsis NRT15 nitrate transporter causes defective root-to-shoot nitrate transport The Plant Cell 202514-2528
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Li JY Fu YL Pike SM Bao J Tian W Zhang Y Chen CZ Zhang Y Li HM Huang J Li LG Schroeder J Gassmann W Gong JM(2010) The arabidopsis nitrate transporter NRT18 functions in nitrate removal from the xylem sap and mediates cadmiumtolerance The Plant Cell 22 1633-1646
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Luo JK Sun SB Jia LJ Chen W Shen QR (2006) The mechanism of nitrate accumulation in Pakchoi [BrassicacampestrisLsspChinensis (L)] Plant and Soil 282291-300
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Wang YY Tsay YF (2011) Arabidopsis nitrate transporter NRT19 is important in phloem nitrate transport The Plant Cell 231945-1957
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wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Reviewer PDF
- ONE
- TWO
- THREE
- FOUR
- FIVE
- SIX
- SEVEN
- T-ONE
- T-TWO
- Parsed Citations
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b
c
aa a a
0
10
20
30
a
b bb
0
1000
2000
3000
4000
b
a
0
10
20
30
NU
E b
ased
on
b
iom
ass
(gg
)
col-
0
nrt
15
-3
nrt
18
-2
Ws
vha
-a2
vha
-a3
avp
1
clca-2 N
UE
bas
ed o
n
bio
mas
s (g
g)
col-0 vha-a2 vha-a3 avp1
a
b
0
1000
2000
3000
4000
Ws clca-2
NO
3
- co
nce
ntr
atio
n
(μg
g-1
FW
)
NO
3
- co
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ntr
atio
n
(μg
g-1
FW
)
A B
C D
Figure 6 Increased NO3- shootsroots
ratio essentially contributed to enhancing
NUE in A thaliana
Plants grown hydroponically were
sampled for further analysis at seedling
stage and at harvest Conditions for
hydroponics culture and characteristics
of the various A thaliana genotypes are
defined in Materials and Methods The
wild-type Columbia-0 (col-0) plants
were used as control for V-ATPase
mutants (vha-a2 and vha-a3) V-PPase
mutants (avp1) nrt15-3 mutants and
nrt18-2 mutants A thaliana wild-type
Wassilewskija (Ws) plants were used as
control for clca-2 mutants
Differences in NUE based on biomass
(A) and NO3- concentration (C) are
shown for the wild-type (col-0) and
mutants nrt15-3 nrt18-2 vha-a2 vha-
a3 and avp1
Differences of NUE based on biomass
(B) and NO3-concentration (D) are
shown between A thaliana wild-type
plants Ws and clca-2 Different letters at
the top of the histogram bars denote
significant differences between the
Arabidopsis genotypes (Plt005) Vertical
bars on the figures indicate SD (n=6) wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsAndrews M (1986) The partitioning of nitrate assimilation between root and shoot of higher plants Plant Cell Environment 9511-519
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Angeli AD Monachello D Ephritikhine G Frachisse JM Thomine S Gambale F Barbier-Brygoo H (2006) The nitrateprotonantiporter AtCLCa mediates nitrate accumulation in plant vacuoles Nature 442 939-942
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Arteca RN Arteca JM (2000) A novel method for growing Arabidopsis thaliana plants hydroponically Physiol Plantarum 108 188-193Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brux A Liu TY Krebs M Stierhof YD Lohmann JU Miersch O Wasternack C Sschumacher K (2008) Reduced V-ATPPase activityin the trans-Golgi network causes oxylipin-dependent hypocotyl growth inhibition in arabidopsis The Plant Cell 20 1088-1100
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen CZ Lv XF Li JY Yi HY Gong JM (2012) Arabidopsis NRT15 is another essential component in the regulation of nitratereallocation and stress tolerance Plant Physiology 159 1582-1590
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen XP Cui ZL Fan MS Vitousek P Zhao M Ma WQ Wang ZL Zhang WJ Yan XY Yang JC Deng XP Gao Q Zhang Q Guo SWRen J Li SQ Ye YL Wang ZH Huang JL Tang QY Sun YX Peng XL Zhang JW He MR Zhu YJ Xue JQ Wang GL Wu L An N WuLQ Ma L Zhang WF Zhang FS (2014) Producing more grain with lower environmental costs Nature 514 486-489
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiu CC Lin CS Hsia AP Su RC Lin HL Tsay YF (2004) Mutation of a nitrate transporter AtNRT14 results in a reduced petiolenitrate content and altered leaf development Plant Cell Physiology 45 1139-1148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chopin F Orsel M Dorbe MF Chardon F Truong HN Miller AJ Krapp A Daniel-VedeleF (2007) The Arabidopsis ATNRT27 nitratetransporter controls nitrate content in seeds Plant Cell 19 1590-1602
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Daniel-Vedele F Filleur S CabocheM (1998) Nitrate transport a key step in nitrate assimilation Current Opinion in Plant Biology 1235-239
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dechorgnat J Nguyen CT Armengaud P Jossier M Diatloff E Filleur S Daniel-Vedele F (2011) From the soil to the seedsthe longjourney of nitrate in plants Journal of Experimental Botany 62 1349-1359
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Fan SC Lin CS Hsu PK Lin SH TsayYF (2009) The Arabidopsis nitrate transporter NRT17 expressed in phloem is responsiblefor source-to-sink remobilization of nitrate Plant Cell 212750-2761
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Fan XR Jia L Li Y Smith SJ Miller AJ Shen QR (2007) Comparing nitrate storage and remobilization in two rice cultivars thatdiffer in their nitrogen use efficiency Journal of Experimental Botany 58(7)1729-1740
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gaxiola RA Li JS Undurraga S Dang LM Allen GJ Alper SL Flink GR (2001) Drought and salt tolerant plants result from overexpression of the AVP1 H+-pump PNAS 98(20) 11444-11449
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Glass ADM Britto DT Kaiser BN Kinghorn JR Kronzucker HJ Kumar A Okamoto M Rawat S Siddiqi MY Unkles SE Vidmar JJ(2002) The regulation of nitrate and ammonium transport systems in plants Journal of Experimental Botany 53855-864
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gong JM Lee DA Schroeder JI (2003) Long-distance root-to-shoot transport of phytochelatins and cadmium in Arabidopsis PNAS100 10118-10123
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Granstedt RC Huffaker RC (1982) Identification of the leaf vacuole as a major nitrate storage pool Plant Physiology70410-413Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han YL Liao Q Yu Y Song HX Liu Q Rong XM Gu JD Lepo JE Guan CY Zhang ZH (2015a) Nitrate reutilization mechanisms inthe tonoplast of two Brassica napus genotypes with different nitrogen use efficiency Acta Physiologia Planturum 3742
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han YL Liu Q Gu JD Gong JM Guan CY Lepo JE Rong XR Song HX Zhang ZH (2015b) V-ATPase and V-PPase at the Tonoplastaffect NO3- Content in Brassica napus by Controlling Distribution of NO3- between the Cytoplasm and Vacuole Journal of PlantGrowth Regulation 2015 34 22-34
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harper AL Trick M Higgins J Fraser F Clissold L Wells R Hattori C Werner P Bancroft I (2012) Associative transcriptomics oftraits in the polyploidy crop species Brassica napus Nature Biotechnology 30 798-802
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huang J Zhang Y Peng JS Zhong C Yi HY Ow DW Gong JM (2012) Fission yeast HMT1 lowers seed Cadmium throughphytochelatin-dependent vacuolar sequestration in Arabidopsis Plant Physiology 158(4) 1779-1788
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krapp A David LC Chardin C Cirin T Marmagne A Leprince AC Chaillou S Ferrario-Mery S Meyer C Daniel-VedeleF (2014)Nitrate transport and signaling in Arabidopsis Journal of Experiment Botany 65789-798
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krebs M Beyhl D Gorlich E Al-Rasheid KA Marten I Stierhof YD Hedrich R Schumacher K (2010) Arabidopsis V-ATPase activityat the tonoplast is required for efficient nutrient storage but not for sodium accumulation PNAS1073251-3256
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leran S Varala K Boyer JC Chiurazzi M Crawford N Daniel-Vedele J David L Rebecca D (2014) A unified nomenclature ofNITRATE TRANSPORTER 1PEPTIDE TRANSPORTER family members in plants Trends in Plant Science 19(1) 5-9
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lin SH Kou HF Canivenc G Lin CS Lepetit M Hsu PK Tillard P Lin HL Wang YY Tsai CB Gojon A Tsay YF (2008) Mutation ofthe Arabidopsis NRT15 nitrate transporter causes defective root-to-shoot nitrate transport The Plant Cell 202514-2528
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li JY Fu YL Pike SM Bao J Tian W Zhang Y Chen CZ Zhang Y Li HM Huang J Li LG Schroeder J Gassmann W Gong JM(2010) The arabidopsis nitrate transporter NRT18 functions in nitrate removal from the xylem sap and mediates cadmiumtolerance The Plant Cell 22 1633-1646
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li JS Yang HB Peer WA Richter G Blakeslee J Bandyopadhyay A Titapiwantakun B Undurraga S Khodakovskaya M RichardsE Krizek B Murphy AS Gilroy S Gaxiola R (2005) Arabidopsis H+-PPase AVP1 regulates auxin-mediated organ developmentScience 310 (121) 121-125
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Luo JK Sun SB Jia LJ Chen W Shen QR (2006) The mechanism of nitrate accumulation in Pakchoi [BrassicacampestrisLsspChinensis (L)] Plant and Soil 282291-300
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ma JF Ueno D Zhao FJ McGrath SP (2005) Subcellular localization of Cd and Zn in the leaves of a Cd-hyperaccumulating ecotypeof ThlaspicaerulescensPlanta 220 731-736
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Martinoia E Massonneau A Frangne N (2000) Transport processes of solutes across the vacuolar membrane of higher plantsPlant Cell Physiology 41(11)1175-1186
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Martinoia E Heck U Wiemken A (1981) Vacuoles as storage compartments for nitrate in barley leaves Nature 289 292-294Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Miller AJ Smith SJ (2008) Cytosolic nitrate ion homeostasis could it have a role in sensing nitrogen status Annals ofBotany101485-489
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Robert S Zouhar J Carter C Raikhel N (2007) Isolation of intact vacuoles from Arabidopsis rosette leaf-derived protoplastsNature Protocols 2(2)259-262
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schroeder JL Delhaize E Frommer WB Guerinot ML Harrison MJ Herrera-Estrella L Horie T Kochian LV Munns R NishizawaNK Tsay YF Sanders D (2013) Using membrane transporters to improve crops for sustainable food production Nature 497 60-66
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi WM Xu WF Li SM Zhao XQ Dong GQ (2010) Responses of two rice cultivars differing in seedling-stage nitrogen useefficiency to growth under low-nitrogen conditions Plant and Soil 326291-302
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shen QR Tang L Xu YC (2003) A review on the behavior of nitrate in vacuoles of plants ActaPedologicaSinica40(3)465-470Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Smirnoff N Stewart G (1985) Nitrate assimilation and translocation by higher plants Comparative physiology and ecologicalconsequences Physiologia Plantarum 64 133-140
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang TF Sun XC Hu CX Tan QL Zhao XH (2013) Genotypic differences in nitrate uptake translocation and assimilation of twoChinese cabbage cultivars [Brassica campestris L ssp Chinesnsis (L)] Plant Physiology and Biochemistry7014-20
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang Z Fan XR Li Qing Feng HM Miller AJ Shen QR Xu GH (2012) Knockdown of a rice stellar nitrate transporter alters long-distance translocation but not root influx Plant Physiology 1602052-2063
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Vogeli-Lange R Wagner GJ (1990) Subcellular localization of cadmium and cadmium-binding peptides in tobacco leavesimplication of a transport function for cadmium-binding peptides Plant Physiology 921086-1093
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang GL Ding GD Li L Cai HM Ye XS Zou J Xu FS (2014) Identification and characterization of improved nitrogen efficiency ininterspecific hybridized new-type Brassica napus Annals of Botany 114(3) 549-559
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Wang YY Tsay YF (2011) Arabidopsis nitrate transporter NRT19 is important in phloem nitrate transport The Plant Cell 231945-1957
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wege S Angeli AD Droillard MJ Kroniewicz L Merlot S Cornu D Gambale F Martinoia E Barbier-Brygoo H Thomine SLeonhardt N Filleur S (2014) Phosphorylation of the vacuolar anion exchanger AtCLCa is required for the stomatal response toabscisic acid Science Signalling 7(333) ra65
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wilkinson S Bacon MA Davies WJ (2007) Nitrate signaling to stomatal and growing leaves interactions with soil drying ABA andxylem sap pH in maize Journal of Experimental Botany 58(7) 1705-1716
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xu GH Fan XR Miller AJ (2012) Plant nitrogen assimilation and use efficiency The Annual Review of Plant Biology 63153-182Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yandeau-Nelson MD Laurens L Shi Z Xia H Smith AM Guiltinan MJ (2011) Starch-Branching Enzyme IIa Is Required for ProperDiurnal Cycling of Starch in Leaves of Maize [OA] Plant Physiology 156479-490
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang GB Yi HY Gong JM (2014) The Arabidopsis EthyleneJasmonic acid-NRT signaling module coordinates nitrate reallocationand the trade-off between growth and environmental adaption The Plant Cell 26(10)3984-3998
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Song HX Liu Qing Rong XM Xie GX Peng JW Zhang YP (2009) Study on differences of nitrogen efficiency andnitrogen response in different oilseed rape (Brassica napus L) varieties Asian Journal of Crop Science 1(2)105-112
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2010) Nitrogen redistribution characteristics ofoilseed rape varieties with different nitrogen use efficiencies during later growth period Communications in Soil Science andPlant Analysis 41(14) 1693-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Huang HT Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2012) Research advances on nitratenitrogen reutilization by proton pump of tonoplast and its relation to nitrogen use efficiency Australian Journal of Crop Science6(9) 1377-1382
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhu ZJ Qian YR Pfeiffer W (2001) Effect of nitrogen form on the activity of tonoplast pyrophosphatase in tomato roots ActaBotanica Sinica 43(11) 1146-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Reviewer PDF
- ONE
- TWO
- THREE
- FOUR
- FIVE
- SIX
- SEVEN
- T-ONE
- T-TWO
- Parsed Citations
-
Vascular tissues
Vacuole
Cytoplasm
V-PPase
H+
V-ATPase
1 H+
CLCa
2 NO3- 2 NO3
-
Xy
lem
NO
3-
Ph
loem
NRT18
Unloading
NO3-
NRT15
Loading
NO3-
Shoot
Root
En
erg
y
Figure 7 Simplified model for NO3- long-distance transport in xylem of
vascular tissues
Long-distance transport is regulated by NO3- distribution between the vacuole
and the cytosol in root tissues Red lines display the route for regulation
pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsAndrews M (1986) The partitioning of nitrate assimilation between root and shoot of higher plants Plant Cell Environment 9511-519
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Angeli AD Monachello D Ephritikhine G Frachisse JM Thomine S Gambale F Barbier-Brygoo H (2006) The nitrateprotonantiporter AtCLCa mediates nitrate accumulation in plant vacuoles Nature 442 939-942
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Arteca RN Arteca JM (2000) A novel method for growing Arabidopsis thaliana plants hydroponically Physiol Plantarum 108 188-193Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brux A Liu TY Krebs M Stierhof YD Lohmann JU Miersch O Wasternack C Sschumacher K (2008) Reduced V-ATPPase activityin the trans-Golgi network causes oxylipin-dependent hypocotyl growth inhibition in arabidopsis The Plant Cell 20 1088-1100
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen CZ Lv XF Li JY Yi HY Gong JM (2012) Arabidopsis NRT15 is another essential component in the regulation of nitratereallocation and stress tolerance Plant Physiology 159 1582-1590
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen XP Cui ZL Fan MS Vitousek P Zhao M Ma WQ Wang ZL Zhang WJ Yan XY Yang JC Deng XP Gao Q Zhang Q Guo SWRen J Li SQ Ye YL Wang ZH Huang JL Tang QY Sun YX Peng XL Zhang JW He MR Zhu YJ Xue JQ Wang GL Wu L An N WuLQ Ma L Zhang WF Zhang FS (2014) Producing more grain with lower environmental costs Nature 514 486-489
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiu CC Lin CS Hsia AP Su RC Lin HL Tsay YF (2004) Mutation of a nitrate transporter AtNRT14 results in a reduced petiolenitrate content and altered leaf development Plant Cell Physiology 45 1139-1148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chopin F Orsel M Dorbe MF Chardon F Truong HN Miller AJ Krapp A Daniel-VedeleF (2007) The Arabidopsis ATNRT27 nitratetransporter controls nitrate content in seeds Plant Cell 19 1590-1602
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Daniel-Vedele F Filleur S CabocheM (1998) Nitrate transport a key step in nitrate assimilation Current Opinion in Plant Biology 1235-239
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dechorgnat J Nguyen CT Armengaud P Jossier M Diatloff E Filleur S Daniel-Vedele F (2011) From the soil to the seedsthe longjourney of nitrate in plants Journal of Experimental Botany 62 1349-1359
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Fan SC Lin CS Hsu PK Lin SH TsayYF (2009) The Arabidopsis nitrate transporter NRT17 expressed in phloem is responsiblefor source-to-sink remobilization of nitrate Plant Cell 212750-2761
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Fan XR Jia L Li Y Smith SJ Miller AJ Shen QR (2007) Comparing nitrate storage and remobilization in two rice cultivars thatdiffer in their nitrogen use efficiency Journal of Experimental Botany 58(7)1729-1740
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gaxiola RA Li JS Undurraga S Dang LM Allen GJ Alper SL Flink GR (2001) Drought and salt tolerant plants result from overexpression of the AVP1 H+-pump PNAS 98(20) 11444-11449
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Glass ADM Britto DT Kaiser BN Kinghorn JR Kronzucker HJ Kumar A Okamoto M Rawat S Siddiqi MY Unkles SE Vidmar JJ(2002) The regulation of nitrate and ammonium transport systems in plants Journal of Experimental Botany 53855-864
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gong JM Lee DA Schroeder JI (2003) Long-distance root-to-shoot transport of phytochelatins and cadmium in Arabidopsis PNAS100 10118-10123
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Granstedt RC Huffaker RC (1982) Identification of the leaf vacuole as a major nitrate storage pool Plant Physiology70410-413Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han YL Liao Q Yu Y Song HX Liu Q Rong XM Gu JD Lepo JE Guan CY Zhang ZH (2015a) Nitrate reutilization mechanisms inthe tonoplast of two Brassica napus genotypes with different nitrogen use efficiency Acta Physiologia Planturum 3742
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han YL Liu Q Gu JD Gong JM Guan CY Lepo JE Rong XR Song HX Zhang ZH (2015b) V-ATPase and V-PPase at the Tonoplastaffect NO3- Content in Brassica napus by Controlling Distribution of NO3- between the Cytoplasm and Vacuole Journal of PlantGrowth Regulation 2015 34 22-34
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harper AL Trick M Higgins J Fraser F Clissold L Wells R Hattori C Werner P Bancroft I (2012) Associative transcriptomics oftraits in the polyploidy crop species Brassica napus Nature Biotechnology 30 798-802
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huang J Zhang Y Peng JS Zhong C Yi HY Ow DW Gong JM (2012) Fission yeast HMT1 lowers seed Cadmium throughphytochelatin-dependent vacuolar sequestration in Arabidopsis Plant Physiology 158(4) 1779-1788
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krapp A David LC Chardin C Cirin T Marmagne A Leprince AC Chaillou S Ferrario-Mery S Meyer C Daniel-VedeleF (2014)Nitrate transport and signaling in Arabidopsis Journal of Experiment Botany 65789-798
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krebs M Beyhl D Gorlich E Al-Rasheid KA Marten I Stierhof YD Hedrich R Schumacher K (2010) Arabidopsis V-ATPase activityat the tonoplast is required for efficient nutrient storage but not for sodium accumulation PNAS1073251-3256
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leran S Varala K Boyer JC Chiurazzi M Crawford N Daniel-Vedele J David L Rebecca D (2014) A unified nomenclature ofNITRATE TRANSPORTER 1PEPTIDE TRANSPORTER family members in plants Trends in Plant Science 19(1) 5-9
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lin SH Kou HF Canivenc G Lin CS Lepetit M Hsu PK Tillard P Lin HL Wang YY Tsai CB Gojon A Tsay YF (2008) Mutation ofthe Arabidopsis NRT15 nitrate transporter causes defective root-to-shoot nitrate transport The Plant Cell 202514-2528
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li JY Fu YL Pike SM Bao J Tian W Zhang Y Chen CZ Zhang Y Li HM Huang J Li LG Schroeder J Gassmann W Gong JM(2010) The arabidopsis nitrate transporter NRT18 functions in nitrate removal from the xylem sap and mediates cadmiumtolerance The Plant Cell 22 1633-1646
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li JS Yang HB Peer WA Richter G Blakeslee J Bandyopadhyay A Titapiwantakun B Undurraga S Khodakovskaya M RichardsE Krizek B Murphy AS Gilroy S Gaxiola R (2005) Arabidopsis H+-PPase AVP1 regulates auxin-mediated organ developmentScience 310 (121) 121-125
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Luo JK Sun SB Jia LJ Chen W Shen QR (2006) The mechanism of nitrate accumulation in Pakchoi [BrassicacampestrisLsspChinensis (L)] Plant and Soil 282291-300
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ma JF Ueno D Zhao FJ McGrath SP (2005) Subcellular localization of Cd and Zn in the leaves of a Cd-hyperaccumulating ecotypeof ThlaspicaerulescensPlanta 220 731-736
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Martinoia E Massonneau A Frangne N (2000) Transport processes of solutes across the vacuolar membrane of higher plantsPlant Cell Physiology 41(11)1175-1186
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Martinoia E Heck U Wiemken A (1981) Vacuoles as storage compartments for nitrate in barley leaves Nature 289 292-294Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Miller AJ Smith SJ (2008) Cytosolic nitrate ion homeostasis could it have a role in sensing nitrogen status Annals ofBotany101485-489
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Robert S Zouhar J Carter C Raikhel N (2007) Isolation of intact vacuoles from Arabidopsis rosette leaf-derived protoplastsNature Protocols 2(2)259-262
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schroeder JL Delhaize E Frommer WB Guerinot ML Harrison MJ Herrera-Estrella L Horie T Kochian LV Munns R NishizawaNK Tsay YF Sanders D (2013) Using membrane transporters to improve crops for sustainable food production Nature 497 60-66
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi WM Xu WF Li SM Zhao XQ Dong GQ (2010) Responses of two rice cultivars differing in seedling-stage nitrogen useefficiency to growth under low-nitrogen conditions Plant and Soil 326291-302
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shen QR Tang L Xu YC (2003) A review on the behavior of nitrate in vacuoles of plants ActaPedologicaSinica40(3)465-470Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Smirnoff N Stewart G (1985) Nitrate assimilation and translocation by higher plants Comparative physiology and ecologicalconsequences Physiologia Plantarum 64 133-140
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang TF Sun XC Hu CX Tan QL Zhao XH (2013) Genotypic differences in nitrate uptake translocation and assimilation of twoChinese cabbage cultivars [Brassica campestris L ssp Chinesnsis (L)] Plant Physiology and Biochemistry7014-20
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang Z Fan XR Li Qing Feng HM Miller AJ Shen QR Xu GH (2012) Knockdown of a rice stellar nitrate transporter alters long-distance translocation but not root influx Plant Physiology 1602052-2063
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Vogeli-Lange R Wagner GJ (1990) Subcellular localization of cadmium and cadmium-binding peptides in tobacco leavesimplication of a transport function for cadmium-binding peptides Plant Physiology 921086-1093
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang GL Ding GD Li L Cai HM Ye XS Zou J Xu FS (2014) Identification and characterization of improved nitrogen efficiency ininterspecific hybridized new-type Brassica napus Annals of Botany 114(3) 549-559
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Wang YY Tsay YF (2011) Arabidopsis nitrate transporter NRT19 is important in phloem nitrate transport The Plant Cell 231945-1957
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wege S Angeli AD Droillard MJ Kroniewicz L Merlot S Cornu D Gambale F Martinoia E Barbier-Brygoo H Thomine SLeonhardt N Filleur S (2014) Phosphorylation of the vacuolar anion exchanger AtCLCa is required for the stomatal response toabscisic acid Science Signalling 7(333) ra65
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wilkinson S Bacon MA Davies WJ (2007) Nitrate signaling to stomatal and growing leaves interactions with soil drying ABA andxylem sap pH in maize Journal of Experimental Botany 58(7) 1705-1716
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xu GH Fan XR Miller AJ (2012) Plant nitrogen assimilation and use efficiency The Annual Review of Plant Biology 63153-182Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yandeau-Nelson MD Laurens L Shi Z Xia H Smith AM Guiltinan MJ (2011) Starch-Branching Enzyme IIa Is Required for ProperDiurnal Cycling of Starch in Leaves of Maize [OA] Plant Physiology 156479-490
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang GB Yi HY Gong JM (2014) The Arabidopsis EthyleneJasmonic acid-NRT signaling module coordinates nitrate reallocationand the trade-off between growth and environmental adaption The Plant Cell 26(10)3984-3998
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Song HX Liu Qing Rong XM Xie GX Peng JW Zhang YP (2009) Study on differences of nitrogen efficiency andnitrogen response in different oilseed rape (Brassica napus L) varieties Asian Journal of Crop Science 1(2)105-112
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2010) Nitrogen redistribution characteristics ofoilseed rape varieties with different nitrogen use efficiencies during later growth period Communications in Soil Science andPlant Analysis 41(14) 1693-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Huang HT Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2012) Research advances on nitratenitrogen reutilization by proton pump of tonoplast and its relation to nitrogen use efficiency Australian Journal of Crop Science6(9) 1377-1382
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhu ZJ Qian YR Pfeiffer W (2001) Effect of nitrogen form on the activity of tonoplast pyrophosphatase in tomato roots ActaBotanica Sinica 43(11) 1146-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Reviewer PDF
- ONE
- TWO
- THREE
- FOUR
- FIVE
- SIX
- SEVEN
- T-ONE
- T-TWO
- Parsed Citations
-
Table 1 Comparison of NUE between the two B napus genotype
aNUE values were calculated based on either total biomass (shoot root and grains) per total N
uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype
(814)
Different letters associated with data represent significant differences at P<005 n=3
bGenotypes
Physiological parameters aNUE
Biomass
(g plant-1)
Grain yield
(g plant-1)
Total N in plant
(g plant-1)
Based on biomass
(gg-1)
Based on grain yield
(gg-1)
H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a
L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsAndrews M (1986) The partitioning of nitrate assimilation between root and shoot of higher plants Plant Cell Environment 9511-519
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Angeli AD Monachello D Ephritikhine G Frachisse JM Thomine S Gambale F Barbier-Brygoo H (2006) The nitrateprotonantiporter AtCLCa mediates nitrate accumulation in plant vacuoles Nature 442 939-942
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Arteca RN Arteca JM (2000) A novel method for growing Arabidopsis thaliana plants hydroponically Physiol Plantarum 108 188-193Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brux A Liu TY Krebs M Stierhof YD Lohmann JU Miersch O Wasternack C Sschumacher K (2008) Reduced V-ATPPase activityin the trans-Golgi network causes oxylipin-dependent hypocotyl growth inhibition in arabidopsis The Plant Cell 20 1088-1100
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen CZ Lv XF Li JY Yi HY Gong JM (2012) Arabidopsis NRT15 is another essential component in the regulation of nitratereallocation and stress tolerance Plant Physiology 159 1582-1590
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen XP Cui ZL Fan MS Vitousek P Zhao M Ma WQ Wang ZL Zhang WJ Yan XY Yang JC Deng XP Gao Q Zhang Q Guo SWRen J Li SQ Ye YL Wang ZH Huang JL Tang QY Sun YX Peng XL Zhang JW He MR Zhu YJ Xue JQ Wang GL Wu L An N WuLQ Ma L Zhang WF Zhang FS (2014) Producing more grain with lower environmental costs Nature 514 486-489
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiu CC Lin CS Hsia AP Su RC Lin HL Tsay YF (2004) Mutation of a nitrate transporter AtNRT14 results in a reduced petiolenitrate content and altered leaf development Plant Cell Physiology 45 1139-1148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chopin F Orsel M Dorbe MF Chardon F Truong HN Miller AJ Krapp A Daniel-VedeleF (2007) The Arabidopsis ATNRT27 nitratetransporter controls nitrate content in seeds Plant Cell 19 1590-1602
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Daniel-Vedele F Filleur S CabocheM (1998) Nitrate transport a key step in nitrate assimilation Current Opinion in Plant Biology 1235-239
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dechorgnat J Nguyen CT Armengaud P Jossier M Diatloff E Filleur S Daniel-Vedele F (2011) From the soil to the seedsthe longjourney of nitrate in plants Journal of Experimental Botany 62 1349-1359
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Fan SC Lin CS Hsu PK Lin SH TsayYF (2009) The Arabidopsis nitrate transporter NRT17 expressed in phloem is responsiblefor source-to-sink remobilization of nitrate Plant Cell 212750-2761
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Fan XR Jia L Li Y Smith SJ Miller AJ Shen QR (2007) Comparing nitrate storage and remobilization in two rice cultivars thatdiffer in their nitrogen use efficiency Journal of Experimental Botany 58(7)1729-1740
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gaxiola RA Li JS Undurraga S Dang LM Allen GJ Alper SL Flink GR (2001) Drought and salt tolerant plants result from overexpression of the AVP1 H+-pump PNAS 98(20) 11444-11449
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Glass ADM Britto DT Kaiser BN Kinghorn JR Kronzucker HJ Kumar A Okamoto M Rawat S Siddiqi MY Unkles SE Vidmar JJ(2002) The regulation of nitrate and ammonium transport systems in plants Journal of Experimental Botany 53855-864
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gong JM Lee DA Schroeder JI (2003) Long-distance root-to-shoot transport of phytochelatins and cadmium in Arabidopsis PNAS100 10118-10123
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Granstedt RC Huffaker RC (1982) Identification of the leaf vacuole as a major nitrate storage pool Plant Physiology70410-413Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han YL Liao Q Yu Y Song HX Liu Q Rong XM Gu JD Lepo JE Guan CY Zhang ZH (2015a) Nitrate reutilization mechanisms inthe tonoplast of two Brassica napus genotypes with different nitrogen use efficiency Acta Physiologia Planturum 3742
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han YL Liu Q Gu JD Gong JM Guan CY Lepo JE Rong XR Song HX Zhang ZH (2015b) V-ATPase and V-PPase at the Tonoplastaffect NO3- Content in Brassica napus by Controlling Distribution of NO3- between the Cytoplasm and Vacuole Journal of PlantGrowth Regulation 2015 34 22-34
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harper AL Trick M Higgins J Fraser F Clissold L Wells R Hattori C Werner P Bancroft I (2012) Associative transcriptomics oftraits in the polyploidy crop species Brassica napus Nature Biotechnology 30 798-802
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huang J Zhang Y Peng JS Zhong C Yi HY Ow DW Gong JM (2012) Fission yeast HMT1 lowers seed Cadmium throughphytochelatin-dependent vacuolar sequestration in Arabidopsis Plant Physiology 158(4) 1779-1788
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krapp A David LC Chardin C Cirin T Marmagne A Leprince AC Chaillou S Ferrario-Mery S Meyer C Daniel-VedeleF (2014)Nitrate transport and signaling in Arabidopsis Journal of Experiment Botany 65789-798
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krebs M Beyhl D Gorlich E Al-Rasheid KA Marten I Stierhof YD Hedrich R Schumacher K (2010) Arabidopsis V-ATPase activityat the tonoplast is required for efficient nutrient storage but not for sodium accumulation PNAS1073251-3256
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leran S Varala K Boyer JC Chiurazzi M Crawford N Daniel-Vedele J David L Rebecca D (2014) A unified nomenclature ofNITRATE TRANSPORTER 1PEPTIDE TRANSPORTER family members in plants Trends in Plant Science 19(1) 5-9
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lin SH Kou HF Canivenc G Lin CS Lepetit M Hsu PK Tillard P Lin HL Wang YY Tsai CB Gojon A Tsay YF (2008) Mutation ofthe Arabidopsis NRT15 nitrate transporter causes defective root-to-shoot nitrate transport The Plant Cell 202514-2528
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li JY Fu YL Pike SM Bao J Tian W Zhang Y Chen CZ Zhang Y Li HM Huang J Li LG Schroeder J Gassmann W Gong JM(2010) The arabidopsis nitrate transporter NRT18 functions in nitrate removal from the xylem sap and mediates cadmiumtolerance The Plant Cell 22 1633-1646
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li JS Yang HB Peer WA Richter G Blakeslee J Bandyopadhyay A Titapiwantakun B Undurraga S Khodakovskaya M RichardsE Krizek B Murphy AS Gilroy S Gaxiola R (2005) Arabidopsis H+-PPase AVP1 regulates auxin-mediated organ developmentScience 310 (121) 121-125
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Luo JK Sun SB Jia LJ Chen W Shen QR (2006) The mechanism of nitrate accumulation in Pakchoi [BrassicacampestrisLsspChinensis (L)] Plant and Soil 282291-300
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ma JF Ueno D Zhao FJ McGrath SP (2005) Subcellular localization of Cd and Zn in the leaves of a Cd-hyperaccumulating ecotypeof ThlaspicaerulescensPlanta 220 731-736
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Martinoia E Massonneau A Frangne N (2000) Transport processes of solutes across the vacuolar membrane of higher plantsPlant Cell Physiology 41(11)1175-1186
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Martinoia E Heck U Wiemken A (1981) Vacuoles as storage compartments for nitrate in barley leaves Nature 289 292-294Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Miller AJ Smith SJ (2008) Cytosolic nitrate ion homeostasis could it have a role in sensing nitrogen status Annals ofBotany101485-489
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Robert S Zouhar J Carter C Raikhel N (2007) Isolation of intact vacuoles from Arabidopsis rosette leaf-derived protoplastsNature Protocols 2(2)259-262
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schroeder JL Delhaize E Frommer WB Guerinot ML Harrison MJ Herrera-Estrella L Horie T Kochian LV Munns R NishizawaNK Tsay YF Sanders D (2013) Using membrane transporters to improve crops for sustainable food production Nature 497 60-66
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi WM Xu WF Li SM Zhao XQ Dong GQ (2010) Responses of two rice cultivars differing in seedling-stage nitrogen useefficiency to growth under low-nitrogen conditions Plant and Soil 326291-302
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shen QR Tang L Xu YC (2003) A review on the behavior of nitrate in vacuoles of plants ActaPedologicaSinica40(3)465-470Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Smirnoff N Stewart G (1985) Nitrate assimilation and translocation by higher plants Comparative physiology and ecologicalconsequences Physiologia Plantarum 64 133-140
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang TF Sun XC Hu CX Tan QL Zhao XH (2013) Genotypic differences in nitrate uptake translocation and assimilation of twoChinese cabbage cultivars [Brassica campestris L ssp Chinesnsis (L)] Plant Physiology and Biochemistry7014-20
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang Z Fan XR Li Qing Feng HM Miller AJ Shen QR Xu GH (2012) Knockdown of a rice stellar nitrate transporter alters long-distance translocation but not root influx Plant Physiology 1602052-2063
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Vogeli-Lange R Wagner GJ (1990) Subcellular localization of cadmium and cadmium-binding peptides in tobacco leavesimplication of a transport function for cadmium-binding peptides Plant Physiology 921086-1093
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang GL Ding GD Li L Cai HM Ye XS Zou J Xu FS (2014) Identification and characterization of improved nitrogen efficiency ininterspecific hybridized new-type Brassica napus Annals of Botany 114(3) 549-559
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Wang YY Tsay YF (2011) Arabidopsis nitrate transporter NRT19 is important in phloem nitrate transport The Plant Cell 231945-1957
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wege S Angeli AD Droillard MJ Kroniewicz L Merlot S Cornu D Gambale F Martinoia E Barbier-Brygoo H Thomine SLeonhardt N Filleur S (2014) Phosphorylation of the vacuolar anion exchanger AtCLCa is required for the stomatal response toabscisic acid Science Signalling 7(333) ra65
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wilkinson S Bacon MA Davies WJ (2007) Nitrate signaling to stomatal and growing leaves interactions with soil drying ABA andxylem sap pH in maize Journal of Experimental Botany 58(7) 1705-1716
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xu GH Fan XR Miller AJ (2012) Plant nitrogen assimilation and use efficiency The Annual Review of Plant Biology 63153-182Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yandeau-Nelson MD Laurens L Shi Z Xia H Smith AM Guiltinan MJ (2011) Starch-Branching Enzyme IIa Is Required for ProperDiurnal Cycling of Starch in Leaves of Maize [OA] Plant Physiology 156479-490
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang GB Yi HY Gong JM (2014) The Arabidopsis EthyleneJasmonic acid-NRT signaling module coordinates nitrate reallocationand the trade-off between growth and environmental adaption The Plant Cell 26(10)3984-3998
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Song HX Liu Qing Rong XM Xie GX Peng JW Zhang YP (2009) Study on differences of nitrogen efficiency andnitrogen response in different oilseed rape (Brassica napus L) varieties Asian Journal of Crop Science 1(2)105-112
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2010) Nitrogen redistribution characteristics ofoilseed rape varieties with different nitrogen use efficiencies during later growth period Communications in Soil Science andPlant Analysis 41(14) 1693-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Huang HT Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2012) Research advances on nitratenitrogen reutilization by proton pump of tonoplast and its relation to nitrogen use efficiency Australian Journal of Crop Science6(9) 1377-1382
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhu ZJ Qian YR Pfeiffer W (2001) Effect of nitrogen form on the activity of tonoplast pyrophosphatase in tomato roots ActaBotanica Sinica 43(11) 1146-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Reviewer PDF
- ONE
- TWO
- THREE
- FOUR
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- T-TWO
- Parsed Citations
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Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate
between high (H) and low (L) nitrogen use efficiency genotypes of B napus
aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at
1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the
4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering
Different letters between the H and L genotypes denote a significant difference at Plt005 level
Data are means plusmnSD (n=6)
Genotypes
Seedling stage Flowering stage
Chlorophyll
contenta(SP
AD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
Chlorophyll
content (SPAD
readings)
Intercellular
CO2
concentration
(micromol
CO2mol-1)
Photosynthetic
rate (micromol
CO2 m-2 s-1)
H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a
L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsAndrews M (1986) The partitioning of nitrate assimilation between root and shoot of higher plants Plant Cell Environment 9511-519
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Angeli AD Monachello D Ephritikhine G Frachisse JM Thomine S Gambale F Barbier-Brygoo H (2006) The nitrateprotonantiporter AtCLCa mediates nitrate accumulation in plant vacuoles Nature 442 939-942
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Arteca RN Arteca JM (2000) A novel method for growing Arabidopsis thaliana plants hydroponically Physiol Plantarum 108 188-193Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brux A Liu TY Krebs M Stierhof YD Lohmann JU Miersch O Wasternack C Sschumacher K (2008) Reduced V-ATPPase activityin the trans-Golgi network causes oxylipin-dependent hypocotyl growth inhibition in arabidopsis The Plant Cell 20 1088-1100
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen CZ Lv XF Li JY Yi HY Gong JM (2012) Arabidopsis NRT15 is another essential component in the regulation of nitratereallocation and stress tolerance Plant Physiology 159 1582-1590
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen XP Cui ZL Fan MS Vitousek P Zhao M Ma WQ Wang ZL Zhang WJ Yan XY Yang JC Deng XP Gao Q Zhang Q Guo SWRen J Li SQ Ye YL Wang ZH Huang JL Tang QY Sun YX Peng XL Zhang JW He MR Zhu YJ Xue JQ Wang GL Wu L An N WuLQ Ma L Zhang WF Zhang FS (2014) Producing more grain with lower environmental costs Nature 514 486-489
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiu CC Lin CS Hsia AP Su RC Lin HL Tsay YF (2004) Mutation of a nitrate transporter AtNRT14 results in a reduced petiolenitrate content and altered leaf development Plant Cell Physiology 45 1139-1148
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chopin F Orsel M Dorbe MF Chardon F Truong HN Miller AJ Krapp A Daniel-VedeleF (2007) The Arabidopsis ATNRT27 nitratetransporter controls nitrate content in seeds Plant Cell 19 1590-1602
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Daniel-Vedele F Filleur S CabocheM (1998) Nitrate transport a key step in nitrate assimilation Current Opinion in Plant Biology 1235-239
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dechorgnat J Nguyen CT Armengaud P Jossier M Diatloff E Filleur S Daniel-Vedele F (2011) From the soil to the seedsthe longjourney of nitrate in plants Journal of Experimental Botany 62 1349-1359
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Fan SC Lin CS Hsu PK Lin SH TsayYF (2009) The Arabidopsis nitrate transporter NRT17 expressed in phloem is responsiblefor source-to-sink remobilization of nitrate Plant Cell 212750-2761
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Fan XR Jia L Li Y Smith SJ Miller AJ Shen QR (2007) Comparing nitrate storage and remobilization in two rice cultivars thatdiffer in their nitrogen use efficiency Journal of Experimental Botany 58(7)1729-1740
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gaxiola RA Li JS Undurraga S Dang LM Allen GJ Alper SL Flink GR (2001) Drought and salt tolerant plants result from overexpression of the AVP1 H+-pump PNAS 98(20) 11444-11449
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Glass ADM Britto DT Kaiser BN Kinghorn JR Kronzucker HJ Kumar A Okamoto M Rawat S Siddiqi MY Unkles SE Vidmar JJ(2002) The regulation of nitrate and ammonium transport systems in plants Journal of Experimental Botany 53855-864
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gong JM Lee DA Schroeder JI (2003) Long-distance root-to-shoot transport of phytochelatins and cadmium in Arabidopsis PNAS100 10118-10123
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Granstedt RC Huffaker RC (1982) Identification of the leaf vacuole as a major nitrate storage pool Plant Physiology70410-413Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han YL Liao Q Yu Y Song HX Liu Q Rong XM Gu JD Lepo JE Guan CY Zhang ZH (2015a) Nitrate reutilization mechanisms inthe tonoplast of two Brassica napus genotypes with different nitrogen use efficiency Acta Physiologia Planturum 3742
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han YL Liu Q Gu JD Gong JM Guan CY Lepo JE Rong XR Song HX Zhang ZH (2015b) V-ATPase and V-PPase at the Tonoplastaffect NO3- Content in Brassica napus by Controlling Distribution of NO3- between the Cytoplasm and Vacuole Journal of PlantGrowth Regulation 2015 34 22-34
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harper AL Trick M Higgins J Fraser F Clissold L Wells R Hattori C Werner P Bancroft I (2012) Associative transcriptomics oftraits in the polyploidy crop species Brassica napus Nature Biotechnology 30 798-802
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huang J Zhang Y Peng JS Zhong C Yi HY Ow DW Gong JM (2012) Fission yeast HMT1 lowers seed Cadmium throughphytochelatin-dependent vacuolar sequestration in Arabidopsis Plant Physiology 158(4) 1779-1788
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krapp A David LC Chardin C Cirin T Marmagne A Leprince AC Chaillou S Ferrario-Mery S Meyer C Daniel-VedeleF (2014)Nitrate transport and signaling in Arabidopsis Journal of Experiment Botany 65789-798
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krebs M Beyhl D Gorlich E Al-Rasheid KA Marten I Stierhof YD Hedrich R Schumacher K (2010) Arabidopsis V-ATPase activityat the tonoplast is required for efficient nutrient storage but not for sodium accumulation PNAS1073251-3256
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leran S Varala K Boyer JC Chiurazzi M Crawford N Daniel-Vedele J David L Rebecca D (2014) A unified nomenclature ofNITRATE TRANSPORTER 1PEPTIDE TRANSPORTER family members in plants Trends in Plant Science 19(1) 5-9
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lin SH Kou HF Canivenc G Lin CS Lepetit M Hsu PK Tillard P Lin HL Wang YY Tsai CB Gojon A Tsay YF (2008) Mutation ofthe Arabidopsis NRT15 nitrate transporter causes defective root-to-shoot nitrate transport The Plant Cell 202514-2528
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li JY Fu YL Pike SM Bao J Tian W Zhang Y Chen CZ Zhang Y Li HM Huang J Li LG Schroeder J Gassmann W Gong JM(2010) The arabidopsis nitrate transporter NRT18 functions in nitrate removal from the xylem sap and mediates cadmiumtolerance The Plant Cell 22 1633-1646
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li JS Yang HB Peer WA Richter G Blakeslee J Bandyopadhyay A Titapiwantakun B Undurraga S Khodakovskaya M RichardsE Krizek B Murphy AS Gilroy S Gaxiola R (2005) Arabidopsis H+-PPase AVP1 regulates auxin-mediated organ developmentScience 310 (121) 121-125
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Luo JK Sun SB Jia LJ Chen W Shen QR (2006) The mechanism of nitrate accumulation in Pakchoi [BrassicacampestrisLsspChinensis (L)] Plant and Soil 282291-300
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ma JF Ueno D Zhao FJ McGrath SP (2005) Subcellular localization of Cd and Zn in the leaves of a Cd-hyperaccumulating ecotypeof ThlaspicaerulescensPlanta 220 731-736
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Martinoia E Massonneau A Frangne N (2000) Transport processes of solutes across the vacuolar membrane of higher plantsPlant Cell Physiology 41(11)1175-1186
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Martinoia E Heck U Wiemken A (1981) Vacuoles as storage compartments for nitrate in barley leaves Nature 289 292-294Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Miller AJ Smith SJ (2008) Cytosolic nitrate ion homeostasis could it have a role in sensing nitrogen status Annals ofBotany101485-489
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Robert S Zouhar J Carter C Raikhel N (2007) Isolation of intact vacuoles from Arabidopsis rosette leaf-derived protoplastsNature Protocols 2(2)259-262
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schroeder JL Delhaize E Frommer WB Guerinot ML Harrison MJ Herrera-Estrella L Horie T Kochian LV Munns R NishizawaNK Tsay YF Sanders D (2013) Using membrane transporters to improve crops for sustainable food production Nature 497 60-66
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi WM Xu WF Li SM Zhao XQ Dong GQ (2010) Responses of two rice cultivars differing in seedling-stage nitrogen useefficiency to growth under low-nitrogen conditions Plant and Soil 326291-302
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shen QR Tang L Xu YC (2003) A review on the behavior of nitrate in vacuoles of plants ActaPedologicaSinica40(3)465-470Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Smirnoff N Stewart G (1985) Nitrate assimilation and translocation by higher plants Comparative physiology and ecologicalconsequences Physiologia Plantarum 64 133-140
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang TF Sun XC Hu CX Tan QL Zhao XH (2013) Genotypic differences in nitrate uptake translocation and assimilation of twoChinese cabbage cultivars [Brassica campestris L ssp Chinesnsis (L)] Plant Physiology and Biochemistry7014-20
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang Z Fan XR Li Qing Feng HM Miller AJ Shen QR Xu GH (2012) Knockdown of a rice stellar nitrate transporter alters long-distance translocation but not root influx Plant Physiology 1602052-2063
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Vogeli-Lange R Wagner GJ (1990) Subcellular localization of cadmium and cadmium-binding peptides in tobacco leavesimplication of a transport function for cadmium-binding peptides Plant Physiology 921086-1093
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang GL Ding GD Li L Cai HM Ye XS Zou J Xu FS (2014) Identification and characterization of improved nitrogen efficiency ininterspecific hybridized new-type Brassica napus Annals of Botany 114(3) 549-559
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Wang YY Tsay YF (2011) Arabidopsis nitrate transporter NRT19 is important in phloem nitrate transport The Plant Cell 231945-1957
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wege S Angeli AD Droillard MJ Kroniewicz L Merlot S Cornu D Gambale F Martinoia E Barbier-Brygoo H Thomine SLeonhardt N Filleur S (2014) Phosphorylation of the vacuolar anion exchanger AtCLCa is required for the stomatal response toabscisic acid Science Signalling 7(333) ra65
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wilkinson S Bacon MA Davies WJ (2007) Nitrate signaling to stomatal and growing leaves interactions with soil drying ABA andxylem sap pH in maize Journal of Experimental Botany 58(7) 1705-1716
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xu GH Fan XR Miller AJ (2012) Plant nitrogen assimilation and use efficiency The Annual Review of Plant Biology 63153-182Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yandeau-Nelson MD Laurens L Shi Z Xia H Smith AM Guiltinan MJ (2011) Starch-Branching Enzyme IIa Is Required for ProperDiurnal Cycling of Starch in Leaves of Maize [OA] Plant Physiology 156479-490
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang GB Yi HY Gong JM (2014) The Arabidopsis EthyleneJasmonic acid-NRT signaling module coordinates nitrate reallocationand the trade-off between growth and environmental adaption The Plant Cell 26(10)3984-3998
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Song HX Liu Qing Rong XM Xie GX Peng JW Zhang YP (2009) Study on differences of nitrogen efficiency andnitrogen response in different oilseed rape (Brassica napus L) varieties Asian Journal of Crop Science 1(2)105-112
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2010) Nitrogen redistribution characteristics ofoilseed rape varieties with different nitrogen use efficiencies during later growth period Communications in Soil Science andPlant Analysis 41(14) 1693-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Huang HT Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2012) Research advances on nitratenitrogen reutilization by proton pump of tonoplast and its relation to nitrogen use efficiency Australian Journal of Crop Science6(9) 1377-1382
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhu ZJ Qian YR Pfeiffer W (2001) Effect of nitrogen form on the activity of tonoplast pyrophosphatase in tomato roots ActaBotanica Sinica 43(11) 1146-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Reviewer PDF
- ONE
- TWO
- THREE
- FOUR
- FIVE
- SIX
- SEVEN
- T-ONE
- T-TWO
- Parsed Citations
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Parsed CitationsAndrews M (1986) The partitioning of nitrate assimilation between root and shoot of higher plants Plant Cell Environment 9511-519
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Angeli AD Monachello D Ephritikhine G Frachisse JM Thomine S Gambale F Barbier-Brygoo H (2006) The nitrateprotonantiporter AtCLCa mediates nitrate accumulation in plant vacuoles Nature 442 939-942
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Arteca RN Arteca JM (2000) A novel method for growing Arabidopsis thaliana plants hydroponically Physiol Plantarum 108 188-193Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Brux A Liu TY Krebs M Stierhof YD Lohmann JU Miersch O Wasternack C Sschumacher K (2008) Reduced V-ATPPase activityin the trans-Golgi network causes oxylipin-dependent hypocotyl growth inhibition in arabidopsis The Plant Cell 20 1088-1100
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Chen CZ Lv XF Li JY Yi HY Gong JM (2012) Arabidopsis NRT15 is another essential component in the regulation of nitratereallocation and stress tolerance Plant Physiology 159 1582-1590
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Chen XP Cui ZL Fan MS Vitousek P Zhao M Ma WQ Wang ZL Zhang WJ Yan XY Yang JC Deng XP Gao Q Zhang Q Guo SWRen J Li SQ Ye YL Wang ZH Huang JL Tang QY Sun YX Peng XL Zhang JW He MR Zhu YJ Xue JQ Wang GL Wu L An N WuLQ Ma L Zhang WF Zhang FS (2014) Producing more grain with lower environmental costs Nature 514 486-489
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Chiu CC Lin CS Hsia AP Su RC Lin HL Tsay YF (2004) Mutation of a nitrate transporter AtNRT14 results in a reduced petiolenitrate content and altered leaf development Plant Cell Physiology 45 1139-1148
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Chopin F Orsel M Dorbe MF Chardon F Truong HN Miller AJ Krapp A Daniel-VedeleF (2007) The Arabidopsis ATNRT27 nitratetransporter controls nitrate content in seeds Plant Cell 19 1590-1602
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Daniel-Vedele F Filleur S CabocheM (1998) Nitrate transport a key step in nitrate assimilation Current Opinion in Plant Biology 1235-239
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Dechorgnat J Nguyen CT Armengaud P Jossier M Diatloff E Filleur S Daniel-Vedele F (2011) From the soil to the seedsthe longjourney of nitrate in plants Journal of Experimental Botany 62 1349-1359
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Fan SC Lin CS Hsu PK Lin SH TsayYF (2009) The Arabidopsis nitrate transporter NRT17 expressed in phloem is responsiblefor source-to-sink remobilization of nitrate Plant Cell 212750-2761
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Fan XR Jia L Li Y Smith SJ Miller AJ Shen QR (2007) Comparing nitrate storage and remobilization in two rice cultivars thatdiffer in their nitrogen use efficiency Journal of Experimental Botany 58(7)1729-1740
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Gaxiola RA Li JS Undurraga S Dang LM Allen GJ Alper SL Flink GR (2001) Drought and salt tolerant plants result from overexpression of the AVP1 H+-pump PNAS 98(20) 11444-11449
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wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Glass ADM Britto DT Kaiser BN Kinghorn JR Kronzucker HJ Kumar A Okamoto M Rawat S Siddiqi MY Unkles SE Vidmar JJ(2002) The regulation of nitrate and ammonium transport systems in plants Journal of Experimental Botany 53855-864
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gong JM Lee DA Schroeder JI (2003) Long-distance root-to-shoot transport of phytochelatins and cadmium in Arabidopsis PNAS100 10118-10123
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Granstedt RC Huffaker RC (1982) Identification of the leaf vacuole as a major nitrate storage pool Plant Physiology70410-413Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han YL Liao Q Yu Y Song HX Liu Q Rong XM Gu JD Lepo JE Guan CY Zhang ZH (2015a) Nitrate reutilization mechanisms inthe tonoplast of two Brassica napus genotypes with different nitrogen use efficiency Acta Physiologia Planturum 3742
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han YL Liu Q Gu JD Gong JM Guan CY Lepo JE Rong XR Song HX Zhang ZH (2015b) V-ATPase and V-PPase at the Tonoplastaffect NO3- Content in Brassica napus by Controlling Distribution of NO3- between the Cytoplasm and Vacuole Journal of PlantGrowth Regulation 2015 34 22-34
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harper AL Trick M Higgins J Fraser F Clissold L Wells R Hattori C Werner P Bancroft I (2012) Associative transcriptomics oftraits in the polyploidy crop species Brassica napus Nature Biotechnology 30 798-802
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huang J Zhang Y Peng JS Zhong C Yi HY Ow DW Gong JM (2012) Fission yeast HMT1 lowers seed Cadmium throughphytochelatin-dependent vacuolar sequestration in Arabidopsis Plant Physiology 158(4) 1779-1788
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krapp A David LC Chardin C Cirin T Marmagne A Leprince AC Chaillou S Ferrario-Mery S Meyer C Daniel-VedeleF (2014)Nitrate transport and signaling in Arabidopsis Journal of Experiment Botany 65789-798
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krebs M Beyhl D Gorlich E Al-Rasheid KA Marten I Stierhof YD Hedrich R Schumacher K (2010) Arabidopsis V-ATPase activityat the tonoplast is required for efficient nutrient storage but not for sodium accumulation PNAS1073251-3256
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leran S Varala K Boyer JC Chiurazzi M Crawford N Daniel-Vedele J David L Rebecca D (2014) A unified nomenclature ofNITRATE TRANSPORTER 1PEPTIDE TRANSPORTER family members in plants Trends in Plant Science 19(1) 5-9
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lin SH Kou HF Canivenc G Lin CS Lepetit M Hsu PK Tillard P Lin HL Wang YY Tsai CB Gojon A Tsay YF (2008) Mutation ofthe Arabidopsis NRT15 nitrate transporter causes defective root-to-shoot nitrate transport The Plant Cell 202514-2528
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li JY Fu YL Pike SM Bao J Tian W Zhang Y Chen CZ Zhang Y Li HM Huang J Li LG Schroeder J Gassmann W Gong JM(2010) The arabidopsis nitrate transporter NRT18 functions in nitrate removal from the xylem sap and mediates cadmiumtolerance The Plant Cell 22 1633-1646
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li JS Yang HB Peer WA Richter G Blakeslee J Bandyopadhyay A Titapiwantakun B Undurraga S Khodakovskaya M RichardsE Krizek B Murphy AS Gilroy S Gaxiola R (2005) Arabidopsis H+-PPase AVP1 regulates auxin-mediated organ developmentScience 310 (121) 121-125
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Luo JK Sun SB Jia LJ Chen W Shen QR (2006) The mechanism of nitrate accumulation in Pakchoi [BrassicacampestrisLsspChinensis (L)] Plant and Soil 282291-300
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ma JF Ueno D Zhao FJ McGrath SP (2005) Subcellular localization of Cd and Zn in the leaves of a Cd-hyperaccumulating ecotypeof ThlaspicaerulescensPlanta 220 731-736
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Martinoia E Massonneau A Frangne N (2000) Transport processes of solutes across the vacuolar membrane of higher plantsPlant Cell Physiology 41(11)1175-1186
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Martinoia E Heck U Wiemken A (1981) Vacuoles as storage compartments for nitrate in barley leaves Nature 289 292-294Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Miller AJ Smith SJ (2008) Cytosolic nitrate ion homeostasis could it have a role in sensing nitrogen status Annals ofBotany101485-489
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Robert S Zouhar J Carter C Raikhel N (2007) Isolation of intact vacuoles from Arabidopsis rosette leaf-derived protoplastsNature Protocols 2(2)259-262
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schroeder JL Delhaize E Frommer WB Guerinot ML Harrison MJ Herrera-Estrella L Horie T Kochian LV Munns R NishizawaNK Tsay YF Sanders D (2013) Using membrane transporters to improve crops for sustainable food production Nature 497 60-66
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi WM Xu WF Li SM Zhao XQ Dong GQ (2010) Responses of two rice cultivars differing in seedling-stage nitrogen useefficiency to growth under low-nitrogen conditions Plant and Soil 326291-302
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shen QR Tang L Xu YC (2003) A review on the behavior of nitrate in vacuoles of plants ActaPedologicaSinica40(3)465-470Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Smirnoff N Stewart G (1985) Nitrate assimilation and translocation by higher plants Comparative physiology and ecologicalconsequences Physiologia Plantarum 64 133-140
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang TF Sun XC Hu CX Tan QL Zhao XH (2013) Genotypic differences in nitrate uptake translocation and assimilation of twoChinese cabbage cultivars [Brassica campestris L ssp Chinesnsis (L)] Plant Physiology and Biochemistry7014-20
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang Z Fan XR Li Qing Feng HM Miller AJ Shen QR Xu GH (2012) Knockdown of a rice stellar nitrate transporter alters long-distance translocation but not root influx Plant Physiology 1602052-2063
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Vogeli-Lange R Wagner GJ (1990) Subcellular localization of cadmium and cadmium-binding peptides in tobacco leavesimplication of a transport function for cadmium-binding peptides Plant Physiology 921086-1093
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang GL Ding GD Li L Cai HM Ye XS Zou J Xu FS (2014) Identification and characterization of improved nitrogen efficiency ininterspecific hybridized new-type Brassica napus Annals of Botany 114(3) 549-559
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Wang YY Tsay YF (2011) Arabidopsis nitrate transporter NRT19 is important in phloem nitrate transport The Plant Cell 231945-1957
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wege S Angeli AD Droillard MJ Kroniewicz L Merlot S Cornu D Gambale F Martinoia E Barbier-Brygoo H Thomine SLeonhardt N Filleur S (2014) Phosphorylation of the vacuolar anion exchanger AtCLCa is required for the stomatal response toabscisic acid Science Signalling 7(333) ra65
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wilkinson S Bacon MA Davies WJ (2007) Nitrate signaling to stomatal and growing leaves interactions with soil drying ABA andxylem sap pH in maize Journal of Experimental Botany 58(7) 1705-1716
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xu GH Fan XR Miller AJ (2012) Plant nitrogen assimilation and use efficiency The Annual Review of Plant Biology 63153-182Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yandeau-Nelson MD Laurens L Shi Z Xia H Smith AM Guiltinan MJ (2011) Starch-Branching Enzyme IIa Is Required for ProperDiurnal Cycling of Starch in Leaves of Maize [OA] Plant Physiology 156479-490
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang GB Yi HY Gong JM (2014) The Arabidopsis EthyleneJasmonic acid-NRT signaling module coordinates nitrate reallocationand the trade-off between growth and environmental adaption The Plant Cell 26(10)3984-3998
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Song HX Liu Qing Rong XM Xie GX Peng JW Zhang YP (2009) Study on differences of nitrogen efficiency andnitrogen response in different oilseed rape (Brassica napus L) varieties Asian Journal of Crop Science 1(2)105-112
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2010) Nitrogen redistribution characteristics ofoilseed rape varieties with different nitrogen use efficiencies during later growth period Communications in Soil Science andPlant Analysis 41(14) 1693-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Huang HT Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2012) Research advances on nitratenitrogen reutilization by proton pump of tonoplast and its relation to nitrogen use efficiency Australian Journal of Crop Science6(9) 1377-1382
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhu ZJ Qian YR Pfeiffer W (2001) Effect of nitrogen form on the activity of tonoplast pyrophosphatase in tomato roots ActaBotanica Sinica 43(11) 1146-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Reviewer PDF
- ONE
- TWO
- THREE
- FOUR
- FIVE
- SIX
- SEVEN
- T-ONE
- T-TWO
- Parsed Citations
-
Glass ADM Britto DT Kaiser BN Kinghorn JR Kronzucker HJ Kumar A Okamoto M Rawat S Siddiqi MY Unkles SE Vidmar JJ(2002) The regulation of nitrate and ammonium transport systems in plants Journal of Experimental Botany 53855-864
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gong JM Lee DA Schroeder JI (2003) Long-distance root-to-shoot transport of phytochelatins and cadmium in Arabidopsis PNAS100 10118-10123
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Granstedt RC Huffaker RC (1982) Identification of the leaf vacuole as a major nitrate storage pool Plant Physiology70410-413Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han YL Liao Q Yu Y Song HX Liu Q Rong XM Gu JD Lepo JE Guan CY Zhang ZH (2015a) Nitrate reutilization mechanisms inthe tonoplast of two Brassica napus genotypes with different nitrogen use efficiency Acta Physiologia Planturum 3742
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han YL Liu Q Gu JD Gong JM Guan CY Lepo JE Rong XR Song HX Zhang ZH (2015b) V-ATPase and V-PPase at the Tonoplastaffect NO3- Content in Brassica napus by Controlling Distribution of NO3- between the Cytoplasm and Vacuole Journal of PlantGrowth Regulation 2015 34 22-34
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harper AL Trick M Higgins J Fraser F Clissold L Wells R Hattori C Werner P Bancroft I (2012) Associative transcriptomics oftraits in the polyploidy crop species Brassica napus Nature Biotechnology 30 798-802
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huang J Zhang Y Peng JS Zhong C Yi HY Ow DW Gong JM (2012) Fission yeast HMT1 lowers seed Cadmium throughphytochelatin-dependent vacuolar sequestration in Arabidopsis Plant Physiology 158(4) 1779-1788
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krapp A David LC Chardin C Cirin T Marmagne A Leprince AC Chaillou S Ferrario-Mery S Meyer C Daniel-VedeleF (2014)Nitrate transport and signaling in Arabidopsis Journal of Experiment Botany 65789-798
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krebs M Beyhl D Gorlich E Al-Rasheid KA Marten I Stierhof YD Hedrich R Schumacher K (2010) Arabidopsis V-ATPase activityat the tonoplast is required for efficient nutrient storage but not for sodium accumulation PNAS1073251-3256
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leran S Varala K Boyer JC Chiurazzi M Crawford N Daniel-Vedele J David L Rebecca D (2014) A unified nomenclature ofNITRATE TRANSPORTER 1PEPTIDE TRANSPORTER family members in plants Trends in Plant Science 19(1) 5-9
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lin SH Kou HF Canivenc G Lin CS Lepetit M Hsu PK Tillard P Lin HL Wang YY Tsai CB Gojon A Tsay YF (2008) Mutation ofthe Arabidopsis NRT15 nitrate transporter causes defective root-to-shoot nitrate transport The Plant Cell 202514-2528
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li JY Fu YL Pike SM Bao J Tian W Zhang Y Chen CZ Zhang Y Li HM Huang J Li LG Schroeder J Gassmann W Gong JM(2010) The arabidopsis nitrate transporter NRT18 functions in nitrate removal from the xylem sap and mediates cadmiumtolerance The Plant Cell 22 1633-1646
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li JS Yang HB Peer WA Richter G Blakeslee J Bandyopadhyay A Titapiwantakun B Undurraga S Khodakovskaya M RichardsE Krizek B Murphy AS Gilroy S Gaxiola R (2005) Arabidopsis H+-PPase AVP1 regulates auxin-mediated organ developmentScience 310 (121) 121-125
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Luo JK Sun SB Jia LJ Chen W Shen QR (2006) The mechanism of nitrate accumulation in Pakchoi [BrassicacampestrisLsspChinensis (L)] Plant and Soil 282291-300
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ma JF Ueno D Zhao FJ McGrath SP (2005) Subcellular localization of Cd and Zn in the leaves of a Cd-hyperaccumulating ecotypeof ThlaspicaerulescensPlanta 220 731-736
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Martinoia E Massonneau A Frangne N (2000) Transport processes of solutes across the vacuolar membrane of higher plantsPlant Cell Physiology 41(11)1175-1186
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Martinoia E Heck U Wiemken A (1981) Vacuoles as storage compartments for nitrate in barley leaves Nature 289 292-294Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Miller AJ Smith SJ (2008) Cytosolic nitrate ion homeostasis could it have a role in sensing nitrogen status Annals ofBotany101485-489
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Robert S Zouhar J Carter C Raikhel N (2007) Isolation of intact vacuoles from Arabidopsis rosette leaf-derived protoplastsNature Protocols 2(2)259-262
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schroeder JL Delhaize E Frommer WB Guerinot ML Harrison MJ Herrera-Estrella L Horie T Kochian LV Munns R NishizawaNK Tsay YF Sanders D (2013) Using membrane transporters to improve crops for sustainable food production Nature 497 60-66
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi WM Xu WF Li SM Zhao XQ Dong GQ (2010) Responses of two rice cultivars differing in seedling-stage nitrogen useefficiency to growth under low-nitrogen conditions Plant and Soil 326291-302
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shen QR Tang L Xu YC (2003) A review on the behavior of nitrate in vacuoles of plants ActaPedologicaSinica40(3)465-470Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Smirnoff N Stewart G (1985) Nitrate assimilation and translocation by higher plants Comparative physiology and ecologicalconsequences Physiologia Plantarum 64 133-140
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang TF Sun XC Hu CX Tan QL Zhao XH (2013) Genotypic differences in nitrate uptake translocation and assimilation of twoChinese cabbage cultivars [Brassica campestris L ssp Chinesnsis (L)] Plant Physiology and Biochemistry7014-20
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang Z Fan XR Li Qing Feng HM Miller AJ Shen QR Xu GH (2012) Knockdown of a rice stellar nitrate transporter alters long-distance translocation but not root influx Plant Physiology 1602052-2063
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Vogeli-Lange R Wagner GJ (1990) Subcellular localization of cadmium and cadmium-binding peptides in tobacco leavesimplication of a transport function for cadmium-binding peptides Plant Physiology 921086-1093
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang GL Ding GD Li L Cai HM Ye XS Zou J Xu FS (2014) Identification and characterization of improved nitrogen efficiency ininterspecific hybridized new-type Brassica napus Annals of Botany 114(3) 549-559
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title wwwplantphysiolorgon May 17 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Wang YY Tsay YF (2011) Arabidopsis nitrate transporter NRT19 is important in phloem nitrate transport The Plant Cell 231945-1957
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wege S Angeli AD Droillard MJ Kroniewicz L Merlot S Cornu D Gambale F Martinoia E Barbier-Brygoo H Thomine SLeonhardt N Filleur S (2014) Phosphorylation of the vacuolar anion exchanger AtCLCa is required for the stomatal response toabscisic acid Science Signalling 7(333) ra65
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wilkinson S Bacon MA Davies WJ (2007) Nitrate signaling to stomatal and growing leaves interactions with soil drying ABA andxylem sap pH in maize Journal of Experimental Botany 58(7) 1705-1716
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xu GH Fan XR Miller AJ (2012) Plant nitrogen assimilation and use efficiency The Annual Review of Plant Biology 63153-182Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yandeau-Nelson MD Laurens L Shi Z Xia H Smith AM Guiltinan MJ (2011) Starch-Branching Enzyme IIa Is Required for ProperDiurnal Cycling of Starch in Leaves of Maize [OA] Plant Physiology 156479-490
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang GB Yi HY Gong JM (2014) The Arabidopsis EthyleneJasmonic acid-NRT signaling module coordinates nitrate reallocationand the trade-off between growth and environmental adaption The Plant Cell 26(10)3984-3998
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Song HX Liu Qing Rong XM Xie GX Peng JW Zhang YP (2009) Study on differences of nitrogen efficiency andnitrogen response in different oilseed rape (Brassica napus L) varieties Asian Journal of Crop Science 1(2)105-112
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2010) Nitrogen redistribution characteristics ofoilseed rape varieties with different nitrogen use efficiencies during later growth period Communications in Soil Science andPlant Analysis 41(14) 1693-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Huang HT Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2012) Research advances on nitratenitrogen reutilization by proton pump of tonoplast and its relation to nitrogen use efficiency Australian Journal of Crop Science6(9) 1377-1382
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhu ZJ Qian YR Pfeiffer W (2001) Effect of nitrogen form on the activity of tonoplast pyrophosphatase in tomato roots ActaBotanica Sinica 43(11) 1146-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Reviewer PDF
- ONE
- TWO
- THREE
- FOUR
- FIVE
- SIX
- SEVEN
- T-ONE
- T-TWO
- Parsed Citations
-
Luo JK Sun SB Jia LJ Chen W Shen QR (2006) The mechanism of nitrate accumulation in Pakchoi [BrassicacampestrisLsspChinensis (L)] Plant and Soil 282291-300
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ma JF Ueno D Zhao FJ McGrath SP (2005) Subcellular localization of Cd and Zn in the leaves of a Cd-hyperaccumulating ecotypeof ThlaspicaerulescensPlanta 220 731-736
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Martinoia E Massonneau A Frangne N (2000) Transport processes of solutes across the vacuolar membrane of higher plantsPlant Cell Physiology 41(11)1175-1186
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Martinoia E Heck U Wiemken A (1981) Vacuoles as storage compartments for nitrate in barley leaves Nature 289 292-294Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Miller AJ Smith SJ (2008) Cytosolic nitrate ion homeostasis could it have a role in sensing nitrogen status Annals ofBotany101485-489
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Robert S Zouhar J Carter C Raikhel N (2007) Isolation of intact vacuoles from Arabidopsis rosette leaf-derived protoplastsNature Protocols 2(2)259-262
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schroeder JL Delhaize E Frommer WB Guerinot ML Harrison MJ Herrera-Estrella L Horie T Kochian LV Munns R NishizawaNK Tsay YF Sanders D (2013) Using membrane transporters to improve crops for sustainable food production Nature 497 60-66
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shi WM Xu WF Li SM Zhao XQ Dong GQ (2010) Responses of two rice cultivars differing in seedling-stage nitrogen useefficiency to growth under low-nitrogen conditions Plant and Soil 326291-302
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shen QR Tang L Xu YC (2003) A review on the behavior of nitrate in vacuoles of plants ActaPedologicaSinica40(3)465-470Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Smirnoff N Stewart G (1985) Nitrate assimilation and translocation by higher plants Comparative physiology and ecologicalconsequences Physiologia Plantarum 64 133-140
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang TF Sun XC Hu CX Tan QL Zhao XH (2013) Genotypic differences in nitrate uptake translocation and assimilation of twoChinese cabbage cultivars [Brassica campestris L ssp Chinesnsis (L)] Plant Physiology and Biochemistry7014-20
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang Z Fan XR Li Qing Feng HM Miller AJ Shen QR Xu GH (2012) Knockdown of a rice stellar nitrate transporter alters long-distance translocation but not root influx Plant Physiology 1602052-2063
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Vogeli-Lange R Wagner GJ (1990) Subcellular localization of cadmium and cadmium-binding peptides in tobacco leavesimplication of a transport function for cadmium-binding peptides Plant Physiology 921086-1093
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang GL Ding GD Li L Cai HM Ye XS Zou J Xu FS (2014) Identification and characterization of improved nitrogen efficiency ininterspecific hybridized new-type Brassica napus Annals of Botany 114(3) 549-559
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Wang YY Tsay YF (2011) Arabidopsis nitrate transporter NRT19 is important in phloem nitrate transport The Plant Cell 231945-1957
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Wege S Angeli AD Droillard MJ Kroniewicz L Merlot S Cornu D Gambale F Martinoia E Barbier-Brygoo H Thomine SLeonhardt N Filleur S (2014) Phosphorylation of the vacuolar anion exchanger AtCLCa is required for the stomatal response toabscisic acid Science Signalling 7(333) ra65
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wilkinson S Bacon MA Davies WJ (2007) Nitrate signaling to stomatal and growing leaves interactions with soil drying ABA andxylem sap pH in maize Journal of Experimental Botany 58(7) 1705-1716
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xu GH Fan XR Miller AJ (2012) Plant nitrogen assimilation and use efficiency The Annual Review of Plant Biology 63153-182Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yandeau-Nelson MD Laurens L Shi Z Xia H Smith AM Guiltinan MJ (2011) Starch-Branching Enzyme IIa Is Required for ProperDiurnal Cycling of Starch in Leaves of Maize [OA] Plant Physiology 156479-490
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang GB Yi HY Gong JM (2014) The Arabidopsis EthyleneJasmonic acid-NRT signaling module coordinates nitrate reallocationand the trade-off between growth and environmental adaption The Plant Cell 26(10)3984-3998
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Song HX Liu Qing Rong XM Xie GX Peng JW Zhang YP (2009) Study on differences of nitrogen efficiency andnitrogen response in different oilseed rape (Brassica napus L) varieties Asian Journal of Crop Science 1(2)105-112
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2010) Nitrogen redistribution characteristics ofoilseed rape varieties with different nitrogen use efficiencies during later growth period Communications in Soil Science andPlant Analysis 41(14) 1693-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Huang HT Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2012) Research advances on nitratenitrogen reutilization by proton pump of tonoplast and its relation to nitrogen use efficiency Australian Journal of Crop Science6(9) 1377-1382
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhu ZJ Qian YR Pfeiffer W (2001) Effect of nitrogen form on the activity of tonoplast pyrophosphatase in tomato roots ActaBotanica Sinica 43(11) 1146-1149
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wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
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Wang YY Tsay YF (2011) Arabidopsis nitrate transporter NRT19 is important in phloem nitrate transport The Plant Cell 231945-1957
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wege S Angeli AD Droillard MJ Kroniewicz L Merlot S Cornu D Gambale F Martinoia E Barbier-Brygoo H Thomine SLeonhardt N Filleur S (2014) Phosphorylation of the vacuolar anion exchanger AtCLCa is required for the stomatal response toabscisic acid Science Signalling 7(333) ra65
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wilkinson S Bacon MA Davies WJ (2007) Nitrate signaling to stomatal and growing leaves interactions with soil drying ABA andxylem sap pH in maize Journal of Experimental Botany 58(7) 1705-1716
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xu GH Fan XR Miller AJ (2012) Plant nitrogen assimilation and use efficiency The Annual Review of Plant Biology 63153-182Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yandeau-Nelson MD Laurens L Shi Z Xia H Smith AM Guiltinan MJ (2011) Starch-Branching Enzyme IIa Is Required for ProperDiurnal Cycling of Starch in Leaves of Maize [OA] Plant Physiology 156479-490
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang GB Yi HY Gong JM (2014) The Arabidopsis EthyleneJasmonic acid-NRT signaling module coordinates nitrate reallocationand the trade-off between growth and environmental adaption The Plant Cell 26(10)3984-3998
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Song HX Liu Qing Rong XM Xie GX Peng JW Zhang YP (2009) Study on differences of nitrogen efficiency andnitrogen response in different oilseed rape (Brassica napus L) varieties Asian Journal of Crop Science 1(2)105-112
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2010) Nitrogen redistribution characteristics ofoilseed rape varieties with different nitrogen use efficiencies during later growth period Communications in Soil Science andPlant Analysis 41(14) 1693-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang ZH Huang HT Song HX Liu Q Rong XM Peng JW Xie GX Zhang YP Guan CY (2012) Research advances on nitratenitrogen reutilization by proton pump of tonoplast and its relation to nitrogen use efficiency Australian Journal of Crop Science6(9) 1377-1382
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhu ZJ Qian YR Pfeiffer W (2001) Effect of nitrogen form on the activity of tonoplast pyrophosphatase in tomato roots ActaBotanica Sinica 43(11) 1146-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
wwwplantphysiolorgon May 17 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
- Parsed Citations
- Reviewer PDF
- ONE
- TWO
- THREE
- FOUR
- FIVE
- SIX
- SEVEN
- T-ONE
- T-TWO
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