1 the lsm1-7 complex differentially regulates arabidopsis...
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THE LSM1-7 COMPLEX DIFFERENTIALLY REGULATES ARABIDOPSIS 1
TOLERANCE TO ABIOTIC STRESS CONDITIONS BY PROMOTING 2
SELECTIVE mRNA DECAPPING 3
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Carlos Perea-Resa1*, Cristian Carrasco-López1*, Rafael Catalá1, Veronika 6
Turečková2, Ondrej Novak2, Weiping Zhang3, Leslie Sieburth3, José Manuel 7
Jiménez-Gómez4,5 and Julio Salinas1,† 8
9 1Departamento de Biología Medioambiental, Centro Investigaciones Biológicas, CSIC, 10
28040 Madrid, Spain 11 2Laboratory of Growth Regulators, Institute of Experimental Botany Academy of 12
Sciences of the Czech Republic, v.v.i. & Palacký University, 78371 Olomouc, Czech 13
Republic 14 3Department of Biology, University of Utah, Salt Lake City, UT 84112, USA 15 4Department of Plant Breeding and Genetics, Max Planck Institute for Plant Breeding 16
Research, 50829 Cologne, Germany 17 5INRA, Institut Jean-Pierre Bourgin, UMR 1318, 78026 Versailles, France. 18
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21 * These authors contributed equally to this work 22
23 †Corresponding author: [email protected] 24
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Short title: LSM1-7 promotes selective mRNA decapping 26
Synopsis: A decapping activator complex ensures the adequacy and specificity of the 27
response of Arabidopsis to different abiotic stresses by promoting selective mRNA 28
degradation. 29
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The author responsible for distribution of materials integral to the findings presented in 31
this article in accordance with the policy described in the Instructions for Authors 32
(www.plant cell.org) is: Julio Salinas ([email protected]) 33
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ABSTRACT 35
In eukaryotes, the decapping machinery is highly conserved and plays an essential role 36
in controlling mRNA stability, a key step in the regulation of gene expression. Yet, the 37
role of mRNA decapping in shaping gene expression profiles in response to 38
environmental cues and the operating molecular mechanisms are poorly understood. 39
Here, we provide genetic and molecular evidence that a component of the decapping 40
machinery, the LSM1-7 complex, plays a critical role in plant tolerance to abiotic 41
stresses. Our results demonstrate that, depending on the stress, the complex from 42
Arabidopsis thaliana interacts with different selected stress-inducible transcripts 43
targeting them for decapping and subsequent degradation. This interaction ensures the 44
correct turnover of the target transcripts and, consequently, the appropriate patterns of 45
downstream stress-responsive gene expression that are required for plant adaptation. 46
Remarkably, among the selected target transcripts of the LSM1-7 complex are those 47
encoding NCED3 and NCED5, two key enzymes in ABA biosynthesis. We demonstrate 48
that the complex modulates ABA levels in Arabidopsis exposed to cold and high salt by 49
differentially controlling NCED3 and NCED5 mRNA turnover, which represents a new 50
layer of regulation in ABA biosynthesis in response to abiotic stress. Our findings 51
uncover an unanticipated functional plasticity of the mRNA decapping machinery to 52
modulate the relationship between plants and their environment. 53
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INTRODUCTION 55
Plants have evolved sophisticated mechanisms to perceive and rapidly respond to the 56
numerous abiotic stresses to which they are continuously challenged. A decisive 57
component of all stress responses is the ability to reprogram transcriptomes. 58
Transcriptome reprogramming in response to adverse environments has been generally 59
considered as a complex process mainly based on the modulation of transcriptional 60
activity of stress-related genes (Yamaguchi-Shinozaki and Shinozaki, 2006; Gujjar et 61
al., 2014; Nakashima et al., 2014). Recent data, however, indicate that 62
posttranscriptional mechanisms also influential to make fine and timely adjustments of 63
the plant transcriptomes to unfavorable environments (Guerra et al., 2015). The control 64
of mRNA stability, in particular, is critical for the regulation of gene expression in 65
response to abiotic stress. Stability determinants for intrinsically unstable eukaryotic 66
mRNAs include the 3’ poly (A) tail and the 5’ cap. Transcript abundance is then fine-67
tuned by a major pathway of cytoplasmic mRNA degradation involving deadenylation, 68
decapping and subsequent exoribonuclease 5’-3’ activity (Parker, 2012). mRNA 69
decapping is achieved in distinct cytoplasmic foci named processing bodies (P-bodies) 70
by the action of the decapping complex, a protein conglomerate highly conserved 71
among eukaryotes (Coller and Parker, 2004; Fillman and Lykke-Andersen, 2005; Xu et 72
al., 2006; Goeres et al., 2007). This complex includes the decapping enzyme DCP2 and 73
its activators DCP1, DCP5, VCS, DHH1, PAT1 and the LSM1-7 complex (Coller and 74
Parker, 2004; Fillman and Lykke-Andersen, 2005; Xu et al., 2006; Goeres et al., 2007; 75
Xu and Chua, 2009). mRNA decapping has been shown to play important roles in 76
development (Xu et al., 2006; Goeres et al., 2007; Xu and Chua, 2009). By contrast, the 77
role of the decapping complex in abiotic stress signaling is barely documented. To date, 78
only DCP1 and DCP5 have been described to associate under drought conditions to 79
promote drought tolerance in Arabidopsis (Xu and Chua, 2012). Whether mRNA 80
decapping is involved in regulating eukaryotic responses to other abiotic stresses and 81
the molecular mechanisms operating in mRNA decapping during such responses is 82
largely unknown. 83
The SM-like proteins (LSMs) are implicated in numerous aspects of RNA metabolism 84
in eukaryotes. The Arabidopsis genome contains eleven LSM genes encoding eight 85
central, highly evolutionarily conserved LSM proteins (LSM1-LSM8) (Perea-Resa et 86
al., 2012). LSM1, LSM3 and LSM6 are each duplicated and code for pairs of 87
functionally redundant proteins (LSM1A, B; LSM3A, B; LSM6A, B). In Arabidopsis, 88
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as in yeast and animals, the eight conserved LSM proteins are organized in two 89
heteroheptameric ring complexes, LSM1-7 and LSM2-8, specifically localized in the 90
cytoplasm and nucleus, and defined by the subunits LSM1 and LSM8, respectively. The 91
LSM2-8 complex functions in pre-mRNA splicing through U6 snRNA stabilization, and 92
ensures normal Arabidopsis development (Perea-Resa et al., 2012). Moreover, 93
alternative splicing analysis in lsm4 and sad1/lsm5 mutants uncovered that this complex 94
acts as a positive regulator of salt tolerance in Arabidopsis (Zhang et al., 2011; Cui et 95
al., 2014). The LSM1-7 complex is involved in accurate mRNA turnover by promoting 96
decapping and subsequent 5’-3’ degradation, and is required for the formation of P-97
bodies (Perea-Resa et al., 2012). In the yeast Saccharomyces cerevisiae, the LSM1-7 98
complex has been described to operate through the interaction of LSM1 with 99
oligoadenylated mRNAs that are then targeted for degradation (Chowdhury et al., 100
2007). The analysis of an Arabidopsis lsm1a lsm1b double mutant defective in LSM1A 101
and LSM1B expression unveiled that LSM1 proteins are essential for the assembly of 102
the LSM cytoplasmic complex and that this complex is needed for correct plant 103
development (Perea-Resa et al., 2012). The participation of the LSM1-7 decapping 104
activator complex in abiotic stress responses, however, has not yet been established in 105
any organism. Here, we demonstrate that this complex regulates Arabidopsis tolerance 106
to freezing, drought and high salt by modulating the transcriptome reprogramming that 107
takes place in response to these adverse conditions. More important, RNA 108
immunoprecipitation (RIP) assays revealed that, depending on the abiotic stress to 109
which the plant is subjected, the LSM cytoplasmic complex targets selected stress-110
inducible transcripts for decapping and degradation, thus controlling their levels and, 111
therefore, ensuring the adequate transcriptomic response. Interestingly, among the 112
selected mRNAs that are differentially targeted for decapping in response to low 113
temperature, water deficiency and high salt are those encoding NCED3 and NCED5, 114
two key enzymes in ABA biosynthesis. We show that, as a consequence, the LSM1-7 115
complex determines the appropriate levels of ABA in Arabidopsis plants exposed to 116
different abiotic stresses. Our finding thus reveal a previously unrecognized layer of 117
regulation in the biosynthesis of this phytohormone and uncover unexpected functional 118
plasticity of the mRNA decapping machinery in influencing the responses of plants to 119
their environment. 120
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RESULTS 122
Arabidopsis LSM1 proteins localize to P-bodies in response to abiotic stress 123
The molecular characterization of Arabidopsis LSM1 genes uncovered that they were 124
responsive to low temperature. Quantitative RT-PCR (qPCR) assays showed that 125
LSM1A and LSM1B transcripts accumulated in response to 4ºC, reaching a peak of 126
accumulation after 1 day of treatment (Figure 1 A). This accumulation was detected 127
broadly through the adult Arabidopsis plant (Supplemental Figure 1 A). Transcripts 128
corresponding to LSM2-7 proteins exhibited similar cold-induced accumulation as 129
LSM1 mRNAs (Supplemental Figure 1 B). LSM transcripts did not accumulate, 130
however, in plants exposed to other related abiotic stresses, such as drought (55% PEG) 131
or high salt (150 mM NaCl) (Supplemental Figure 1 C). We concluded that the 132
expression of genes encoding the Arabidopsis cytoplasmic LSM complex is positively 133
regulated by low temperature. 134
Since LSM1A and LSM1B transcripts accumulated in response to low temperature, we 135
assessed whether this accumulation was followed by an increase of the corresponding 136
proteins. Immunoblot experiments using Arabidopsis plants containing genomic LSM1-137
GFP fusions driven by the corresponding LSM1 promoters (LSM1PRO) (Perea-Resa et 138
al., 2012) showed that LSM1A-GFP and LSM1B-GFP proteins were notably more 139
abundant after some days of cold exposure (Figure 1 B). Consistent with the expression 140
results, water and salt stresses did not alter the levels of LSM1 proteins (Supplemental 141
Figure 2 A). Therefore, concomitantly with the accumulation of their transcripts, the 142
levels of LSM1 proteins also increase in response to low temperature. 143
Previous data revealed that heat treatment promotes the localization of LSM1A and 144
LSM1B to P-bodies (Perea-Resa et al., 2012). We decided to investigate if LSM1 145
proteins also localize to these foci under cold, drought or high salt conditions by 146
examining the distribution of green fluorescence in root cells from the LSM1APRO-147
LSM1A-GFP and LSM1BPRO-LSM1B-GFP plants. In according with earlier results 148
(Perea-Resa et al., 2012), confocal microscopy analysis indicated that, at 20ºC, GFP 149
activity was in both cases broadly distributed in the cytoplasm (Figure 1 C). However, 150
when transgenic plants were exposed to 4ºC, water deficiency, or salt stress, LSM1A-151
GFP and LSM1B-GFP fusion proteins were found aggregated in discrete cytoplasmic 152
spots that resembled P-bodies. After cycloheximide treatment, which causes loss of P-153
bodies (Sheth and Parker, 2003), no cytoplasmic foci were observed in any case, 154
suggesting that the detected LSM1-GFP spots indeed corresponded to P-bodies (Figures 155
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1C and Supplemental Figure 2 B). The identity of these foci was confirmed by 156
colocalization studies with DCP1, a protein that accumulates in P-bodies (Xu et al., 157
2006; Weber et al., 2008; Motomura et al., 2015). The examination of LSM1APRO-158
LSM1A-GFP and LSM1BPRO-LSM1B-GFP plants cotransformed with a 35S-RED 159
FLUORESCENT PROTEIN (RFP)-DCP1 fusion (Perea-Resa et al., 2012) demonstrated 160
that, in fact, LSM1A-GFP and LSM1B-GFP colocalized with RFP-DCP1 in root cells 161
exposed to 4ºC (Figure 1 D). Identical results were obtained when LSM1APRO-LSM1A-162
GFP and LSM1BPRO-LSM1B-GFP transgenic plants were subjected to drought or high 163
salt (Supplemental Figure 2 C), providing evidence that LSM1 proteins localize to P-164
bodies in response to abiotic stress. 165
166
LSM1 proteins have been reported to be crucial for P-body integrity under heat stress 167
conditions (Perea-Resa et al., 2012). We examined whether they were also required for 168
P-body formation in response to low temperature. With this aim, the subcellular 169
localization of DCP2 and VCS, two P-body markers (Sheth and Parker, 2003; Xu et al., 170
2006), was compared in Col-0 (WT) and lsm1a lsm1b double mutant seedlings 171
containing 35S-GFP-DCP2 or 35S-GFP-VCS fusions (Perea-Resa et al., 2012) exposed 172
to 4ºC. While in WT plants both proteins accumulated in P-bodies, a mostly dispersed 173
cytosolic signal was observed in lsm1a lsm1b mutants (Figure 1 E), showing that, in 174
addition to accumulating in P-bodies, LSM1 proteins are also essential for the formation 175
of these cytoplasmic foci in response to low temperature. Similar results were obtained 176
when plants were subjected to water and salt stresses (Supplemental Figure 2 D), 177
demonstrating that LSM1 proteins also localize to P-bodies under these abiotic stresses, 178
and are necessary for the assembly of these foci in response to water deficiency and 179
high salt. 180
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The LSM1-7 complex regulates Arabidopsis tolerance to abiotic stress 182
To further characterize the role of LSM1 proteins and, therefore, of the LSM 183
cytoplasmic complex in plant adaptation to abiotic stress, we evaluated their possible 184
implication in Arabidopsis tolerance to freezing, drought and salinity. Freezing 185
tolerance was analyzed in non-acclimated and cold-acclimated (7 d, 4ºC) lsm1a lsm1b 186
mutants exposed for 6 h to different freezing temperatures (Catalá et al., 2014). Non-187
acclimated mutants presented a similar capacity to tolerate freezing as the WT, the LT50 188
(temperature that causes 50% lethality) values being in both cases around -4.5ºC 189
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(Supplemental Figure 3 A). By contrast, the freezing tolerance of cold-acclimated lsm1a 190
lsm1b mutants was significantly higher than that of WT plants. In this case, the 191
determined LT50 values were -11ºC and -9ºC, respectively (Figure 2A). 192
Drought tolerance was examined in lsm1a lsm1b seedlings transferred to plates 193
containing 25% PEG (Verslues et al., 2006). After one week, they exhibited 194
significantly enhanced tolerance compared with WT seedlings as revealed by the 195
quantification of their fresh weights and lateral roots (Figure 2 B). Tolerance to high salt 196
was assayed in lsm1a lsm1b seedlings grown one further week on plates containing 150 197
mM NaCl (Verslues et al., 2006). Mutants displayed lower fresh weights and shorter 198
main roots than WT seedlings (Figure 2 C), manifesting more sensitivity to salt stress. It 199
is worth noting that lsm1a lsm1b plants grown on soil showed identical drought-tolerant 200
and salt-sensitive tolerance phenotypes (Supplemental Figures 3 B and 3 C). 201
In all cases, lsm1a lsm1b mutants complemented with either LSM1APRO-LSM1A-GFP 202
(c-lsm1a) or LSM1BPRO-LSM1B-GFP (c-lsm1b) fusions (Perea-Resa et al., 2012) had 203
WT capacity to cold acclimate and to tolerate water deficiency and high salt (Figure 2 204
and Supplemental Figure 3), establishing that the stress-tolerance phenotypes of lsm1a 205
lsm1b were a direct consequence of the absence of LSM1A and LSM1B expression. 206
Together, these data provided genetic evidence that the LSM1-7 complex is 207
differentially involved in plant tolerance to abiotic stresses. It negatively regulates the 208
ability of Arabidopsis to cold acclimate and tolerate drought, but functions as a positive 209
regulator of salt tolerance. 210
211
The Arabidopsis LSM cytoplasmic complex differentially regulates gene 212
expression in response to abiotic stress 213
Since the Arabidopsis LSM1-7 complex influences gene expression by promoting RNA 214
decapping and decay (Perea-Resa et al., 2012), we examined whether it might affect 215
gene expression in response to abiotic stresses as a first step to understanding how it 216
differentially regulates plant tolerance to environmental challenges. High-throughput 217
RNA sequencing (RNAseq) was used to estimate the effect of lsm1a and lsm1b 218
mutations on the transcriptomes of Arabidopsis plants subjected to cold, drought or 219
high-salt conditions. For this, we sequenced cDNA libraries prepared from stress-220
treated lsm1a lsm1b and WT plants. The resulting reads (±12 Mbp/sample) were 221
mapped to the Arabidopsis genome (TAIR10 version) and gene expression changes in 222
the double mutant evaluated. In each treatment, the top 1000 upregulated and top 1000 223
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downregulated genes in lsm1a lsm1b, based on fold-change ratios with respect to their 224
corresponding controls, were considered for analysis. The expression levels of the top 225
1000 upregulated genes in mutant plants exposed for 24 h to 4ºC were increased at least 226
3.4-fold compared with the WT (Supplemental Data Set 1). Remarkably, 53.3% of these 227
genes (533) have been reported to be induced (≥2 fold) in response to cold (Kilian et al., 228
2007) (Supplemental Data Set 2). Many of them, moreover, have been associated with 229
the development of cold acclimation (Vogel et al., 2005; Cuevas et al., 2008; Miura and 230
Furumoto, 2013; Shi et al., 2015) and, therefore, could account for the tolerance 231
phenotype of the double mutant. The upregulation of some of these genes, including 232
LEA5, LEA7, ZAT12, NCED3, WRKY33, RD28, ERF53, PR5, WRKY46 and NCED5, in 233
lsm1a lsm1b was verified by qPCR, validating the RNAseq results (Figure 3 A). 234
Compared with the WT, the top 1000 downregulated genes in cold-treated mutants 235
displayed a decreased expression of at least 2.1-fold (Supplemental Data Set 3). Of 236
them, 311 (31.1%) have been described to be cold-induced (Kilian et al., 2007) 237
(Supplemental Data Set 4), but none as functioning as a negative regulator of cold 238
acclimation. Representative cold-inducible genes that were downregulated in lsm1a 239
lsm1b were also validated by qPCR (Supplemental Figure 4 A). 240
Under conditions of water deficiency (10 h, 55% PEG), the transcript levels of the top 241
1000 upregulated genes in lsm1a lsm1b plants were found to be higher, by at least 2-242
fold, than in the WT (Supplemental Data Set 5). Interestingly, 372 of these genes 243
(37.2%) have been shown to be induced (≥2 fold) in response to drought (Kilian et al., 244
2007) (Supplemental Data Set 6) and some of them, such as ABR1, ANAC019 or 245
ERF53, to have a positive role in Arabidopsis tolerance to drought (Tran et al., 2004; 246
Pandey et al., 2005; Cheng et al., 2012). qPCR experiments confirmed the upregulation 247
in lsm1a lsm1b plants of these and other genes that promote drought tolerance (Figure 3 248
B), which would account for the drought tolerance phenotype of the mutants and 249
validate the RNAseq data. Otherwise, the top 1000 downregulated genes in mutant 250
plants subjected to drought exhibited at least 1.9-fold lower expression than in WT 251
plants (Supplemental Data Set 7). Among them, 32.6% (326) have been previously 252
shown to be induced by water deficiency (Kilian et al., 2007) (Supplemental Data Set 253
8), although none acting as negative regulator of drought tolerance. These results were 254
also validated by analyzing representative drought-inducible genes that were 255
downregulated in lsm1a lsm1b using qPCR (Supplemental Figure 4 B). 256
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When comparing the transcriptome profiles of mutant and WT plants exposed to salt 257
stress (10 h, 150 mM NaCl), the expression levels of the top 1000 upregulated genes in 258
lsm1a lsm1b were at least 2.4-fold higher than in the WT (Supplemental Data Set 9). In 259
this case, 47% of the genes (470) have been reported to be salt-induced (≥2-fold) 260
(Kilian et al., 2007) (Supplemental Data Set 10), and several of them, such as 261
ANAC092, ATGSTU17 or AHK5, to act as negative regulators of salt tolerance in 262
Arabidopsis (Balazadeh et al., 2010; Chen et al., 2012; Pham et al., 2012). The 263
upregulation of these and other salt-responsive genes was verified by qPCR, validating 264
the RNAseq data (Figure 3 C). The top 1000 downregulated genes in mutant plants 265
showed at least 2.1-fold lower expression levels than in the WT (Supplemental Data Set 266
11). Strikingly, 457 (45.7%) of these genes were salt-induced (Kilian et al., 2007) 267
(Supplemental Data Set 12) and some of them, including DFR, PHI1 or CNI1 have been 268
described to be positive regulators of Arabidopsis tolerance to high salt (Cui et al., 269
2014; Peng et al., 2014). qPCR experiments confirmed that the expression of these and 270
other salt-regulated genes was lower in lsm1a lsm1b than in WT plants after salt 271
treatment, validating once again the RNAseq data (Figure 3 D). The altered expression 272
of the negative and positive regulators of salt tolerance described above was fully 273
consistent with the salt-sensitive phenotype exhibited by the double mutant. 274
In all cases, when RNAseq experiments were validated by qPCR we found a strong, 275
statistically significant correlation (Pearson r ≥ 0.909; P-value ≤ 0.0001) between the 276
fold-change results obtained from both kinds of analysis (Supplemental Figure 5). 277
Consistent with the close relationship existing between plant responses to low 278
temperature, water deficiency and high salt (Kilian et al., 2007), several genes were 279
regulated by the LSM1-7 complex in response to more than one stress condition. 280
Nonetheless, most LSM1-7-regulated genes were stress specific (Supplemental Figure 6 281
and Supplemental Data Sets 1-12). c-lsm1a plants presented WT expression patterns for 282
all validated genes (Figure 3). Overall, these results indicated that the Arabidopsis LSM 283
cytoplasmic complex regulates Arabidopsis tolerance to abiotic stresses by differentially 284
modulating stress-responsive gene expression. 285
286
The Arabidopsis LSM1-7 complex regulates the turnover of selected stress-287
dependent transcripts in response to abiotic stresses 288
Considering the capacity of the LSM cytoplasmic complex to control transcript turnover 289
through the interaction of LSM1 with target mRNAs, promoting their decapping and 290
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subsequent degradation (Chowdhury et al., 2007), we reasoned that it could 291
differentially regulate gene expression in response to abiotic stresses by inducing the 292
decay of selected transcripts in a stress-dependent manner. To investigate this idea, we 293
first identified direct targets of the complex under low temperature, drought and high 294
salt conditions by means of RIP assays. These assays were performed with c-lsm1a 295
plants grown under control conditions or exposed to 4ºC, 55% PEG or 150 mM NaCl. 296
Transcripts interacting with LSM1A-GFP were co-immunoprecipitated (co-IP) with 297
anti-GFP antibody and then identified by qPCR. Arabidopsis transgenic plants 298
expressing a GFP-tagged GRP7 protein (Streitner et al., 2012) were also used in RIP 299
experiments as a control for the specificity of the detected interactions. Given the 300
function of the LSM1-7 complex, we expected some of its direct targets to be among 301
the mRNAs upregulated in the stress-treated lsm1a lsm1b mutants. Targets, therefore, 302
were searched for among the cold-, drought- and salt-inducible transcripts whose levels 303
we had confirmed were upregulated in lsm1a lsm1b mutants in response to low 304
temperature, water stress and high salt, respectively (Figure 3A-C). As anticipated, 305
several cold-induced transcripts, including LEA7, ZAT12, NCED3, WRKY33, RD28, 306
ERF53, WRKY46, and NCED5 were significantly enriched (≥2 fold) in co-IP RNA 307
samples from cold-treated c-lsm1a plants but not in the RNA samples from GRP7-GFP 308
plants (Figure 4 A), providing evidence that they were direct targets of the Arabidopsis 309
LSM cytoplasmic complex in response to low temperature. Some mRNAs, such as 310
ZAT12, NCED3, RD28, ERF53, and NCED5, were already significantly detected, 311
although at lower levels, in co-IP RNA samples recovered from unstressed c-lsm1a 312
plants (Figure 4A), suggesting that they were also targets of the complex under control 313
conditions. 314
Compared to those obtained from GRP7-GFP plants, co-IP RNA samples obtained 315
from c-lsm1a plants exposed to water deficiency revealed a specific and significant 316
enrichment in ABR1, ANAC019, RD28, ERF53, and WRKY46 transcripts (Figure 4 B). 317
Consistent with the results obtained when looking for targets of the complex under cold 318
conditions (Figure 4A), RD28 and ERF53 mRNAs were also found to be significantly 319
detected in co-IP RNA samples from control c-lsm1a plants (Figure 4B). In the case of 320
RNA samples collected from c-lsm1a and GRP7-GFP plants subjected to high salt, RIP 321
assays uncovered a specific and significant enrichment of ANAC092, AHK5, ERF53, 322
and NCED5 mRNAs in c-lsm1a samples (Figure 4 C). ERF53 and NCED5 transcripts 323
were also significantly detected in co-IP RNA samples from non-stressed c-lsm1a plants 324
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(Figure 4C). 325
326
As anticipated from the function of the LSM cytoplasmic complex in mRNA 327
degradation by promoting the decapping of its targets (Perea-Resa et al., 2012), rapid 328
amplification of cDNA ends (RACE)-qPCR experiments revealed that LEA7, ZAT12, 329
NCED3, WRKY33, RD28, ERF53, WRKY46, and NCED5 messengers accumulated in 330
their capped forms in lsm1a lsm1b mutants compared with WT plants exposed to low 331
temperature (Figure 5 A). Additionally, the levels of capped ZAT12, NCED3, RD28, 332
ERF53, and NCED5 transcripts were higher in mutant than in WT plants grown under 333
control conditions (Figure 5A). RACE-qPCR experiments with RNAs from water-334
stressed lsm1a lsm1b and WT plants showed that capped ABR1, ANAC019, RD28, 335
ERF53, and WRKY46 messengers were significantly increased in the mutants. 336
Moreover, as expected, RD28 and ERF53 transcripts were also augmented in their 337
capped forms in unstressed mutants (Figure 5 B). The levels of capped ANAC092, 338
AHK5, ERF53 and NCED5 mRNAs were clearly higher in lsm1a lsm1b than in WT 339
plants exposed to NaCl (Figure 5 C). Furthermore, capped ERF53 and NCED5 340
transcripts also accumulated in mutants grown under standard conditions (Figure 5C). 341
The capped forms of all mRNAs analyzed were unaltered in c-lsm1a plants (Figure 5). 342
All of these results strongly suggested that the LSM1-7 complex regulates the turnover 343
of different selected stress-responsive target transcripts, including both specific and 344
non-specific ones, in response to different abiotic stresses. The existence of non-specific 345
target mRNAs, such as RD28, ERF53, WRKY46 and NCED5, has been already 346
evidenced (Figures 4 and 5). The existence of specific targets was established by 347
assessing the affinity of the complex for LEA7, ZAT12, NCED3, WRKY33, ABR1, 348
ANAC019, ANAC092 and AHK5 transcripts under different unfavorable conditions. 349
Thus, RIP experiments showed that LEA7, ZAT12, NCED3 and WRKY33, direct targets 350
of the LSM cytoplasmic complex in response to low temperature (Figures 4A and 5A), 351
were not enriched in co-IP RNA samples from c-lsm1a plants exposed to water 352
deficiency and high salt (Figure 6). In turn, ABR1 and ANAC019, direct targets of the 353
complex in response to drought (Figures 4B and 5B), were not recovered with LSM1-354
GFP in response to cold and salt stresses (Figure 6). Similarly, ANAC092 and AHK5, 355
direct targets of the complex in response to high salt (Figures 4C and 5C), were not 356
enriched in co-IP RNA samples from c-lsm1a plants subjected to 4ºC and drought 357
(Figure 6). In addition, as expected from the RIP assays, the levels of capped LEA7, 358
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ZAT12, NCED3 and WRKY33 messengers did not increase in lsm1a lsm1b mutants 359
under drought and high salt conditions (Figure 7 A), and ABR1 and ANAC019 mRNAs 360
did not accumulate in their capped forms in mutant plants in response to low 361
temperature and salt stress (Figure 7 B). Moreover, capped ANAC092 and AHK5 362
transcripts did not increase in lsm1a lsm1b in response to cold and water deficiency 363
(Figure 7 C). In all cases, c-lsm1a plants presented equivalent levels of capped mRNAs 364
as WT plants (Figure 7). 365
Together, our data indicated that the Arabidopsis LSM1-7 complex differentially 366
regulates gene expression in response to abiotic stresses, and consequently plant 367
tolerance to these challenging situations, by modulating in each case the decay of 368
selected, both specific and non-specific, stress-responsive transcripts. The fact that some 369
of these mRNAs are, furthermore, direct targets of the complex at 20ºC, suggests that it 370
also regulates stress-responsive gene expression under control conditions. 371
372
The Arabidopsis LSM cytoplasmic complex regulates ABA biosynthesis in 373
response to abiotic stress 374
Interestingly, among the mRNAs whose decay was differentially regulated by the 375
Arabidopsis LSM1-7 complex in response to abiotic stresses were those encoding 376
NCED3 and NCED5, two key enzymes in the biosynthesis of the phytohormone 377
abscisic acid (ABA) that plays a critical role in plant responses to abiotic stresses (Jia et 378
al., 2002; Cuevas et al., 2008; Wu et al., 2009; Frey et al., 2012). In fact, while NCED3 379
transcripts were direct targets of the complex specifically after cold treatment (Figures 6 380
and 7A), those of NCED5 were direct targets under both cold and high-salt conditions 381
(Figures 4A, 4C, 5A and 5C) but not following drought (Supplemental Figure 7). These 382
data strongly suggested that the LSM1-7 complex might differentially regulate ABA 383
biosynthesis in Arabidopsis in response to abiotic stresses. To test this prediction, we 384
measured the ABA content of WT and lsm1a lsm1b mutant plants grown under control 385
conditions or exposed to low temperature (4ºC), water deficiency (55% PEG), or high 386
salt (150 mM NaCl). When exposed to low temperature, ABA levels increased in both 387
WT and lsm1a lsm1b mutants but the increase was significantly higher in mutants than 388
in WT plants (Figure 8 A). Unstressed lsm1a lsm1b mutants also showed higher levels 389
of ABA than WT plants (Figure 8A). These levels, however, were much lower than 390
those detected after cold treatment, paralleling the lower binding affinity of the LSM 391
cytoplasmic complex for NCED3 and NCED5 mRNAs at 20ºC compared with 4ºC 392
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(Figures 4A and 5A). c-lsm1a plants exhibited similar ABA content as WT plants 393
(Figure 8A), providing evidence that the Arabidopsis LSM1-7 complex attenuates ABA 394
biosynthesis in response to low temperature and, to a lesser extent, under standard 395
conditions by promoting the decay of NCED3 and NCED5 messengers. 396
ABA levels also increased very prominently in Arabidopsis WT plants exposed to water 397
stress or high salt (Figure 8 B). In the case of lsm1a lsm1b mutants, water deficiency 398
caused an accumulation of ABA similar to that of WT plants. By contrast, salt stress 399
provoked an accumulation of ABA that was significantly higher than in WT plants. c-400
lsm1a plants exhibited similar ABA content as the WT in response to both drought and 401
salt stresses (Figure 8B). Therefore, as predicted from the expression and RIP analyses, 402
the LSM cytoplasmic complex also mediates the biosynthesis of ABA in Arabidopsis 403
plants exposed to salt stress but it is not involved in ABA biosynthesis in response to 404
water stress. Collectively, these findings demonstrated that the Arabidopsis LSM1-7 405
complex guarantees adequate levels of ABA in Arabidopsis plants exposed to different 406
abiotic stresses by differentially regulating the decay of NCED3 and NCED5 transcripts. 407
408
DISCUSSION 409
The control of mRNA stability is a key step in the regulation of gene expression. 410
However, the effect of mRNA decapping on plant transcriptome reprogramming in 411
response to abiotic stress, and its importance to stress tolerance are largely unknown. 412
Here, we show that the LSM1-7 decapping activator complex serves as an integration 413
node of regulatory pathways mediating plant tolerance to abiotic stresses. It regulates 414
Arabidopsis tolerance to freezing, drought and high salt by interacting with selected, 415
specific and nonspecific, stress-inducible transcripts under each stress condition to 416
promote their decapping and subsequent degradation, which ultimately ensures the 417
appropriate patterns of downstream stress-responsive gene expression. Interestingly, 418
one of the regulatory pathways mediated by the LSM1-7 complex is the one leading to 419
ABA biosynthesis. We demonstrate that this complex modulates the levels of ABA in 420
response to adverse environmental situations through the differential regulation of 421
NCED3 and NCED5 mRNA turnover. 422
The expression analysis described in this work revealed that LSM1-7 genes are 423
differentially regulated in response to abiotic stresses. LSM1-7 mRNAs accumulate in 424
response to cold but not in response to drought or high salt. The mechanisms underlying 425
this regulation are still under investigation. Consistent with the expression results, an 426
14
increase in LSM1A and LSM1B protein levels was observed only in plants exposed to 427
low temperature. Remarkably, however, Arabidopsis LSM1 proteins were found to 428
localize to P-bodies in response to the different stresses assayed, indicating that the 429
molecular mechanisms mediating LSM1 localization to these foci do not depend on 430
protein levels. In this way, DCP1 has also been reported to localize to P-bodies under 431
stress conditions even though its levels do not increase (Motomura et al., 2015). These 432
findings suggest that the localization of LSM1 to P-bodies, like that of DCP1 and other 433
Arabidopsis RNA-decay related proteins (Merret et al., 2013; Motomura et al., 2015), is 434
induced in plants exposed to abiotic stresses. Moreover, our results show that LSM1 435
proteins are required for P-body formation under challenging situations, indicating that 436
the assembly of these foci is also promoted by adverse environmental conditions. How 437
abiotic stresses regulate the cytoplasmic dynamics of proteins involved in RNA 438
degradation and, ultimately, P-body formation is poorly understood. Phosphorylation of 439
different components of human and Arabidopsis mRNA decapping machineries by 440
mitogen-activated protein kinases (MPKs) during stress responses seems to be 441
necessary for their cytoplasmic localization and for P-body assembly (Rzeczkowski et 442
al., 2011; Roux et al., 2015). The sequence of LSM1 proteins (Perea-Resa et al., 2012) 443
contains a consensus motif S/T-P for phosphorylation by MPK. It is, therefore, tempting 444
to speculate that the subcellular localization of LSM1 proteins could be regulated by 445
MPKs in response to abiotic stresses. 446
Our results demonstrate that the LSM1-7 complex differentially regulates Arabidopsis 447
tolerance to challenging environmental situations. In fact, the complex restrains the 448
plant’s capacity to cold acclimate and tolerate drought while promoting its tolerance to 449
high salt. The implication of a single component of the mRNA decay apparatus in a 450
range of abiotic stress responses has not been shown in any system, and uncovers the 451
enormous potential of this apparatus to precisely modulate the adaptation of a given 452
organism to its surroundings. The LSM cytoplasmic complex, therefore, seems to serve 453
as a regulatory node where pathways mediating abiotic stress responses converge and 454
integrate to guarantee the precise development of Arabidopsis tolerance to freezing, 455
drought and salinity. Our results, furthermore, provide evidence that the LSM1-7 456
complex operates in Arabidopsis tolerance to abiotic stresses by modulating stress-457
responsive gene expression. Hundreds of genes are specifically regulated by the 458
complex under low temperature, water deficiency or high salt. Moreover, the complex 459
also regulates the expression of many genes in response to more than one abiotic stress, 460
15
which indicates that it mediates gene expression under different unfavorable situations 461
via specific and shared signaling pathways. Interestingly, numerous specific and 462
nonspecific genes that are regulated by the LSM cytoplasmic complex under each stress 463
condition have been described to have a role in plant tolerance to the relevant condition, 464
substantiating a major and differential function for the complex in plant tolerance to 465
abiotic stresses by ensuring appropriate patterns of stress-responsive gene expression. 466
DCP5 has also been shown to act in abiotic stress tolerance through the regulation of 467
stress-related gene expression. It enhances Arabidopsis tolerance to dehydration by 468
modulating numerous dehydration-responsive genes (Xu and Chua, 2012). Whether 469
DCP5 and/or other components of the Arabidopsis mRNA decay apparatus have the 470
ability, similar to the LSM1-7 complex, to differentially regulate gene expression in a 471
stress-dependent manner remains unknown. 472
The role of the LSM cytoplasmic complex in the transcriptome reprogramming that 473
takes place in Arabidopsis when subjected to abiotic stresses is a consequence of its 474
capacity to modulate the turnover of stress-selected target transcripts. In fact, by 475
analyzing the interaction between mRNAs whose levels are attenuated by the complex 476
in response to abiotic stresses and the LSM1 protein, we have been able to identify 477
transcripts that are direct targets of the complex when Arabidopsis plants are exposed to 478
low temperature, drought or high salt, and may act as positive or negative regulators of 479
gene expression. Stress-selected targets are then decapped and subsequently degraded. It 480
is noteworthy that most of them are specific for each stress while others, however, are 481
selected by the complex in response to various adverse conditions. Modulating the 482
differential accumulation of the selected specific and nonspecific target transcripts, 483
therefore, ensures adequate stress-responsive gene expression under each abiotic stress, 484
and ultimately contributes to the capacity of the LSM1-7 complex to regulate 485
Arabidopsis tolerance to freezing, drought and salinity. The Arabidopsis decapping 486
activator DCP5 has also been reported to select target transcripts in response to an 487
individual abiotic stress, namely water deficiency (Xu and Chua, 2012). Our results, 488
nonetheless, unveil a more intricate scenario when considering the regulation of gene 489
expression and plant adaptation by the mRNA decay apparatus under unfavorable 490
circumstances. They provide evidence that one component can select different targets 491
depending on the stress situations, highlighting a previously unknown regulatory 492
mechanism of mRNA turnover during stress responses. 493
16
Remarkably, among the selected stress-dependent transcripts that are triggered by the 494
Arabidopsis LSM cytoplasmic complex for decapping in response to abiotic stresses, 495
we found those encoding NCED3 and NCED5, two key enzymes in ABA biosynthesis 496
(Tan et al., 2003; Frey et al., 2012). NCED3 and NCED5 messengers are direct targets 497
of the complex under low temperature but not under drought conditions. Under salt 498
stress only NCED5 is targeted by the LSM1-7 complex. ABA is biosynthesized de novo 499
in plants subjected to cold, drought or salinity, and plays a pivotal role in plant 500
responses to abiotic stresses (Jia et al., 2002; Cuevas et al., 2008; Wu et al., 2009). In 501
Arabidopsis, the accumulation of ABA after abiotic stress exposure seems to be the 502
result of NCED3 and NCED5 expression (Tan et al., 2003; Frey et al., 2012). So far, 503
available data had indicated that this expression is regulated at the transcriptional level 504
(Barrero et al., 2006; Cuevas et al., 2008; Jiang et al., 2012). The results reported here 505
show that the mRNA decay machinery, and in particular the decapping apparatus, has a 506
crucial function in establishing the levels of NCED3 and NCED5 mRNAs when plants 507
are exposed to stressful circumstances, indicating that these levels are also regulated by 508
post-transcriptional mechanisms. Our findings, therefore, reveal a layer of regulation of 509
ABA biosynthesis in response to abiotic stresses. Consistent with its role in 510
differentially modulating the turnover of NCED3 and NCED5 transcripts under adverse 511
environmental circumstances, our data also demonstrate that the LSM cytoplasmic 512
complex contributes to establish the appropriate levels of ABA under cold and high salt 513
conditions. It is not involved, however, in modulating the accumulation of ABA caused 514
by water deficiency. 515
Based on the results described in this work, a hypothetical model for the function of the 516
Arabidopsis LSM1-7 complex in plant adaptation to abiotic stresses is presented in 517
Figure 9. In response to low temperature, drought and salinity, the complex would 518
preferentially localize to P-bodies. There, depending on the stress, the complex would 519
interact with selected transcripts promoting their decapping and subsequent 5’-3’ 520
degradation. The vast majority of selected transcripts are stress specific; others, 521
however, interact with the complex under more than one stress condition. In most cases, 522
target transcripts would correspond to stress-inducible genes, and some of them, 523
moreover, encode proteins involved in regulating, positively or negatively, Arabidopsis 524
tolerance to abiotic stresses. The degradation of the LSM1-7 target transcripts selected 525
in response to a given abiotic stress would have, in turn, a substantial effect on the 526
downstream stress-regulated gene expression that would contribute to shape the 527
17
transcriptome reprogramming required for plant adaptation to that stress. In conclusion, 528
the results presented here reveal that the LSM1-7 decapping activator complex plays a 529
critical role in plant adaptation to abiotic stresses by modulating the turnover of selected 530
stress-specific and nonspecific target transcripts, and, consequently, stress-regulated 531
gene expression. Identifying the molecular mechanisms whereby the LSM cytoplasmic 532
complex selects different targets depending on the stress conditions is a clear goal of 533
future studies. 534
535
18
METHODS 536
Plant Materials, Growth Conditions, Treatments and Tolerance Assays 537
Arabidopsis Columbia (Col-0) ecotype was used in all experiments. The lsm1a lsm1b 538
double mutants, transgenic lines LSM1APRO-LSM1A-GFP (c-lsm1a) and LSM1BPRO-539
LSM1B-GFP (c-lsm1b), lines LSM1APRO-LSM1A-GFP and LSM1BPRO-LSM1B-GFP 540
expressing 35S-DCP1-RFP construct, and lsm1a lsm1b lines expressing the 35S-GFP-541
DCP2 or 35S-GFP-VCS constructs were previously described (Perea-Resa et al., 2012). 542
The Arabidopsis transgenic line expressing the GRP7PRO-GRP7-GFP fusion (Streitner 543
et al., 2012) was kindly provided by Dorothee Staiger (University of Bielefeld, 544
Germany). Plants were grown at 20ºC under long-day photoperiod (16 h of cool-white 545
fluorescent light, photon flux of 90 µmol m-2s-1) in pots containing a mixture of organic 546
substrate and vermiculite (3:1 v/v) or in Petri dishes containing Murashige and Skoog 547
medium supplemented with 1% sucrose (GM) and solidified with 0.8% (w/v) agar. 548
Low-temperature treatment for gene expression and immunoblot analyses was 549
performed by transferring plants growing in Petri dishes or on soil under control 550
conditions to a growth chamber set to 4°C for different periods of time under long-day 551
photoperiods with a photon flux of 40 mmol m-2s-1. For the rest of experiments, plants 552
were always exposed to 4ºC for 24 h. In all cases, water and salt stress treatments were 553
accomplished by transferring plants growing in Petri dishes under standard conditions to 554
plates containing GM medium supplemented with 55% PEG or 150 mM NaCl, 555
respectively, for 10 h. For immunoblot analysis, water and salt stress treatments were 556
also carried out for 24 h. Tolerance to freezing temperatures was determined on 2-week-557
old plants grown on soil as described (Catalá et al., 2014). In vitro tolerance to water 558
(25% PEG) and salt (150 mM NaCl) stresses was assayed on 7-day-old seedlings grown 559
on GM medium as reported (Verslues et al., 2006). The tolerance of 2-week-old plants 560
growing on soil to these stresses was estimated as the number of surviving individuals 561
after 10 days of water deprivation and 5 days of re-watering, or after watering with 250 562
mM NaCl for 10 days. In all cases, data reported are expressed as means of three 563
independent experiments with 50 plants each. 564
565
Microscopic Analysis 566
Subcellular localization of LSM1A-GFP, LSM1B-GFP, DCP1-RFP, GFP-DCP2 and 567
GFP-VCS fusion proteins was performed by confocal microscopy in roots from 6-day-568
old transgenic seedlings grown under control or subjected to stress conditions as 569
19
described above. Treatments with cycloheximide were performed by incubating stressed 570
seedlings in GM liquid medium supplemented with 200 µg/ml cycloheximide for 2 h at 571
20ºC or 4ºC. Microscopy images were collected using a Confocal Laser Spectral 572
microscope TCS SP5 (Leica Mycrosystem, Wetzlar, Germany). The excitation lines for 573
imaging GFP and RFP fusions were 488 and 561 nm, respectively. 574
575
ABA Measurements 576
ABA levels were determined in 20 mg of 2-week-old WT, lsm1a lsm1b and c-lsm1a 577
plants grown under control or stress conditions as described (Turečková et al., 2009). 578
All experiments were repeated as four biological replicates employing 10 pmol stable 579
isotope-labelled standard to validate the LC-MS method. 580
581
Immunoblot Analysis 582
Total protein was extracted from 2-week-old Arabidopsis LSM1APRO-LSM1A-GFP, 583
LSM1BPRO-LSM1B-GFP and WT plants grown under control or stress conditions as 584
reported (Catalá et al., 2014). Monoclonal anti-GFP (ab290; GR158277-1, Abcam) was 585
used as primary antibody, and horseradish peroxidase-conjugated anti-rabbit as 586
secondary antibody. Coomassie staining of the large subunit of Rubisco was used as a 587
loading control. All assays were carried out in triplicate employing three independent 588
protein samples. 589
590
Gene Expression Analysis and RNAseq Experiments 591
For gene expression, qPCR experiments were performed as described (Catalá et al., 592
2014). In all cases, the relative expression values were determined using AT4G24610 as 593
a control (Czechowski et al., 2005). Primers used are listed in Supplemental Data Set 594
13. All reactions were performed in triplicate employing three independent RNA 595
samples. 596
For RNAseq experiments, total RNA was extracted from 2-week-old WT and lsm1a 597
lsm1b plants subjected to stress conditions (see above) using TRIzol® Reagent (Life 598
Technologies) and cleaned with RNeasy Plant Mini Kit (Quiagen). cDNA libraries were 599
generated from 3 independent RNA preparations each. RNA quality, library preparation 600
and subsequent sequencing were performed by the staff of the Beijing Genome Institute 601
(BGI). RNA-seq reads were aligned to the TAIR10 Col-0 reference genome using 602
20
TopHat2 (Kim et al., 2013) with default parameters. Uniquely mapped reads 603
(Supplemental Data Set 14) were counted per representative gene model (excluding 604
introns) according to the TAIR10 annotation using custom R scripts. Only genes with 605
rpkm greater than 1 in at least one sample were used for differential expression analysis 606
between WT and lsm1a lsm1b plants in each condition using DEseq2 (Love et al., 607
2014). This package internally estimates size factors for each sample, calculates 608
dispersion for each gene and then fits a negative binomial GLM to detect differentially 609
expressed genes taking into account the size factors and dispersion values. 610
611
Capped mRNA Analysis and RIP Experiments 612
Capped transcript levels were determined essentially as reported (Perea-Resa et al., 613
2012). Total RNA from 2-week-old plants grown on GM plates under control or stress 614
conditions (see above) was employed. Capped mRNAs were quantified by qRT-PCR 615
using reverse specific-gene primers (Supplemental Data Set 13) and the 5´RACE Inner 616
Primer included in the First Choice RLM-RACE kit (Ambion). The levels of capped 617
transcripts were corrected with respect to the levels of their corresponding total 618
transcripts, determined by qPCR using gene-specific primers (Supplemental Data Set 619
13), and represented relative to the levels in WT plants under control conditions for 620
each gene. In all cases, reactions were performed in triplicate employing three 621
independent RNA samples. 622
In vivo RIP experiments were carried out essentially as described (Streitner et al., 2012). 623
Extracts from 2-week-old c-lsm1a and GRP7-GFP plants grown on GM plates under 624
control or stress conditions (see above) were incubated overnight at 4ºC with GFP-Trap 625
beads (Chromotek). Beads were subsequently washed three times prior to RNA 626
extraction using TRIzol® Reagent (Life Technologies). RNAs resulting from inputs, 627
prepared in parallel, and from coprecipitated samples, were subsequently quantified by 628
qPCR (see above) using gene-specific primers (Supplemental Data Set 13). Transcript 629
levels in coprecipitated samples from c-lsm1a plants were corrected with respect to their 630
corresponding input values and represented relative to their levels in coprecipitated 631
samples from GRP7-GFP plants for each gene. All assays were performed in triplicate 632
employing three independent RNA samples. 633
634
Statistical Analysis 635
21
Data sets were analyzed using Prism 6 software (GraphPad Software Inc., USA). 636
Comparisons between two groups were made using Student's t‐test. Comparisons 637
between multiple groups were made using one‐way or two-way analysis of variance 638
(ANOVA) test depending whether one or two different variables were considered, 639
respectively. Correlation plots were computed using Prism 6 software (GraphPad 640
Software Inc., USA) from log2-transformed values and shown the relationship between 641
qRT-PCR results (X-axis) and the corresponding data from RNA-seq (y-axis). 642
643
Accession Numbers 644
Sequence data from this article can be found in the GeneBank/EMBL data libraries 645
under the accession numbers listed in Supplemental Data Set 15. The full name of the 646
genes mentioned in this article are also included in Supplemental Data Set 15. The 647
complete genome-wide data from this publication have been submitted to the GEO 648
database (http://www.ncbi.nlm.nih.gov/geo/) and assigned the identifier accession 649
GSE70491. 650
651
Supplemental Data 652
Supplemental Figure 1. Expression analysis of Arabidopsis LSM1-7 genes in response 653
to abiotic stresses. 654
Supplemental Figure 2. Arabidopsis LSM1 proteins do not accumulate in response to 655
drought and salinity but localize to P-bodies under these abiotic stresses. 656
Supplemental Figure 3. Constitutive freezing tolerance and tolerance of drought and 657
salinity of lsm1a lsm1b plants grown on soil. 658
Supplemental Figure 4. Expression levels of genes downregulated in lsm1a lsm1b 659
plants in response to cold and drought. 660
Supplemental Figure 5. Correlation between RNAseq and qPCR results. 661
Supplemental Figure 6. Venn diagrams of de-regulated transcripts in lsm1a lsm1b 662
plants under cold, drought and salt stresses. 663
Supplemental Figure 7. The Arabidopsis LSM1-7 complex differentially interacts with 664
and promotes the decapping of NCED5 transcripts in response to drought and high salt. 665
Supplemental Data Set 1. Top 1000 upregulated genes in lsm1a lsm1b mutants in 666
response to low temperature. 667
Supplemental Data Set 2. Cold-inducible genes upregulated in lsm1a lsm1b mutants in 668
response to low temperature. 669
22
Supplemental Data Set 3. Top 1000 downregulated genes in lsm1a lsm1b mutants in 670
response to low temperature. 671
Supplemental Data Set 4. Cold-inducible genes downregulated in lsm1a lsm1b 672
mutants in response to low temperature. 673
Supplemental Data Set 5. Top 1000 upregulated genes in lsm1a lsm1b mutants in 674
response to drought. 675
Supplemental Data Set 6. Drought-inducible genes upregulated in lsm1a lsm1b 676
mutants in response to drought. 677
Supplemental Data Set 7. Top 1000 downregulated genes in lsm1a lsm1b mutants in 678
response to drought. 679
Supplemental Data Set 8. Drought-inducible genes downregulated in lsm1a lsm1b 680
mutants in response to drought. 681
Supplemental Data Set 9. Top 1000 upregulated genes in lsm1a lsm1b mutants in 682
response to salt stress. 683
Supplemental Data Set 10. Salt-inducible genes upregulated in lsm1a lsm1b mutants in 684
response to salt stress. 685
Supplemental Data Set 11. Top 1000 downregulated genes in lsm1a lsm1b mutants in 686
response to salt stress. 687
Supplemental Data Set 12. Salt-inducible genes downregulated in lsm1a lsm1b 688
mutants in response to salt stress. 689
Supplemental Data Set 13. Oligonucleotides used in this work. 690
Supplemental Data Set 14. Total mapped and unmapped reads obtained for each 691
sample by RNAseq experiments. 692
Supplemental Data Set 15. List of accession numbers appearing in this study. 693
694
ACKNOWLEDGEMENTS 695
We thank D. Staiger for the LSMGRP7PRO-GRP7-GFP Arabidopsis transgenic line, and 696
J.J. Sanchez-Serrano, J.A. Jarillo and R. Solano for discussions and comments. This 697
work was supported by grants BIO2010-17545 and BIO2013-47788-R from MINECO 698
to J.S., GA14-34792S from CSF to O. N. and MCB-1022435 from NSF to L.S. R.C. is 699
supported by a JAE-DOC contract from the CSIC and C.C. is recipient of a FPI 700
fellowship from MINECO. 701
702
AUTHOR CONTRIBUTIONS 703
23
C.P., C.C., O.N., L.S., and J.S. conceived and designed the experiments. C.P., C.C., 704
R.C., V.T., and W.Z. performed the experiments. C.P., C.C., R.C., O.N., L.S., J.M.M., 705
and J.S. analyzed the data. C.P. and J.S. wrote the paper. 706
707
24
REFERENCES 708
Balazadeh, S., Siddiqui, H., Allu, A.D., Matallana-Ramirez, L.P., Caldana, C., 709
Mehrnia, M., Zanor, M.I., Köhler, B., and Mueller-Roeber, B. (2010). A gene 710
regulatory network controlled by the NAC transcription factor 711
ANAC092/AtNAC2/ORE1 during salt-promoted senescence. Plant J. 62: 250-264. 712
713
Barrero, J.M., Rodríguez, P.L., Quesada, V., Piqueras, P., Ponce, M.R., and 714
Micol, J.L. (2006). Both abscisic acid (ABA)-dependent and ABA-independent 715
pathways govern the induction of NCED3, AAO3 and ABA1 in response to salt stress. 716
Plant Cell Environ. 29: 2000-2008. 717
718
Catalá, R., López-Cobollo, R., Castellano, M.M., Angosto, T., Alonso, J.M., Ecker, 719
J.R., and Salinas, J. (2014). The Arabidopsis 14-3-3 Protein RARE COLD 720
INDUCIBLE 1A Links Low-Temperature Response and Ethylene Biosynthesis to 721
Regulate Freezing Tolerance and Cold Acclimation. Plant Cell 26: 3326-3342. 722
723
Chen, J.H., Jiang, H.W., Hsieh, E.J., Chen, H.Y., Chien, C.T., Hsieh, H.L., and 724
Lin, T.P. (2012). Drought and Salt Stress Tolerance of an Arabidopsis Glutathione S-725
Transferase U17 Knockout Mutant Are Attributed to the Combined Effect of 726
Glutathione and Abscisic. Acid Plant Physiol. 158: 340-351. 727
728
Cheng, M.C., Hsieh, E.J., Chen, J.H., Chen, H.Y., and Lin, T.P. (2012). Arabidopsis 729
RGLG2, Functioning as a RING E3 Ligase, Interacts with AtERF53 and Negatively 730
Regulates the Plant Drought Stress Response. Plant Physiol. 158: 363-375. 731
732
Chowdhury, A., Mukhopadhyay, J., and Tharun, S. (2007). The decapping activator 733
Lsm1p-7p–Pat1p complex has the intrinsic ability to distinguish between 734
oligoadenylated and polyadenylated RNAs. RNA 13: 998-1016. 735
736
Coller, J., and Parker, R. (2004). Eukaryotic mRNA decapping. Annu. Rev. Biochem. 737
73: 861-890. 738
739
Cuevas, J.C., López-Cobollo, R., Alcázar, R., Zarza, X., Koncz, C., Altabella, T., 740
Salinas, J., Tiburcio, A.F., and Ferrando, A. (2008). Putrescine Is Involved in 741
25
Arabidopsis Freezing Tolerance and Cold Acclimation by Regulating Abscisic Acid 742
Levels in Response to Low Temperature. Plant Physiol. 148: 1094-1105. 743
744
Cui, P., Zhang, S., Ding, F., Ali, S., and Xiong, L. (2014). Dynamic regulation of 745
genome-wide pre-mRNA splicing and stress tolerance by the Sm-like protein LSm5 in 746
Arabidopsis. Genome Biol. 15: 1-18. 747
748
Czechowski, T., Stitt, M., Altmann, T., Udvardi, M.K., and Scheible, W.R. (2005). 749
Genome-wide identification and testing of superior reference genes for transcript 750
normalization in Arabidopsis. Plant Physiol. 139: 5-17. 751
752
Fillman, C., and Lykke-Andersen, J. (2005). RNA decapping inside and outside of 753
processing bodies. Curr. Opin. Cell Biol. 17: 326-331. 754
755
Frey, A., Effroy, D., Lefebvre, V., Seo, M., Perreau, F., Berger, A., Sechet, J., To, 756
A., North, H.M., and Marion-Poll, A. (2012). Epoxycarotenoid cleavage by NCED5 757
fine-tunes ABA accumulation and affects seed dormancy and drought tolerance with 758
other NCED family members. Plant J. 70: 501-512. 759
760
Goeres, D.C., Van Norman, J.M., Zhang, W., Fauver, N.A., Spencer, M.L., and 761
Sieburth, L.E. (2007). Components of the Arabidopsis mRNA decapping complex are 762
required for early seedling development. Plant Cell 19: 1549-1564. 763
764
Guerra, D., Crosatti, C., Khoshro, H.H., Mastrangelo, A.M., Mica, E., and 765
Mazzucotelli, E. (2015). Post-transcriptional and post-translational regulations of 766
drought and heat response in plants, a spider’s web of mechanisms. Front. Plant Sci. 6: 767
1-14. 768
769
Gujjar, R.S., Akhtar, M., and Singh, M. (2014). Transcription factors in abiotic stress 770
tolerance. Ind. J. Plant Physiol. 19: 306-316. 771
772
26
Jia, W., Wang, Y., Zhang, S., and Zhang, J. (2002). Salt-stress-induced ABA 773
accumulation is more sensitively triggered in roots than in shoots. J. Exp. Bot. 53: 774
2201-2206. 775
776
Jiang, Y., Liang, G., and Yu, D. (2012). Activated Expression of WRKY57 Confers 777
Drought Tolerance in Arabidopsis. Mol. Plant 5: 1375-1388. 778
779
Kilian, J., Whitehead, D., Horak, J., Wanke, D., Weinl, S., Batistic, O., D´Angelo, 780
C., Bornberg-Bauer, E., Kudla, J., and Harter, K. (2007). The AtGenExpress global 781
stress expression data set, protocols, evaluation and model data analysis of UV-B light, 782
drought and cold stress responses. Plant J. 50: 347-363. 783
784
Kim, D., Pertea, G., Trapnell, C., Pimentel, H., Kelley, R., and Salzberg, S.L. 785
(2013). TopHat2: accurate alignment of transcriptomes in the presence of insertions, 786
deletions and gene fusions. Genome Biol. 14: R36. 787
788
Love, M.I., Huber, W., and Anders, S. (2014). Moderated estimation of fold change 789
and dispersion for RNA-seq data with DESeq2. Genome Biol. 15: 550. 790
791
Merret, R., Descombin, J., Juan, Y.T., Favory, J.J., Carpentier, M.C., Chaparro, 792
C., Charng, Y., Deragon, J.M., and Bousquet-Antonelli, C. (2013). XRN4 and 793
LARP1 Are Required for a Heat-Triggered mRNA Decay Pathway Involved in Plant 794
Acclimation and Survival during Thermal Stress. Cell Reports 5: 1279-1293. 795
796
Miura, K., and Furumoto, T. (2013). Cold Signaling and Cold Response in Plants. Int. 797
J. Mol. Sci. 14: 5312-5337. 798
799
Motomura, K., Le, Q.T.N., Hamada, T., Kutsuna, N., Mano, S., Nishimura, M., 800
and Watanabe, Y. (2015). Diffuse Decapping Enzyme DCP2 Accumulates in DCP1 801
Foci Under Heat Stress in Arabidopsis thaliana. Plant Cell Physiol. 56: 107-115. 802
803
27
Nakashima, K., Yamaguchi-Shinozaki, K., and Shinozaki, K. (2014). The 804
transcriptional regulatory network in the drought response and its crosstalk in abiotic 805
stress responses including drought, cold, and heat. Front. Plant Sci. 16: 170. 806
807
Pandey, G.H., Grant, J.J., Cheong, Y.H., Kim, B.G., Li, L., and Luan, S. (2005). 808
ABR1, an APETALA2 Domain Transcription Factor That Functions as a Repressor of 809
ABA Response in Arabidopsis. Plant Plysiol. 139: 1185-1193. 810
811
Parker, R. (2012). RNA Degradation in Saccharomyces cerevisae. Genetics 191: 671-812
702. 813
814
Peng, J., Li, Z., Wen, X., Li, W., Shi, H., Yang, L., Zhu, H., and Guo, H. (2014). 815
Salt-Induced Stabilization of EIN3/EIL1 Confers Salinity Tolerance by Deterring ROS 816
Accumulation in Arabidopsis. PLOS Genetics 10: e1004664. 817
818
Perea-Resa, C., Hernández-Verdeja, T., López-Cobollo, R., Castellano, M.M., and 819
Salinas, J. (2012). LSM Proteins Provide Accurate Splicing and Decay of Selected 820
Transcripts to Ensure Normal Arabidopsis Development. Plant Cell 24: 4930-4947. 821
822
Pham, J., Liu, J., Bennett, M.H., Mansfield, J.W., and Desikan, R. (2012), 823
Arabidopsis histidine kinase 5 regulates salt sensitivity and resistance against bacterial 824
and fungal infection. New Phytol. 194: 168-180. 825
826
Roux, M.E., Rasmussen, M.W., Palma, K., Lolle, S., Mateu-Regué, A., Bethke, G., 827
Glazebrook, J., Zhang, W., Sieburth, L., Larsen, M.R., Mundy, J., and Petersen, 828
M. (2015). The mRNA decay factor PAT1 functions in a pathway including MAP 829
kinase 4 and immune receptor SUMM2. EMBO J. 34: 593-608. 830
831
Rzeczkowski, K., Beuerlein, K., Müller, H., Dittrich-Breiholz, O., Schneider, H., 832
Ketter-Buhrow, D., Holtmann, H., and Kracht, M. (2011). C-Jun N-terminal kinase 833
phosphorylates DCP1a to control formation of P bodies. J. Cell Biol. 194: 581-596. 834
835
28
Sheth, U., and Parker, R. (2003). Decapping and Decay of Messenger RNA Occur in 836
Cytoplasmic Processing Bodies. Science 300: 805-808. 837
838
Shi, Y., Ding, Y., and Yang, S. (2015). Cold Signal Transduction and its Interplay with 839
Phytohormones During Cold Acclimation. Plant Cell Physiol. 56: 7-15. 840
841
Streitner, C., Köster, T., Simpson, C.G., Shaw, P., Danisman, S., Brown, J.W.S., 842
and Staiger, D. (2012). An hnRNP-like RNA-binding protein affects alternative 843
splicing by in vivo interaction with transcripts in Arabidopsis thaliana. Nucleic Acis. 844
Res. 40: 11240-11255. 845
846
Tan, B.C., Joseph, L.M., Deng, W.T., Liu, L., Li, Q.B., Cline, K., and McCarty, 847
D.R. (2003). Molecular Characterization of the Arabidopsis 9-cis epoxycarotenoid 848
dioxygenase gene family. Plant J. 35: 44-56. 849
850
Tran, L.S.P., Nakashima, K., Sakuma, Y., Simpson, S.D., Fujita, Y., Maruyama, 851
K., Fujita, M., Seki, M., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2004). 852
Isolation and Functional Analysis of Arabidopsis Stress-Inducible NAC Transcription 853
Factors That Bind to a Drought-Responsive cis-Element in the early responsive to 854
dehydration stress 1 Promoter. Plant Cell 16: 2481-2498. 855
856
Turečková, V., Novák, O., and Strnad, M. (2009). Profiling ABA metabolites in 857
Nicotiana tabacum L leaves by ultra-performance liquid chromatography–electrospray 858
tandem mass spectrometry. Talanta 80: 390-399. 859
860
Verslues, P.E., Agarwal, M., Katiyar-Agarwal, S., Zhu, J., and Zhu, J.K. (2006). 861
Methods and concepts in quantifying resistance to drought, salt and freezing, abiotic 862
stresses that affect plant water status. Plant J. 45: 523-539. 863
864
Vogel, J.T., Zarka, D.G., Van Buskirk, H.A., Fowler, S.G., and Thomashow, M.F. 865
(2005). Roles of the CBF2 and ZAT12 transcription factors in conFigureuring the low 866
temperature transcriptome of Arabidopsis. Plant J. 41: 195-211. 867
868
Weber, C., Nover, L., and Fauth, M. (2008). Plant stress granules and mRNA 869
29
processing bodies are distinct from heat stress granules. Plant J. 56, 517-530. 870
871
Wu, Y., Deng, Z., Lai, J., Zhang, Y., Yang, C., Yin, B., Zhao, Q., Zhang, L., Li, Y., 872
Yang, C., and Xie, Q. (2009). Dual function of Arabidopsis ATAF1 in abiotic and 873
biotic stress responses. Cell Res. 19: 1279-1290. 874
875
Xu, J., Yang, J.Y., Niu, Q.W., and Chua, N.H. (2006). Arabidopsis DCP2, DCP1, and 876
VARICOSE form a decapping complex required for postembryonic development. Plant 877
Cell 18: 3386-3398. 878
879
Xu, J., and Chua, N.H. (2009). Arabidopsis decapping 5 is required for mRNA 880
decapping, P-body formation, and translational repression during postembryonic 881
development. Plant Cell 21: 3270-3279. 882
883
Xu, J., and Chua, N.H. (2012). Dehydration stress activates Arabidopsis MPK6 to 884
signal DCP1 phosphorylation. EMBO J. 31: 1975-1984. 885
886
Yamaguchi-Shinozaki, K., and Shinozaki, K. (2006). Transcriptional regulatory 887
networks in cellular responses and tolerance to dehydration and cold stresses. Annu. 888
Rev. Plant Biol. 57: 781-803. 889
890
Zhang, Z., Zhang, S., Zhang, Y., Wang, X., Li, D., Li, Q., Yue, M., Li, Q., Zhang, 891
Y., Xu, Y., Xue, Y., Chong, K., and Bao, S. (2011). Arabidopsis Floral Initiator SKB1 892
Confers High Salt Tolerance by Regulating Transcription and Pre-mRNA Splicing 893
through Altering Histone H4R3 and Small Nuclear Ribonucleoprotein LSM4 894
Methylation. Plant Cell 23: 396-411. 895
896
30
FIGURE LEGENDS 897
Figure 1. Arabidopsis LSM1 proteins accumulate in response to low temperature 898
and localize to P-bodies. (A) Expression of LSM1A and LSM1B in 2-week-old Col-0 899
plants exposed for the indicated hours (h) or days (d) to 4ºC. Levels, determined by 900
qPCR, are represented as relative to their corresponding values at 0 h. Error bars 901
indicate the standard deviation of the mean (n ≥ 3). In all cases, differences between 902
cold-treated and control (0 h) plants were significant (P ≤ 0.0001), as determined by 903
ANOVA test. (B) Immunoblots showing levels of LSM1A-GFP and LSM1B-GFP in 2-904
week-old transgenic Arabidopsis plants exposed for the indicated times to 4ºC. A lane 905
with Col-0 plants was added in the immunoblot as a negative control. Coomassie 906
staining of the large subunit of Rubisco was used as a loading control. (C) Subcellular 907
localization of LSM1A-GFP and LSM1B-GFP in root tip cells from 6-day-old 908
transgenic Arabidopsis seedlings grown under control conditions (20ºC), or exposed for 909
24 h to 4ºC in the presence or absence of cycloheximide (CHX). Bars = 20 µm. (D) 910
Colocalization of LSM1A-GFP and LSM1B-GFP with RFP-DCP1 in root tip cells from 911
6-day-old transgenic Arabidopsis seedlings exposed for 24 h to 4ºC. Bars = 20 µm. (E) 912
Subcellular localization of GFP-DCP2 and GFP-VCS in root tip cells from 6-day-old 913
Col-0 and lsm1a lsm1b Arabidopsis seedlings grown under control conditions and 914
subsequently exposed for 24 h to 4ºC. Bars = 20 µm. 915
916
Figure 2. The LSM1-7 complex differentially regulates abiotic stress tolerance in 917
Arabidopsis. (A) Freezing tolerance of cold-acclimated 2-week-old Col-0, lsm1a 918
lsm1b, c-lsm1a and c-lsm1b plants (left). Representative cold-acclimated plants 7 d after 919
being exposed to -11ºC for 6 h (right). (B) Drought tolerance of 7-d-old Col-0, lsm1a 920
lsm1b, c-lsm1a and c-lsm1b seedlings (left). Representative seedlings grown on GM or 921
exposed 7 d to 25% PEG (right). (C) Salt tolerance of 7-d-old Col-0, lsm1a lsm1b, c-922
lsm1a and c-lsm1b seedlings (left). Representative seedlings grown on GM or exposed 7 923
d to 150 mM NaCl (right). In all graphs, error bars indicate the standard deviation of the 924
mean (n ≥ 3). Asterisks (*) indicate significant differences (*P ≤ 0.01, **P ≤ 0.001, 925
***P ≤ 0.0001) between lsm1a lsm1b and the other plants, as determined by ANOVA-926
test. No significant differences between Col-0 and c-lsm1a or c-lsm1b plants were 927
observed in any case. 928
929
31
Figure 3. The Arabidopsis LSM1-7 complex differentially regulates gene 930
expression in response to abiotic stresses. (A) Expression of different cold-inducible 931
genes upregulated in lsm1a lsm1b. Levels, determined by qPCR, in 2-week-old Col-0, 932
lsm1a lsm1b and c-lsm1a plants exposed for 24 h to 4ºC are represented relative to their 933
corresponding values in control plants (C). (B) Expression of different drought-934
inducible genes upregulated in lsm1a lsm1b. Levels, determined by qPCR, in 2-week-935
old Col-0, lsm1a lsm1b and c-lsm1a plants exposed for 10 h to 55% PEG are 936
represented relative to their corresponding values in control plants (C). (C, D) 937
Expression of different salt-inducible genes up- (C) or down-regulated (D) in lsm1a 938
lsm1b. Levels, determined by qPCR, in 2-week-old Col-0, lsm1a lsm1b and c-lsm1a 939
plants exposed for 10 h to 150 mM NaCl are represented relative to their corresponding 940
values in control plants (C). In all graphs, error bars indicate the standard deviation of 941
the mean (n ≥ 3). Differences between lsm1a lsm1b and Col-0 or c-lsm1a plants were 942
always significant (*P ≤ 0.01, **P ≤ 0.001, ***P ≤ 0.0001), as determined by ANOVA-943
test. No significant differences between Col-0 and c-lsm1a plants were observed in any 944
case. 945
946
Figure 4. The Arabidopsis LSM1-7 complex interacts with selected target 947
transcripts in response to abiotic stresses. (A-C) RIP assays of 2-week-old c-lsm1a 948
plants grown under control conditions (Control exposed for 24 h to 4ºC (A), 10 h to 949
55% PEG (B), or 10 h to 150 mM NaCl (C), using an anti-GFP antibody. RIP assays of 950
Arabidopsis containing a GRP7PRO-GRP7-GFP fusion grown under control and stress 951
conditions were also carried out as interaction specificity controls. Co-952
immunoprecipitated RNA samples corresponding to different cold- (A), drought- (B) 953
and salt-inducible (C) genes were quantified by qPCR. Transcript levels in c-lsm1a 954
plants were corrected with respect to their corresponding input values and are 955
represented relative to the levels obtained from RIP control assays. In all graphs, error 956
bars indicate the standard deviation of the mean (n ≥ 3). Asterisks (*) indicate 957
significant differences (*P ≤ 0.01, **P ≤ 0.001, ***P ≤ 0.0001) in transcript levels 958
between RIP assays from stressed and control plants, as determined by t-test. 959
960
Figure 5. The Arabidopsis LSM1-7 complex promotes the decapping of selected 961
target transcripts in response to abiotic stresses. (A-C) Capped transcripts in 2-962
week-old Col-0, lsm1a lsm1b and c-lsm1a plants grown under control conditions (C), 963
32
exposed for 24 h to 4ºC (A), 10 h to 55% PEG (B), or 10 h to 150mM NaCl (C). The 964
levels of capped transcripts corresponding to different cold- (A), drought- (B) and salt-965
inducible (C) genes were corrected with respect to the levels of their corresponding total 966
transcripts and are represented relative to control Col-0 plants. In all graphs, error bars 967
indicate the standard deviation of the mean (n ≥ 3). Asterisks (*) indicate significant 968
differences (*P ≤ 0.01, **P ≤ 0.001, ***P ≤ 0.0001) between lsm1a lsm1b and Col-0 or 969
c-lsm1a plants, as determined by ANOVA tests. No significant differences between 970
Col-0 and c-lsm1a plants were observed in any case. 971
972
Figure 6. The interaction between the Arabidopsis LSM1-7 complex and some of 973
its target transcripts depends on the abiotic stress conditions. RIP assays of 2-week-974
old c-lsm1a plants grown under control conditions (Control, exposed for 24 h to 4ºC, 10 975
h to 55% PEG, or 10 h to 150 mM NaCl, using an anti-GFP antibody. RIP assays of 2-976
week-old Arabidopsis plants containing a GRP7PRO-GRP7-GFP fusion grown under 977
control and stressed conditions were also carried out as interaction specificity controls. 978
Co-immunoprecipitated RNA samples corresponding to LEA7, ZAT12, NCED3, 979
WRKY33, ABR1, ANAC019, ANAC092 and AHK5 genes were quantified by qPCR. 980
Transcript levels in c-lsm1a plants were corrected with respect to their corresponding 981
input values and are represented relative to the levels obtained from RIP control assays. 982
In all graphs, error bars indicate the standard deviation of the mean (n ≥ 3). Asterisks (*) 983
indicate significant differences (*P ≤ 0.01, **P ≤ 0.001, ***P ≤ 0.0001) in transcript 984
levels between RIP assays from stressed and control plants, as determined by ANOVA 985
tests. 986
987
Figure 7. Decapping of some target transcripts by the Arabidopsis LSM1-7 988
complex depends on the abiotic stress conditions. (A-C) Capped transcripts in 2-989
week-old Col-0, lsm1a lsm1b and c-lsm1a plants grown under control conditions (C), 990
exposed for 24 h to 4ºC, 10 h to 55% PEG, or 10 h to 150 mM NaCl. The levels of 991
capped transcripts corresponding to LEA7, ZAT12, NCED3 and WRKY33 (A), ABR1 992
and ANAC019 (B), and ANAC092 and AHK5 (C) were corrected with respect to the 993
levels of their corresponding total transcripts and are represented relative to control Col-994
0 plants. In all graphs, error bars indicate the standard deviation of the mean (n ≥ 3). 995
Asterisks (*) indicate significant differences (**P ≤ 0.001, ***P ≤ 0.0001) between 996
lsm1a lsm1b and Col-0 or c-lsm1a plants, as determined by ANOVA tests. No 997
33
significant differences between lsm1a lsm1b and the other plants were found in any 998
case. 999
1000
Figure 8. ABA biosynthesis is differentially regulated by the Arabidopsis LSM1-7 1001
complex in response to abiotic stresses. (A, B) ABA levels in 2-week-old Col-0, 1002
lsm1a lsm1b and c-lsm1a plants grown under control conditions (C), exposed for 24 h to 1003
4ºC (A), 10 h to 55% PEG (B) or 10 h to 150mM NaCl (B). In all graphs, error bars 1004
indicate the standard deviation of the mean (n = 4). Asterisks (*) indicate significant 1005
differences (**P ≤ 0.001, ***P ≤ 0.0001) between lsm1a lsm1b and Col-0 or c-lsm1a 1006
plants, as determined by ANOVA tests. No significant differences between Col-0 and c-1007
lsm1a plants were observed in any case. 1008
1009
Figure 9. Proposed model for the function of LSM1-7 complex in plant responses 1010
to abiotic stresses. In response to abiotic stress conditions, the LSM1-7 complex would 1011
localize to P-bodies where it would interact with selected, specific and non-specific, 1012
mRNA targets to promote their decapping and subsequent decay. This function would 1013
contribute to shape the transcriptome reprogramming required for plant adaptation to 1014
abiotic stresses. Arrowheads and end lines indicate positive and negative regulation, 1015
respectively. 1016
1017
1018
1019
1020
1021
1022
1023
1024
1025
1026
1027
1028
Figure 1. Arabidopsis LSM1 proteins accumulate in response to low temperature and localize to P-bodies. (A) Expression ofLSM1A and LSM1B in 2-week-old Col-0 plants exposed for the indicated hours (h) or days (d) to 4ºC. Levels, determined by qPCR,are represented as relative to their corresponding values at 0h. Error bars indicate the standard deviation of the mean (n ≥ 3). In allcases, differences between cold-treated and control (0h) plants were significant (P ≤ 0.0001), as determined by ANOVA-test. (B)Levels of LSM1A-GFP and LSM1B-GFP in 2-week-old transgenic Arabidopsis plants exposed for the indicated times to 4ºC. A lanewith Col-0 plants was added in the western blot as a negative control. Comassie staining of the large subunit of Rubisco was used asa loading control. (C) Subcellular localization of LSM1A-GFP and LSM1B-GFP in root tip cells from 6-day-old transgenic Arabidopsisseedlings grown under control conditions (20ºC), exposed 24h to 4ºC, or exposed 24h to 4ºC with cycloheximide (CHX). Bars = 20µm. (D) Colocalization of LSM1A-GFP and LSM1B-GFP with RFP-DCP1 in root tip cells from 6-day-old transgenic Arabidopsisseedlings exposed 24h to 4ºC. Bars = 20 µm. (E) Subcellular localization of GFP-DCP2 and GFP-VCS in root tip cells from 6-day-oldCol-0 and lsm1a lsm1b Arabidopsis seedlings grown under control conditions and subsequently exposed 24h to 4ºC. Bars = 20 µm.
20ºC
4ºC
4ºC +
CHX
C
A
0
1
3
5
7
0h 3h 10h 1d 4d 7d
Rel
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pres
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LSM1ALSM1B
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D
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LSM1B-GFP
0h 1d 4d Col-0
LSM1A-GFP
3h 10h4ºC
Rubisco
Rubisco
55 kD
55 kD
41 kD
41 kD
4ºC
LSM1B-GFPLSM1A-GFP
LSM1A-GFP LSM1B-GFP
LSM1A-GFP LSM1B-GFP
GFP-VCS
GFP-DCP2
Col-0
GFP-DCP2
lsm1a lsm1b
GFP-VCS
lsm1a lsm1bCol-0
E
LSM1A-GFP
RFP-DCP1
LSM1A-GFP/RFP-DCP1
LSM1B-GFP
RFP-DCP1
LSM1B-GFP/RFP-DCP1
4ºC
A Freezing Tolerance
-11ºC, 6h
c- lsm1a c-lsm1b
Col-0 lsm1a lsm1b
Surv
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(%)
20
40
60
80
100
0-11-10-9-8-7-6
Temperature (ºC)-12
Col-0lsm1a lsm1bc-lsm1ac-lsm1b
C Salt ToleranceCol-0 lsm1a lsm1bc-lsm1a c-lsm1b
Col-0 lsm1a lsm1b c-lsm1a
150m
M
NaC
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GM
c-lsm1b
Root lenght
Fresh weight
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Mai
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oot L
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EG, 7
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Fresh weight
Lateral roots
Fres
h W
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Drought Tolerance
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Col-0 lsm1a lsm1bc-lsm1a
Late
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oots
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20
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100
c-lsm1b
PEG (25%)
*** ***
**
**
***
***
***
****
Figure 2. The LSM1-7 complex differentially regulates abiotic stress tolerance in Arabidopsis. (A) Freezing tolerance of coldacclimated 2-week-old Col-0, lsm1a lsm1b, c-lsm1a and c-lsm1b plants (left). Representative cold acclimated plants 7d after beingexposed to -11ºC for 6h (right). (B) Drought tolerance of 7-day-old Col-0, lsm1a lsm1b, c-lsm1a and c-lsm1b seedlings (left).Representative seedlings grown on GM or exposed 7d to 25% PEG (right). (C) Salt tolerance of 7-day-old Col-0, lsm1a lsm1b, c-lsm1a and c-lsm1b seedlings (left). Representative seedlings grown on GM or exposed 7d to 150mM NaCl (right). In all graphs, errorbars indicate the standard deviation of the mean (n ≥ 3). Asterisks (*) indicate significant differences (*P ≤ 0.01, **P ≤ 0.001, ***P ≤0.0001) between lsm1a lsm1b and the other plants, as determined by ANOVA-test. No significant differences between Col-0 and c-lsm1a or c-lsm1b plants were observed in any case.
Figure 3. The Arabidopsis LSM1-7 complex differentially regulates gene expression in response to abiotic stresses. (A)Expression of different cold-inducible genes up-regulated in lsm1a lsm1b. Levels, determined by qPCR, in 2-week-old Col-0, lsm1alsm1b and c-lsm1a plants exposed 24h to 4ºC are represented as relative to their corresponding values in control plants (C). (B)Expression of different drought-inducible genes up-regulated in lsm1a lsm1b. Levels, determined by qPCR, in 2-week-old Col-0, lsm1alsm1b and c-lsm1a plants exposed 10h to 55% PEG are represented as relative to their corresponding values in control plants (C). (C,D) Expression of different salt-inducible genes up- (C) or down-regulated (D) in lsm1a lsm1b. Levels, determined by qPCR, in 2-week-old Col-0, lsm1a lsm1b and c-lsm1a plants exposed 10h to 150mM NaCl are represented as relative to their corresponding values incontrol plants (C). In all graphs, error bars indicate the standard deviation of the mean (n ≥ 3). Differences between lsm1a lsm1b andCol-0 or c-lsm1a plants were always significant (*P ≤ 0.01, **P ≤ 0.001, ***P ≤ 0.0001), as determined by ANOVA-test. No significantdifferences between Col-0 and c-lsm1a plants were observed in any case.
A
Rel
ativ
e Ex
pres
sion
(4ºC
/C)
0
20
40
60
400800 Col-0
lsm1a lsm1bc-lsm1a
B
C
D
Rel
ativ
e Ex
pres
sion
(N
aCl/C
)
0
10
20
3040
Rel
ativ
e Ex
pres
sion
(N
aCl/C
)
0
4
8
2040
Col-0lsm1a lsm1bc-lsm1a
Rel
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e Ex
pres
sion
(PE
G/C
)
0
20
40
150Col-0lsm1a lsm1bc-lsm1a
* *****
****
***
***
***
***
***
***
***
***
*****
*
*****
**
**
**
*** **
**
***
**
**
**
**Col-0lsm1a lsm1bc-lsm1a
***
**
***
**
*** ***
***
Figure 4. The Arabidopsis LSM1-7 complex interacts with selected target transcripts in response to abiotic stresses. (A-C)RIP assays on 2-week-old c-lsm1a plants grown under control conditions (Control), exposed 24h to 4ºC (A), 10h to 55% PEG (B), or10h to 150mM NaCl (C), using an anti-GFP antibody. RIP assays on Arabidopsis containing a GRP7PRO-GRP7-GFP fusion grownunder control and stressed conditions were also carried out as interaction specificity controls. Co-immunoprecipitated RNA samplescorresponding to different cold- (A), drought- (B) and salt-inducible (C) genes were quantified by qPCR. Transcript levels in c-lsm1aplants were corrected with respect to their corresponding input values and represented relative to the levels obtained from RIP controlassays. In all graphs, error bars indicate the standard deviation of the mean (n ≥ 3). Asterisks (*) indicate significant differences (*P ≤0.01, **P ≤ 0.001, ***P ≤ 0.0001) in transcript levels between RIP assays from stressed and control plants, as determined by t-test.
C
0
5
10
15 ControlNaCl
Rel
ativ
e tr
ansc
ript l
evel
s (c
-lsm
1a/G
RP7
-GFP
)
A
Rel
ativ
e tr
ansc
ript l
evel
s (c
-lsm
1a/G
RP7
-GFP
)
0
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200Control4ºC
BControl
0
5
10
15
PEG
Rel
ativ
e tr
ansc
ript l
evel
s (c
-lsm
1a/G
RP7
-GFP
)
****
*
**
* ***
***
*
**
****
*
****
**
*
Figure 5. The Arabidopsis LSM1-7 complex promotes the decapping of selected target transcripts in response to abioticstresses. (A-C) Capped transcripts in 2-week-old Col-0, lsm1a lsm1b and c-lsm1a plants grown under control conditions (C), exposed24h to 4ºC (A), 10h to 55% PEG (B), or 10h to 150mM NaCl (C). The levels of capped transcripts corresponding to different cold- (A),drought- (B) and salt-inducible (C) genes were corrected with respect to the levels of their corresponding total transcripts andrepresented relative to control Col-0 plants. In all graphs, error bars indicate the standard deviation of the mean (n ≥ 3). Asterisks (*)indicate significant differences (*P ≤ 0.01, **P ≤ 0.001, ***P ≤ 0.0001) between lsm1a lsm1b and Col-0 or c-lsm1a plants, asdetermined by ANOVA-test. No significant differences between Col-0 and c-lsm1a plants were observed in any case.
B
Rel
ativ
e ca
pped
mR
NA
s
0
2
4
6
8
10
C PEG C PEGC PEGC PEG C PEGC PEG
Col-0lsm1a lsm1bc-lsm1a
C PEGC PEG
***
**
C PEG
***
***
***
C PEG
***
**
C
0
2
6
10
14
Rel
ativ
e ca
pped
mR
NA
s
C NaClC NaCl C NaClC NaCl C NaCl
Col-0lsm1a lsm1bc-lsm1a
C NaCl
**
*
C NaCl
***
*
***
**
A
C 4ºC
Rel
ativ
e ca
pped
mR
NA
s
Col-0lsm1a lsm1bc-lsm1a
0
4
8
12
C 4ºCC 4ºC C 4ºCC 4ºC C 4ºCC 4ºCC 4ºC C 4ºCC 4ºC
***
**
***
**
***
**
**
****** ***
***
***
C NaCl C NaClC NaCl
Rel
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e tr
ansc
ript l
evel
s (c
-lsm
1a/G
RP7
-GFP
)
Control4ºC
PEGNaCl
Cold-specific Drought-specific Salt-specific
0
5
10
15
*
**
**
**
*******
*
Figure 6. The interaction between the Arabidopsis LSM1-7 complex and some of its target transcripts depends on the abioticstress conditions. RIP assays on 2-week-old c-lsm1a plants grown under control conditions (Control), exposed 24h to 4ºC, 10h to55% PEG, or 10h to 150mM NaCl, using an anti-GFP antibody. RIP assays on 2-week-old Arabidopsis plants containing a GRP7PRO-GRP7-GFP fusion grown under control and stressed conditions were also carried out as interaction specificity controls. Co-immunoprecipitated RNA samples corresponding to LEA7, ZAT12, NCED3, WRKY33, ABR1, ANAC019, ANAC092 and AHK5 geneswere quantified by qPCR. Transcript levels in c-lsm1a plants were corrected with respect to their corresponding input values andrepresented relative to the levels obtained from RIP control assays. In all graphs, error bars indicate the standard deviation of themean (n ≥ 3). Asterisks (*) indicate significant differences (*P ≤ 0.01, **P ≤ 0.001, ***P ≤ 0.0001) in transcript levels between RIPassays from stressed and control plants, as determined by ANOVA-test.
Figure 7. Decapping of some target transcripts by the Arabidopsis LSM1-7 complex depends on the abiotic stressconditions. (A-C) Capped transcripts in 2-week-old Col-0, lsm1a lsm1b and c-lsm1a plants grown under control conditions (C),exposed 24h to 4ºC, 10h to 55% PEG, or 10h to 150mM NaCl. The levels of capped transcripts corresponding to LEA7, ZAT12,NCED3 and WRKY33 (A), ABR1 and ANAC019 (B), and ANAC092 and AHK5 (C) genes were corrected with respect to the levels oftheir corresponding total transcripts and represented relative to control Col-0 plants. In all graphs, error bars indicate the standarddeviation of the mean (n ≥ 3). Asterisks (*) indicate significant differences (**P ≤ 0.001, ***P ≤ 0.0001) between lsm1a lsm1b and Col-0or c-lsm1a plants, as determined by ANOVA-test. No significant differences between lsm1a lsm1b and the other plants were found inany case.
CCol-0lsm1a lsm1bc-lsm1a
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Figure 8. ABA biosynthesis is differentially regulated by the Arabidopsis LSM1-7 complex in response to abiotic stresses. (A,B) ABA levels in 2-week-old Col-0, lsm1a lsm1b and c-lsm1a plants grown under control conditions (C), exposed 24h to 4ºC (A), 10hto 55% PEG (B) or 10h to 150mM NaCl (B). In all graphs, error bars indicate the standard deviation of the mean (n = 4). Asterisks (*)indicate significant differences (**P ≤ 0.001, ***P ≤ 0.0001) between lsm1a lsm1b and Col-0 or c-lsm1a plants, as determined byANOVA-test. No significant differences between Col-0 and c-lsm1a plants were observed in any case.
ColdDrought
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Transcriptomereprogramming
DECAPPING OF SELECTED STRESS‐SPECIFIC AND NONSPECIFIC TARGET
TRANSCRIPTS
PLANT ADAPTATION
ACCURATE STRESS‐RESPONSIVE GENE
EXPRESSION
P‐bodies
Figure 9. Proposed model for the function of LSM1-7 complex in plant response to abiotic stresses. In response to abioticstress conditions the complex would localize in P-bodies where would interact with selected, specific and non-specific, mRNA targetsto promote their decapping and subsequent decay. This function would contribute to shape the transcriptome reprogramming requiredfor plant adaptation to abiotic stress. Arrowheads and end lines indicate positive and negative regulation, respectively.
Parsed CitationsBalazadeh, S., Siddiqui, H., Allu, A.D., Matallana-Ramirez, L.P., Caldana, C., Mehrnia, M., Zanor, M.I., Köhler, B., and Mueller-Roeber, B. (2010). A gene regulatory network controlled by the NAC transcription factor ANAC092/AtNAC2/ORE1 during salt-promoted senescence. Plant J. 62: 250-264.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Barrero, J.M., Rodríguez, P.L., Quesada, V., Piqueras, P., Ponce, M.R., and Micol, J.L. (2006). Both abscisic acid (ABA)-dependentand ABA-independent pathways govern the induction of NCED3, AAO3 and ABA1 in response to salt stress. Plant Cell Environ. 29:2000-2008.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Catalá, R., López-Cobollo, R., Castellano, M.M., Angosto, T., Alonso, J.M., Ecker, J.R., and Salinas, J. (2014). The Arabidopsis 14-3-3Protein RARE COLD INDUCIBLE 1A Links Low-Temperature Response and Ethylene Biosynthesis to Regulate FreezingTolerance and Cold Acclimation. Plant Cell 26: 3326-3342.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Chen, J.H., Jiang, H.W., Hsieh, E.J., Chen, H.Y., Chien, C.T., Hsieh, H.L., and Lin, T.P. (2012). Drought and Salt Stress Tolerance ofan Arabidopsis Glutathione S-Transferase U17 Knockout Mutant Are Attributed to the Combined Effect of Glutathione andAbscisic. Acid Plant Physiol. 158: 340-351.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Cheng, M.C., Hsieh, E.J., Chen, J.H., Chen, H.Y., and Lin, T.P. (2012). Arabidopsis RGLG2, Functioning as a RING E3 Ligase,Interacts with AtERF53 and Negatively Regulates the Plant Drought Stress Response. Plant Physiol. 158: 363-375.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Chowdhury, A., Mukhopadhyay, J., and Tharun, S. (2007). The decapping activator Lsm1p-7p-Pat1p complex has the intrinsic abilityto distinguish between oligoadenylated and polyadenylated RNAs. RNA 13: 998-1016.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Coller, J., and Parker, R. (2004). Eukaryotic mRNA decapping. Annu. Rev. Biochem. 73: 861-890.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Cuevas, J.C., López-Cobollo, R., Alcázar, R., Zarza, X., Koncz, C., Altabella, T., Salinas, J., Tiburcio, A.F., and Ferrando, A. (2008).Putrescine Is Involved in Arabidopsis Freezing Tolerance and Cold Acclimation by Regulating Abscisic Acid Levels in Response toLow Temperature. Plant Physiol. 148: 1094-1105.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Cui, P., Zhang, S., Ding, F., Ali, S., and Xiong, L. (2014). Dynamic regulation of genome-wide pre-mRNA splicing and stress toleranceby the Sm-like protein LSm5 in Arabidopsis. Genome Biol. 15: 1-18.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Czechowski, T., Stitt, M., Altmann, T., Udvardi, M.K., and Scheible, W.R. (2005). Genome-wide identification and testing of superiorreference genes for transcript normalization in Arabidopsis. Plant Physiol. 139: 5-17.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Fillman, C., and Lykke-Andersen, J. (2005). RNA decapping inside and outside of processing bodies. Curr. Opin. Cell Biol. 17: 326-331.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Frey, A., Effroy, D., Lefebvre, V., Seo, M., Perreau, F., Berger, A., Sechet, J., To, A., North, H.M., and Marion-Poll, A. (2012).Epoxycarotenoid cleavage by NCED5 fine-tunes ABA accumulation and affects seed dormancy and drought tolerance with otherNCED family members. Plant J. 70: 501-512.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Goeres, D.C., Van Norman, J.M., Zhang, W., Fauver, N.A., Spencer, M.L., and Sieburth, L.E. (2007). Components of the ArabidopsismRNA decapping complex are required for early seedling development. Plant Cell 19: 1549-1564.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Guerra, D., Crosatti, C., Khoshro, H.H., Mastrangelo, A.M., Mica, E., and Mazzucotelli, E. (2015). Post-transcriptional and post-translational regulations of drought and heat response in plants, a spider's web of mechanisms. Front. Plant Sci. 6: 1-14.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Gujjar, R.S., Akhtar, M., and Singh, M. (2014). Transcription factors in abiotic stress tolerance. Ind. J. Plant Physiol. 19: 306-316.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Jia, W., Wang, Y., Zhang, S., and Zhang, J. (2002). Salt-stress-induced ABA accumulation is more sensitively triggered in roots thanin shoots. J. Exp. Bot. 53: 2201-2206.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Jiang, Y., Liang, G., and Yu, D. (2012). Activated Expression of WRKY57 Confers Drought Tolerance in Arabidopsis. Mol. Plant 5:1375-1388.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kilian, J., Whitehead, D., Horak, J., Wanke, D., Weinl, S., Batistic, O., D´Angelo, C., Bornberg-Bauer, E., Kudla, J., and Harter, K.(2007). The AtGenExpress global stress expression data set, protocols, evaluation and model data analysis of UV-B light, droughtand cold stress responses. Plant J. 50: 347-363.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kim, D., Pertea, G., Trapnell, C., Pimentel, H., Kelley, R., and Salzberg, S.L. (2013). TopHat2: accurate alignment of transcriptomesin the presence of insertions, deletions and gene fusions. Genome Biol. 14: R36.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Love, M.I., Huber, W., and Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.Genome Biol. 15: 550.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Merret, R., Descombin, J., Juan, Y.T., Favory, J.J., Carpentier, M.C., Chaparro, C., Charng, Y., Deragon, J.M., and Bousquet-Antonelli, C. (2013). XRN4 and LARP1 Are Required for a Heat-Triggered mRNA Decay Pathway Involved in Plant Acclimation andSurvival during Thermal Stress. Cell Reports 5: 1279-1293.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Miura, K., and Furumoto, T. (2013). Cold Signaling and Cold Response in Plants. Int. J. Mol. Sci. 14: 5312-5337.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Motomura, K., Le, Q.T.N., Hamada, T., Kutsuna, N., Mano, S., Nishimura, M., and Watanabe, Y. (2015). Diffuse Decapping EnzymeDCP2 Accumulates in DCP1 Foci Under Heat Stress in Arabidopsis thaliana. Plant Cell Physiol. 56: 107-115.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Nakashima, K., Yamaguchi-Shinozaki, K., and Shinozaki, K. (2014). The transcriptional regulatory network in the drought responseand its crosstalk in abiotic stress responses including drought, cold, and heat. Front. Plant Sci. 16: 170.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Pandey, G.H., Grant, J.J., Cheong, Y.H., Kim, B.G., Li, L., and Luan, S. (2005). ABR1, an APETALA2 Domain Transcription FactorThat Functions as a Repressor of ABA Response in Arabidopsis. Plant Plysiol. 139: 1185-1193.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Parker, R. (2012). RNA Degradation in Saccharomyces cerevisae. Genetics 191: 671-702.Pubmed: Author and Title
CrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Peng, J., Li, Z., Wen, X., Li, W., Shi, H., Yang, L., Zhu, H., and Guo, H. (2014). Salt-Induced Stabilization of EIN3/EIL1 Confers SalinityTolerance by Deterring ROS Accumulation in Arabidopsis. PLOS Genetics 10: e1004664.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Perea-Resa, C., Hernández-Verdeja, T., López-Cobollo, R., Castellano, M.M., and Salinas, J. (2012). LSM Proteins Provide AccurateSplicing and Decay of Selected Transcripts to Ensure Normal Arabidopsis Development. Plant Cell 24: 4930-4947.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Pham, J., Liu, J., Bennett, M.H., Mansfield, J.W., and Desikan, R. (2012), Arabidopsis histidine kinase 5 regulates salt sensitivity andresistance against bacterial and fungal infection. New Phytol. 194: 168-180.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Roux, M.E., Rasmussen, M.W., Palma, K., Lolle, S., Mateu-Regué, A., Bethke, G., Glazebrook, J., Zhang, W., Sieburth, L., Larsen,M.R., Mundy, J., and Petersen, M. (2015). The mRNA decay factor PAT1 functions in a pathway including MAP kinase 4 and immunereceptor SUMM2. EMBO J. 34: 593-608.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Rzeczkowski, K., Beuerlein, K., Müller, H., Dittrich-Breiholz, O., Schneider, H., Ketter-Buhrow, D., Holtmann, H., and Kracht, M.(2011). C-Jun N-terminal kinase phosphorylates DCP1a to control formation of P bodies. J. Cell Biol. 194: 581-596.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Sheth, U., and Parker, R. (2003). Decapping and Decay of Messenger RNA Occur in Cytoplasmic Processing Bodies. Science 300:805-808.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Shi, Y., Ding, Y., and Yang, S. (2015). Cold Signal Transduction and its Interplay with Phytohormones During Cold Acclimation. PlantCell Physiol. 56: 7-15.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Streitner, C., Köster, T., Simpson, C.G., Shaw, P., Danisman, S., Brown, J.W.S., and Staiger, D. (2012). An hnRNP-like RNA-bindingprotein affects alternative splicing by in vivo interaction with transcripts in Arabidopsis thaliana. Nucleic Acis. Res. 40: 11240-11255.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Tan, B.C., Joseph, L.M., Deng, W.T., Liu, L., Li, Q.B., Cline, K., and McCarty, D.R. (2003). Molecular Characterization of theArabidopsis 9-cis epoxycarotenoid dioxygenase gene family. Plant J. 35: 44-56.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Tran, L.S.P., Nakashima, K., Sakuma, Y., Simpson, S.D., Fujita, Y., Maruyama, K., Fujita, M., Seki, M., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2004). Isolation and Functional Analysis of Arabidopsis Stress-Inducible NAC Transcription Factors That Bind to aDrought-Responsive cis-Element in the early responsive to dehydration stress 1 Promoter. Plant Cell 16: 2481-2498.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Turecková, V., Novák, O., and Strnad, M. (2009). Profiling ABA metabolites in Nicotiana tabacum L leaves by ultra-performanceliquid chromatography-electrospray tandem mass spectrometry. Talanta 80: 390-399.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Verslues, P.E., Agarwal, M., Katiyar-Agarwal, S., Zhu, J., and Zhu, J.K. (2006). Methods and concepts in quantifying resistance todrought, salt and freezing, abiotic stresses that affect plant water status. Plant J. 45: 523-539.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Vogel, J.T., Zarka, D.G., Van Buskirk, H.A., Fowler, S.G., and Thomashow, M.F. (2005). Roles of the CBF2 and ZAT12 transcriptionfactors in conFigureuring the low temperature transcriptome of Arabidopsis. Plant J. 41: 195-211.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Weber, C., Nover, L., and Fauth, M. (2008). Plant stress granules and mRNA processing bodies are distinct from heat stressgranules. Plant J. 56, 517-530.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Wu, Y., Deng, Z., Lai, J., Zhang, Y., Yang, C., Yin, B., Zhao, Q., Zhang, L., Li, Y., Yang, C., and Xie, Q. (2009). Dual function ofArabidopsis ATAF1 in abiotic and biotic stress responses. Cell Res. 19: 1279-1290.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Xu, J., Yang, J.Y., Niu, Q.W., and Chua, N.H. (2006). Arabidopsis DCP2, DCP1, and VARICOSE form a decapping complex requiredfor postembryonic development. Plant Cell 18: 3386-3398.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Xu, J., and Chua, N.H. (2009). Arabidopsis decapping 5 is required for mRNA decapping, P-body formation, and translationalrepression during postembryonic development. Plant Cell 21: 3270-3279.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Xu, J., and Chua, N.H. (2012). Dehydration stress activates Arabidopsis MPK6 to signal DCP1 phosphorylation. EMBO J. 31: 1975-1984.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yamaguchi-Shinozaki, K., and Shinozaki, K. (2006). Transcriptional regulatory networks in cellular responses and tolerance todehydration and cold stresses. Annu. Rev. Plant Biol. 57: 781-803.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Zhang, Z., Zhang, S., Zhang, Y., Wang, X., Li, D., Li, Q., Yue, M., Li, Q., Zhang, Y., Xu, Y., Xue, Y., Chong, K., and Bao, S. (2011).Arabidopsis Floral Initiator SKB1 Confers High Salt Tolerance by Regulating Transcription and Pre-mRNA Splicing throughAltering Histone H4R3 and Small Nuclear Ribonucleoprotein LSM4 Methylation. Plant Cell 23: 396-411.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title