dual role for autophagy in lipid metabolism in arabidopsis · 1 1 research article 2 3 dual role...
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RESEARCH ARTICLE 1
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Dual Role for Autophagy in Lipid Metabolism in Arabidopsis 3
4 Jilian Fan1, Linhui Yu1 and Changcheng Xu2 5
Biology Department, Brookhaven National Laboratory, Upton, NY 11973 6 7
1These authors contributed equally to this work. 8
2Address correspondence to [email protected] 9
10 The author responsible for distribution of materials integral to the findings presented in this 11 article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) 12 is: Changcheng Xu ([email protected]) 13
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Running title: Role of Autophagy in Lipid Metabolism 15 16 One-sentence summary: Autophagy regulates both lipid droplet biogenesis and turnover in 17 plants. 18 19
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ABSTRACT 21
Autophagy is a major catabolic pathway whereby cytoplasmic constituents including lipid 22 droplets (LDs), storage compartments for neutral lipids, are delivered to the lysosome or 23 vacuole for degradation. The autophagic degradation of cytosolic LDs, a process termed 24 lipophagy, has been extensively studied in yeast and mammals, but little is known about the role 25 for autophagy in lipid metabolism in plants. Organisms maintain a basal level of autophagy 26 under favorable conditions and upregulate the autophagic activity under stress including 27 starvation. Here, we demonstrate that Arabidopsis thaliana basal autophagy contributes to 28 triacylglycerol (TAG) synthesis, whereas inducible autophagy contributes to LD degradation. We 29 found that disruption of basal autophagy impedes organellar membrane lipid turnover, and 30 hence fatty acid mobilization from membrane lipids to TAG. We show that lipophagy is induced 31 under starvation as indicated by colocalization of LDs with the autophagic marker and the 32 presence of LDs in vacuoles. We additionally show that lipophagy occurs in a process 33 morphologically resembling microlipophagy and requires the core components of the 34 macroautophagic machinery. Together, this study provides mechanistic insight into lipophagy, 35 and reveals a dual role for autophagy in regulating lipid synthesis and turnover in plants. 36
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Plant Cell Advance Publication. Published on April 29, 2019, doi:10.1105/tpc.19.00170
©2019 American Society of Plant Biologists. All Rights Reserved
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INTRODUCTION 40
Eukaryotic cells employ a highly evolutionarily conserved mechanism named autophagy 41
to deliver cytoplasmic constituents into lysosomes or vacuoles for degradation. This 42
catabolic process enables the removal of obsolete or damaged macromolecules, 43
defective organelles and invading microorganisms and at the same time the recycling of 44
cellular components into needed nutrients, and is therefore essential for homeostasis, 45
development and survival (Jaishy and Abel, 2016; Anding and Baehrecke, 2017; Dikic, 46
2017). Autophagy is functional at basal levels in virtually all cell types under favorable 47
growth conditions, but can also be massively induced by a wide array of developmental 48
and environmental stimuli including nutrient starvation, senescence, pathogens, 49
metabolic stress and many other abiotic and biotic cues (Levine and Kroemer, 2008; 50
Jaishy and Abel, 2016; Anding and Baehrecke, 2017; Wang et al., 2017b; Nakamura 51
and Yoshimori, 2018). The functional importance of both inducible and basal autophagy 52
is well illustrated in plants using autophagy-defective mutants of Arabidopsis thaliana 53
and other plants (Liu et al., 2005; Guiboileau et al., 2012; Yoshimoto, 2012; Minina et 54
al., 2014; Yang and Bassham, 2015; Barros et al., 2017; Elander et al., 2017; Enrique 55
Gomez et al., 2017; Ustun et al., 2017; Wang et al., 2018; Wang et al., 2017). These 56
mutants display early senescence, shortened life span, reduced seed yield, defective 57
reproductive growth and altered phytohormone signaling under normal growth 58
conditions, and are also hypersensitive to nutrient deprivation, oxidative stress, 59
pathogen infection, drought and high salinity, although many mechanistic details 60
underlying these phenotypes and the stress sensitivity remain largely unknown. 61
Two major types of autophagy-related pathways, namely macroautophagy and 62
microautophagy, have been described in yeast, plants and mammals (Yoshimoto, 2012; 63
Noda and Inagaki, 2015; Yang and Bassham, 2015; Antonioli et al., 2017; Elander et 64
al., 2017; Galluzzi et al., 2017; Ustun et al., 2017; Wang et al., 2018). Macroautophagy 65
is the most extensively studied form of autophagy in diverse organisms and is 66
characterized by the formation of a double-membrane structure named an 67
autophagosome. Initial steps in macroautophagy involve the assembly and expansion of 68
an isolated membrane structure called the phagophore. This membrane structure 69
eventually closes to enwrap a portion of cytoplasmic content as cargo. Subsequently, 70
the outer membrane of the autophagosome is fused with the vacuolar membrane to 71
release the inner membrane along with cargo as an autophagic body into the vacuole 72
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for degradation and recycling. Macroautophagy is mediated by a group of proteins 73
encoded by AUTOPHAGY-RELATED (ATG) genes that are highly conserved from 74
yeast to mammals and plants. Among them, ATG3, ATG4, ATG5, ATG7, ATG8, 75
ATG10, ATG12, and ATG16 are components of two ubiquitin-like conjugation systems 76
essential for the formation of autophagosomes, their fusion with the tonoplast and 77
vacuolar degradation (Yoshimoto, 2012; Elander et al., 2017; Ustun et al., 2017; Soto-78
Burgos et al., 2018). These two systems facilitate the formation of ATG8-79
phosphatidylethanolamine (PE) conjugates, whose abundance is often used as 80
indicators of autophagic activity (Suzuki and Ohsumi, 2007; Yoshimoto, 2012). In 81
particular, because ATG8 proteins are membrane-associated, residing in phagophores, 82
autophagosomes and autophagic bodies, GFP-tagged ATG or autophagic cargo 83
proteins are often used to monitor the delivery of autophagosomes to or the breakdown 84
of autophagic cargos in the vacuole or lysosome (Yoshimoto, 2012; Klionsky, 2016; 85
Galluzzi et al., 2017). 86
In contrast to macroautophagy, our understanding of microautophagy in terms of the 87
underlying mechanism, the molecular machinery involved and its functional role is still 88
fragmentary, even in simple model systems such as yeast. At the ultrastructural level, 89
microautophagy involves direct engulfment of cytoplasmic components via tonoplast 90
invagination and subsequent release of the cargo into the vacuolar lumen for 91
degradation (Noda and Inagaki, 2015; Dikic, 2017; Galluzzi et al., 2017; Oku and Sakai, 92
2018). In yeast, various cellular organelles including peroxisomes, mitochondria, the ER 93
and the nucleus were identified as targets of microautophagy (Suzuki and Ohsumi, 94
2007; Reggiori and Klionsky, 2013; Noda and Inagaki, 2015; Galluzzi et al., 2017). 95
Several forms of yeast microautophagy have been shown to require at least some 96
components of the core machinery of macroautophagy (Suzuki and Ohsumi, 2007; 97
Reggiori and Klionsky, 2013; van Zutphen et al., 2014). In plants, microautophagy has 98
been shown to participate in the degradation of cytoplasmic anthocyanin aggregates 99
(Chanoca et al., 2015) and starch granules (Toyooka et al., 2001) and chloroplasts 100
under oxidative stress (Nakamura et al., 2018), but its underlying mechanism remains 101
largely unknown. 102
Recent studies indicate that autophagy is functionally connected to lipid metabolism and 103
storage in diverse model systems (Jaishy and Abel, 2016; Shatz et al., 2016; Wang, 104
2016; Elander et al., 2017; Zechner et al., 2017). Lipids in membranous organelles are 105
used as alternative substrates for energy production via β-oxidation of fatty acids in 106
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mitochondria in mammals (Shatz et al., 2016) and peroxisomes in yeast (Kohlwein, 107
2010) and plants (Graham, 2008) during times of nutrient scarcity. Emerging evidence 108
suggests that rather than directly being used for β-oxidation, fatty acids released from 109
cellular membranes are first stored in the form of triacylglycerol (TAG) in lipid droplets 110
(LDs), and TAG in LDs is then hydrolyzed by a process named lipolysis to supply the 111
cell with fatty acids for the generation of energy (Cabodevilla et al., 2013; Fan et al., 112
2014; Rambold et al., 2015; Fan et al., 2017). In mammalian cells, autophagic digestion 113
of membranous organelles is the major source of fatty acids for TAG synthesis and LD 114
biogenesis under starvation conditions (Rambold et al., 2015; Nguyen et al., 2017). In 115
addition, autophagy plays an important role in the cellular mobilization and degradation 116
of neutral lipids in LDs, in a process termed lipophagy (Wang, 2016; Zechner et al., 117
2017). Recent studies have demonstrated a functional link between lipolysis and 118
lipophagy (Martinez-Lopez et al., 2016; Peng et al., 2016), but the exact contribution of 119
each of these two pathways to LD breakdown remains unknown (Zechner et al., 2017). 120
Mammalian lipophagy depends on the core macroautophagy machinery (Jaishy and 121
Abel, 2016), and is morphologically similar to macroautophagy, and thus referred to as 122
macrolipophagy (Singh et al., 2009). Consequently, disruption of the core ATG genes 123
increases LD accumulation in various organs (Singh et al., 2009). Unlike the situation in 124
mammals, lipophagy in yeast resembles microautophagy and therefore is referred to as 125
microlipophagy (van Zutphen et al., 2014). It has been suggested that microlipophagy 126
plays an important role in maintaining cell viability (van Zutphen et al., 2014) and 127
membrane integrity (Wang et al., 2014) under carbon starvation in yeast, but 128
controversy exists as to whether this process depends on (van Zutphen et al., 2014; 129
Wang et al., 2014) or can occur independently of (Oku et al., 2017), the core ATG 130
proteins. Autophagy-like processes have also been shown to participate in TAG 131
synthesis and/or LD breakdown in microalgae (Zhao et al., 2014; Schwarz et al., 2017) 132
and in rice (Oryza sativa) during anther development (Kurusu et al., 2014). Disruption of 133
autophagy has been shown to affect lipid turnover in maize (Zea mays; McLoughlin et 134
al., 2018) and Arabidopsis thaliana seedling under carbon starvation (Avin-Wittenberg 135
et al., 2015), but whether lipophagy occurs in Arabidopsis and the molecular 136
mechanism involved remain unknown. 137
In plants, as in yeast and mammals, TAG is assembled in the endoplasmic reticulum 138
(ER) and stored in LDs in the cytosol (Chapman and Ohlrogge 2012). In Arabidopsis, 139
phospholipid:diacylglycerol acyltransferase1 (PDAT1) is a key enzyme catalyzing the 140
last step of TAG assembly (Zhang et al., 2009). TAG breakdown is catalyzed by 141
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cytosolic lipases including SUGAR-DEPENDENT1 (SDP1), a patatin domain lipase 142
responsible for the initiation of TAG catabolism (Eastmond, 2006). Disruption of SDP1 143
blocks TAG hydrolysis in germinating seeds (Eastmond, 2006) and in vegetative tissues 144
such as mature leaves and roots (Kelly et al., 2013; Fan et al., 2014), suggesting that 145
cytosolic lipolysis plays a dominant role in TAG breakdown in both seed and non-seed 146
tissues under normal growth conditions. Under extended darkness, TAG levels in 147
leaves of sdp1 mutants increased rapidly and then declined, suggesting an activation of 148
unknown, alternative pathways for TAG hydrolysis under starvation conditions (Fan et 149
al., 2017). 150
Lipid metabolism in photosynthetic tissues such as leaves is geared toward the supply 151
of building blocks for organellar membrane biogenesis and maintenance. As a result, 152
leaf tissues do not accumulate TAG to significant amounts, although they do possess a 153
high capacity for its synthesis and metabolism (Xu and Shanklin 2016). In Arabidopsis, 154
two parallel pathways, compartmentalized in either the ER or the chloroplast, contribute 155
to membrane lipid biosynthesis (Browse and Somerville, 1991; Ohlrogge and Browse, 156
1995). Disruption of either pathway causes drastic changes in lipid metabolism including 157
an increase in fatty acid synthesis and turnover and an accumulation of TAG (Fan et al., 158
2013b; Fan et al., 2015). In the trigalactosyldiacylglycerol1 (tgd1) mutant, a defect in the 159
ER pathway also results in a compensatory increase in the chloroplast pathway activity 160
(Xu et al., 2003; Xu et al., 2005). Similarly, overexpressing PDAT1 draws lipids from the 161
ER pathway to TAG synthesis, causing an increase in the biosynthesis of thylakoid 162
lipids via the chloroplast pathway (Fan et al., 2013a). On the other hand, the plastidic 163
glycerol-3-phosphate acyltransferase1 (act1) mutant is defective in the initial step in the 164
chloroplast pathway of membrane lipid synthesis (Kunst et al., 1988; Xu et al., 2006), 165
and the vast majority of membrane lipids in act1 are assembled via the ER pathway 166
(Kunst et al., 1988). 167
To understand the role of autophagy in lipid metabolism at the mechanistic level, we 168
generated a series of double mutants defective in autophagy in the tgd1, sdp1 or 169
PDAT1-overexpressing-line background. Using these mutants along with transgenic 170
plants coexpressing a LD-targeted, green fluorescent protein (GFP)-tagged OLEOSIN1 171
(OLE1) fusion protein (Fan et al., 2013a) and autophagic or tonoplast markers, we 172
demonstrate here an important role of autophagy in regulating TAG synthesis, 173
membrane lipid turnover and fatty acid synthesis under normal growth conditions, and in 174
mediating LD degradation under starvation. We show that lipophagy occurs in a process 175
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morphologically resembling microautophagy in yeast and requires key core players in 176
macroautophagy. This study demonstrates the functional importance of autophagy in 177
TAG metabolism and storage and the mechanistic basis for lipophagy in plants. 178
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RESULTS 180
Basal Autophagy Contributes to TAG Synthesis in Leaves of Adult Plants 181
To test the role of autophagy in lipid metabolism in plants, we first compared TAG levels 182
in mature seeds, young seedlings and leaves of adult plants between the wild type and 183
two atg mutants defective in ATG2 or ATG5, two core protein components of the 184
macroautophagic machinery. Disruption of autophagy caused small but significant 185
decreases in TAG content in seeds (Figure 1A) and 4-d-old seedlings (Figure 1B). Seed 186
weight was slightly decreased in atg2-1 (169.3 ± 5.3 µg/10 seeds) and atg5-1 (181.3 ± 187
11.7 µg/10 seeds) compared with the wild type (186.7 ± 3.3 µg/10 seeds). TAG levels 188
were low in developing leaves but increased as leaves matured and aged. Compared 189
with the wild type, TAG content was reduced by an average of 29%, 52% and 42% in 190
developing, fully mature and senescing leaves of atg mutants, respectively (Figure 1C). 191
In all tissues examined, there were no significant differences in TAG content between 192
atg2-1 and atg5-1, suggesting the decreased TAG levels in atg mutants are associated 193
with defects in basal autophagy. 194
Mutants defective in the core components of autophagy often display pleiotropic 195
phenotypes including early senescence and defects in nutrient remobilization. 196
Therefore, it is possible that the observed decrease in TAG content in seeds in atg 197
mutants is due to a decrease in resource allocation to seeds rather than to a change in 198
seed TAG metabolism. Similarly, a decreased TAG storage in seeds may also affect 199
TAG content in young seedlings. To test these possibilities, we performed radiotracer 200
labeling experiments using two different labeled substrates, 14C-acetate and 3H2O, 201
which label nascent fatty acids with 14C or 3H during the initial or reduction steps of fatty 202
acid synthesis, respectively (Browse et al., 1981). Under our growth conditions, the 203
incorporation of the radiolabel from14C-acetate or 3H2O into fatty acids of developing 204
embryos was linear for at least 1 h (Supplemental Figure 1). The rate of incorporation of 205 14C or 3H into TAG calculated following 1 h of incubation was similar between wild-type 206
and atg embryos (Supplemental Figure 2). Likewise, there was no significant difference 207
in the rate of radiolabeled TAG accumulation between wild-type and atg seedlings. On 208
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the other hand, the rate of radiolabel incorporation into TAG was significantly reduced in 209
mature and senescing leaves, with the largest effect being observed in mature leaves, 210
the least in developing leaves (Figure 2), mirroring the differences in TAG content in 211
leaves at different ages (Figure 1). Again, leaf TAG levels and rates of radiolabel 212
incorporation into TAG were similar between two atg mutants. 213
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Disruption of Basal Autophagy Compromises Membrane Lipid Turnover 216
The decreased rate of radiolabel incorporation into TAG in atg leaves may be due to a 217
decrease in fatty acid synthesis or a decline in the mobilization of fatty acids from 218
organellar membranes to TAG via autophagy. Rate of fatty acid synthesis can be 219
assessed by measuring the rate of 14C-acetate or 3H2O incorporation into total fatty 220
acids (Browse et al., 1981). As shown in Supplemental Figure 3, growing leaves 221
incorporated 14C from 14C-acetate or 3H from 3H2O into total lipids at a higher rate than 222
did mature and senescing leaves, likely reflecting a higher demand for fatty acids to 223
support membrane expansion and organellar biogenesis during rapid growth. Rates of 224
radiolabel incorporation following 1 h of incubation were similar in wild-type and atg 225
leaves. These results suggest that the decreased TAG synthesis in atg mutants is not 226
due to a decline in the rate of fatty acid synthesis. 227
We next tested whether disruption of autophagy affects membrane lipid turnover. To 228
this end, we first incubated leaves with 14C-acetate for 1 h (pulse). After thoroughly 229
washing with water to remove 14C-acetate, the leaves were incubated in unlabeled 230
solution for an additional 3 d (chase). The radiolabel in leaf total membrane lipids 231
following 1 h of pulse was similar between the wild type and atg mutants (Supplemental 232
Figure 4). Quantification of radioactivity in total membrane lipids showed significant 233
decreases in rates of radiolabeled fatty acid loss, particularly in mature and senescing 234
leaves of atg mutants compared with wild-type leaves of the same age during 3 d of 235
chase (Figure 3). Together, results from pulse-chase labeling experiments suggest that 236
disruption of autophagy results in a decrease in membrane lipid turnover and hence the 237
accumulation of leaf TAG. 238
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The Contribution of Autophagy to TAG Synthesis Differs in Mutants Defective in 240
the Chloroplast or the ER Lipid Biosynthesis Pathway 241
To provide additional evidence for the involvement of autophagy in TAG synthesis and 242
also to test the relative contribution of the chloroplast versus the ER lipid assembly 243
pathway to autophagy-mediated TAG synthesis, we constructed double mutants 244
between tgd1 and atg2-1 or atg5-1. In addition, we crossed the PDAT1 overexpression 245
line 4 in the act1 mutant background (act1/PDAT1-OE4) (Fan et al., 2013a) with atg2-1 246
or atg5-1 and obtained the PDAT1-OE4 line in backgrounds of the wild type or double 247
mutants of atg2-1 act1 or atg5-1 act1. Assays for PDAT activity in microsomal 248
membranes revealed that disruption of autophagy had no significant effect on TAG 249
formation from 14C-labeled PC, whereas the activity was more than 4-fold higher in 250
transgenic plants overexpressing PDAT1 compared with the wild type (Supplemental 251
Figure 5). Analysis of lipid extracts from mature leaves of 5-week-old plants showed 252
that TAG content was higher in tgd1, as expected (Figure 4). Interestingly, there was 253
also a significant increase in TAG in act1 compared with the wild type. Disruption of 254
autophagy caused significant decreases in TAG content in atg2-1 tgd1 and atg5-1 tgd1. 255
TAG levels were 1.9- and 3.5-fold higher in PDAT1-OE4 and act1/PDAT1-OE4, 256
respectively, compared with act1. Importantly, disruption of ATG2 or ATG5 resulted in 257
more than 50% and 70% decreases in TAG content in PDAT1-OE4 and act1/PDAT-258
OE4, respectively. To confirm these results, we crossed atg2-1 or atg5-1 with another 259
independent line act1/PDAT1-OE5 (Fan et al., 2013) and recovered the PDAT1-OE5 in 260
backgrounds of the wild type or double mutants of atg2-1 act1 or atg5-1 act1. Lipid 261
analysis revealed that TAG levels were an average of 46% and 67% lower in 262
atg/PDAT1-OE5 and atg/act1/PDAT1-OE5, compared with PDAT1-OE5 and 263
act1/PDAT1-OE5, respectively (Supplemental Figure 6). 264
Labeling experiments using 14C-acetate revealed that disruption of ATG2 or ATG5 265
caused significant decreases in rates of radiolabel incorporation into total fatty acids in 266
tgd1, PDAT1-OE4 and act1/PDAT1-OE4 (Figure 5A). In addition, rates of the decay in 267
labeled fatty acids of membrane lipids during 3 d of chase following 1 h of pulse with14C-268
acetate (Figure 5B) were significantly lower in atg tgd1, atg PDAT1-OE4 and 269
atg/act1/PDAT1-OE4 than in the wild type, PDAT1-OE4 or act1/PDAT1-OE4. Together, 270
these results suggest that basal autophagy plays an important role in regulating both 271
fatty acid synthesis and membrane lipid turnover, and that the ER lipid biosynthesis 272
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pathway contributes more to autophagy-mediated leaf TAG synthesis than the 273
chloroplast pathway. 274
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Lipophagy Is Induced under Starvation 276
Our data so far indicate that autophagy contributes to TAG synthesis and membrane 277
lipid turnover, but it is not clear whether this mechanism is also involved in the 278
breakdown of TAG stored in LDs. As a first step toward answering this important 279
question, we took advantage of OLE1-GFP overexpressing lines (Fan et al., 2013a). 280
OLE1 is one of the most abundant LD proteins in seeds (Huang, 1996). When 281
ectopically expressed in leaves, OLE1-GFP is specifically targeted to the surface of LDs 282
(Wahlroos et al., 2003), and ectopic expression of OLE1-GFP in the tgd1 (tgd1/OLE1-283
GFP) or wild-type (WT) background (WT/OLE1-GFP) boosted TAG accumulation and 284
induced the formation of clusters of small LDs (Fan et al., 2013a). To facilitate 285
epifluorescence imaging of LD and autophagic dynamics, we generated stable 286
transgenic lines constitutively overexpressing the autophagosome marker ATG8e, one 287
of the nine isoforms of ATG8, fused at the N-terminal with a Discosoma sp red 288
fluorescent protein (DsRed) in the tgd1/OLE1-GFP background (DsRed-289
ATG8e/tgd1/OLEI-GFP). Under normal growth conditions, very few, if any, DsRed-290
ATG8e-labeled punctuate structures, likely representing immature and mature 291
autophagosomes and their precursors (Yoshimoto, 2012), were detected in DsRed-292
ATG8e/tgd1/OLEI-GFP lines (Figure 6). When exposed to extended darkness, a 293
starvation condition known to induce autophagy (Breeze et al., 2011), the number of 294
DsRed-ATG8e-labelded puncta significantly increased (Figure 6 and Supplemental 295
Figure 7), suggesting an increase in autophagic activity. In addition, while the OLE1-296
GFP signals rarely overlapped with DsRed-ATG8e-labeled structures under normal 297
growth conditions, some of the OLE1-GFP signals colocalized with DsRed-ATG8e after 298
3 d of dark treatment (Figure 6). The extent of colocalization was quantified using the 299
Costes image randomization test (Costes et al., 2004). The Pearson’s correlation 300
coefficient (PCC) was determined on 15 image pairs from at least three independent 301
experiments. The average PPC for OLE1-GFP colocalization with DsRed-ATG8e was 302
0.51 ± 0.15 (n = 15) with an average Costes P value of 1.00 ± 0.01 (n =15) (Figure 6), 303
confirming that a subpopulation of LDs colocalize with DsRed-ATG8e-labeled 304
autophagic structures. The relatively low PCC most likely reflects the large difference in 305
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size between the DsRed-labeled structures (less than 100 nm in diameter, Figure 7A) 306
and the OLE1-GFP labeled LD clusters (5 -10 µm in diameter, Figure 7). 307
Under higher magnification, DsRed-ATG8e-labeled autophagic structures were clearly 308
found to be associated with LDs (Figure 7A). Transmission electron microscopy (TEM) 309
analysis of leaves of dark-treated tgd1/OLEI-GFP plants showed that, before dark 310
treatment, LDs were often found to be tightly packed in large clusters with diameters of 311
up to 10 µm (Figure 7B). After dark treatment for 3 d, autophagic vacuoles (AVs) 312
appeared in LD clusters, some of which contained LDs (Figure 7C), which appeared to 313
be partially degraded (Figure 7D). 314
Free GFP is relatively resistant to degradation within the vacuole or lysosome 315
(Yoshimoto, 2012; Klionsky, 2016; Galluzzi et al., 2017). Therefore, if OLE1-GFP-316
coated LDs are degraded in the vacuole, we would expect to observe an increased 317
accumulation of free GFP under dark treatment. To test this possibility, we exposed the 318
WT/OLEI-GFP and tgd1/OLE1-GFP lines to extended darkness. Immunoblot analysis 319
using antibody against GFP showed that free GFP levels markedly increased in both 320
WT/OLEI-GFP and tgd1/OLE1-GFP lines following 3 d of darkness (Figure 8A). 321
Autophagic activity can also be assessed by monitoring the protein level of ATG8-PE 322
(Suzuki and Ohsumi, 2007; Yoshimoto, 2012), which migrates faster on SDS-PAGE in 323
the presence of urea than does the unmodified forms (Chung et al., 2009, 2010). 324
Immunoblot analysis using antibody against ATG8 showed that ATG8-PE conjugates 325
were absent in leaves prior to dark treatment but accumulated after 3 d of darkness 326
(Figure 8A), indicating an overall increase in autophagic activity during dark-induced 327
starvation conditions, as expected. Time-course analysis showed that free GFP levels 328
were low under normal growth conditions but increased steadily during 5 d of darkness, 329
similar to dark-induced accumulation of ATG8-PE (Figure 8B). Importantly, blocking 330
autophagy by disruption of ATG5 largely prevented the accumulation of free GFP in 331
dark-treated WT/OLE1-GFP and tgd1/OLE1-GFP plants (Figure 8A), suggesting that 332
free GFP accumulation is a result of lipophagic activity. ATG5 has been shown to be 333
essential for ATG8 lipidation (Chung et al., 2010). Consistent with this, no ATG8-PE 334
conjugates were detected in atg5-1 leaves following 3 d of dark treatment (Figure 8A). 335
Together, these results provide evidence that lipophagy is induced during dark-induced 336
starvation. 337
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Vacuolar Degradation of LDs Occurs in a Process that Resembles Microlipophagy 339
To dissect the mechanistic basis for lipophagy, we generated transgenic plants 340
coexpressing the tonoplast marker delta tonoplast intrinsic protein (δTIP-DsRed) and 341
OLE1-GFP in the WT background (δTIP-DsRed/OLE1-GFP). When these plants were 342
exposed to dark treatment for 2 d, individual LDs or LD clusters were observed inside 343
(Figures 9A to 9D) or within invagination of (Figures 9E and 9F) δTIP-DsRed-labeled 344
tonoplasts. Analysis of max-intensity projection images of z-stacks acquired by confocal 345
microscopy revealed that the LDs were clearly enclosed by the tonoplast (Figures 9G 346
and 9H). 347
Since typical autophagosomes are small vesicles with diameters of only around 1 µm 348
(Merkulova et al., 2014), the large size of LD clusters in OLEI-GFP transgenic plants 349
(Figure 7, Fan et al., 2013a) may exceed the size capacity of autophagosomes, thereby 350
impeding the recruitment of LD clusters into these structures. Therefore, to further 351
examine the process leading to lipophagy in plants, we took advantage of sdp1 352
mutants, which accumulated small LDs under dark-induced starvation conditions (Fan 353
et al., 2017) and carried out ultrastructural analysis of leaf cells of dark-treated sdp1 354
plants by transmission electron microscopy. Because lipophagy and autophagy 355
appeared to be induced after, but not within, the first 1 d of darkness (Figure 8B), we 356
focused on the subcellular morphological changes in leaf samples between 1 d and 2 d 357
of dark treatment. Consistent with changes in ATG8-PE abundance (Figure 8B), very 358
few autophagic structures were seen after 1 d of darkness (Supplemental Figure 8A). 359
Following 2 d of dark treatment, however, we observed the occurrence of 360
autophagosomes (Supplemental Figures 8B and 8C) and many small vacuoles with 361
diameters of 0.5 to 2 µm (Supplemental Figure 8D). Many of these structures contained 362
autophagic bodies or remnants of cytoplasmic materials, suggesting that they are AVs. 363
Following 1 d of darkness, small LDs with an ~100 nm diameter were frequently seen as 364
spherical structures present in the cytosol (Figure 10A). LDs increased in size after 2 d 365
of dark treatment (Figures 10B to 10F) and were frequently found to be in close contact 366
with AVs (Figure 10C) or within invagination of AV membranes (Figures 10B and 10C) 367
or inside AVs (Figure 10D) or the central vacuole (Figure 10E). Immunoelectron 368
microscopy of dark-treated sdp1 plants with ATG8 antibody revealed the presence of 369
immunogold particles on LDs (Figure 10F). Interestingly, LDs appeared to undergo 370
degradation prior to being fully internalized into AVs, along with other sequestered 371
materials (Figure 10C). Dark treatment in the presence of concanamycin A (concA) also 372
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led to the appearance of LDs in the central vacuole in leaves of wild-type seedlings 373
(Supplemental Figure 9). On the other hand, we did not detect association of 374
macroautophagic membrane structures with LDs as observed during macrolipophagy in 375
mammals (Singh et al., 2009), suggesting that autophagic degradation of LDs during 376
dark-induced starvation in plants is a microlipophagy-like process. Importantly, 377
disruption of ATG2 (Figures 11A and 11B) or ATG5 (Figure 11C and 11D) in sdp1 378
largely blocked the formation of AVs and hence the interaction between LDs and AVs. 379
In atg sdp1 double mutants, most of LDs were still present in the cytosol after 2 d of 380
dark treatment. 381
382
Autophagic Flux Remains Unchanged in sdp1 under Starvation 383
We next tested whether deficiency in cytosolic lipolysis affects autophagy under dark-384
induced starvation in plants as in mammals (Sathyanarayan et al., 2017). To do so, we 385
crossed the wild type or sdp1 with tgd1 overexpressing DsRed-ATG8e and recovered 386
the DsRed-ATG8e line in the wild-type or sdp1 background. As expected, the number 387
of DsRed-ATG8e-labeled puncta increased under dark-induced starvation 388
(Supplemental Figure 10A). Quantitative analysis showed that there was no significant 389
difference in the number of puncta between the wild type and sdp1 after 4 d of 390
darkness. In addition, there was an increase in levels of faster-migrating forms of ATG8-391
PE during dark-induced starvation conditions (Supplemental Figure 10B), and again, 392
there were no discernable differences in levels of starvation-induced ATG8-PE between 393
the wild type and sdp1 mutants. Together, these data suggest that disruption of SDP1 394
does not affect autophagic flux under dark-induced starvation conditions in Arabidopsis. 395
396
Inhibition of Autophagy Increases Leaf TAG Accumulation in sdp1 under 397
Starvation 398
Under dark-induced starvation conditions, TAG accumulated rapidly within the initial 1 d 399
and then started to decline in leaves of sdp1 plants, likely reflecting the induction of 400
lipophagy after dark treatment for 1 d (Fan et al., 2017). To test this possibility, we 401
treated detached leaves of sdp1 mutants with 3-methyladenine (3-MA), a widely used 402
inhibitor of autophagy in mammals (Blommaart et al., 1997) and plants (Yoshimoto, 403
2012). In untreated control leaves, TAG content increased by more than 6-fold during 404
13
the initial 2 d of dark treatment (Figure 12A). Treatment with 3-MA did not affect TAG 405
levels during the initial 2 d of dark incubation, suggesting an involvement of an 406
autophagy-independent mechanism in TAG synthesis. However, TAG content declined 407
after day 2 of dark treatment in the untreated control but continued to increase towards 408
the end of the experiment in 3-MA-treated leaves, such that TAG content was 409
significantly higher at day 3 and day 4 in 3-MA treated leaves compared with the 410
untreated control. These results suggest that autophagy contributes to TAG hydrolysis 411
under severe starvation. 412
413
Lipophagy Requires the Core Components of Autophagy 414
To provide genetic evidence for the induction of lipophagy during dark-induced 415
starvation and also to test whether lipophagy depends on the core autophagic 416
machinery, we generated double mutants between sdp1 and atg2-1 or atg5-1. Under 417
normal growth conditions, TAG levels were lower in leaves of atg sdp1 double mutants 418
compared with sdp1 (Figure 12B). During dark treatment, TAG levels in sdp1 peaked at 419
day 1 following dark exposure and started to decline thereafter (Figure 12 B), consistent 420
with our previous report (Fan et al., 2017). In contrast to sdp1, TAG content in atg sdp1 421
double mutants increased steadily during the first 2 d of dark treatment and remained 422
largely unchanged at day 3. Statistical analysis confirmed that atg sdp1 double mutants 423
accumulated significantly more TAG at day 2 and day 3 following dark treatment, 424
compared with the sdp1 single mutant (Figure 12B). TAG levels remained largely 425
unchanged in the wild type and atg single mutants following dark incubation for 3 d 426
(Supplemental Figure 11). 427
The increased TAG accumulation in atg sdp1 double mutants could result from a 428
decrease in TAG hydrolysis or an increase in the conversion of membrane lipids to 429
TAG. To test these possibilities, we analyzed the changes in levels of total membrane 430
lipids during dark treatment. We detected no significant differences in leaf membrane 431
lipid content among wild type, single and double mutants prior to or during 3 d of 432
darkness (Supplemental Figure 12). Since fatty acid synthesis is completely inactive in 433
the dark (Bao et al., 2000), the absence of change in membrane lipid level supports the 434
hypothesis that disruption of autophagy does not affect membrane lipids to TAG 435
conversion in sdp1 under dark treatment. Total membrane lipid levels were decreased 436
to a similar extent following 3 d of dark treatment in all genotypes analyzed 437
14
(Supplemental Figure 12), apparently because of an increase in fatty acid β-oxidation 438
(Fan et al., 2017). Together, these results suggest that the increased TAG accumulation 439
in atg sdp1 double mutants compared with sdp1 is due to decreased lipophagic activity 440
and that lipophagy relies on the core machinery such as ATG2 and ATG5. 441
442
DISCUSSION 443
We have shown that autophagy plays an important role in organellar membrane 444
turnover, TAG synthesis and LD accumulation under normal growth conditions. 445
Lipophagy, the autophagic degradation of LDs, was induced following extended dark 446
treatment as evident from increased colocalization of LDs and autophagic structures, an 447
increase in accumulation of free GFP derived from OLE1-GFP-coated LDs, the 448
presence of LDs in vacuoles, the association of autophagic marker protein ATG8 with 449
LDs and an increase in TAG levels in atg sdp1 double mutants compared with sdp1. We 450
show that lipophagy occurs in a process resembling microlipophagy as described in 451
yeast and requires the core components of macroautophagy. These results provide 452
mechanistic insight into the role of autophagy in lipid metabolism in plants and lend 453
further support for a critical role of autophagy in quality control of cellular organelles 454
(Yang and Bassham, 2015; Wang et al., 2018), and an immediate role of TAG 455
metabolism in membrane lipid turnover (Fan et al., 2014; Fan et al., 2017). 456
457
Role of Basal Autophagy in TAG Synthesis 458
Our results show that disruption of autophagy impedes membrane lipid turnover and 459
hence TAG synthesis under normal growth conditions. Importantly, the role of basal 460
autophagy in lipid metabolism is tissue and/or development-specific: the contribution of 461
autophagy to TAG synthesis is insignificant in young seedlings, rapidly expanding 462
leaves and developing seeds, but significant in mature and senescing leaves of adult 463
plants. These results are perhaps not surprising because, in contrast to the situation in 464
mature and senescing leaves, organellar membranes in growing cells are newly formed 465
and therefore may not be targeted for autophagy-mediated degradation under normal 466
growth conditions. In developing embryos, fatty acids in membrane lipids are known to 467
be directed to TAG synthesis via acyl editing and headgroup exchange (Bates et al., 468
2012). 469
15
In plants, autophagy has been implicated in the degradation of peroxisomes (Kim et al., 470
2013; Shibata et al., 2013), mitochondria (Li et al., 2014), ER (Liu et al., 2012) and 471
chloroplasts (Ishida et al., 2014), and fatty acids released from membranes of these and 472
other organelles may be used for TAG synthesis (Figure 13). The contribution of 473
autophagy to TAG synthesis is higher in act1 defective in the chloroplast pathway of 474
glycerolipid biosynthesis but lower in tgd1 disrupted in the parallel ER pathway. These 475
results suggest a key role of ER in the provision of fatty acids and/or lipid precursors for 476
autophagy-mediated TAG synthesis. The importance of ER in autophagy-mediated TAG 477
synthesis may reflect not only the role of autophagy in the degradation of this organelle 478
(Liu et al., 2012), but also roles of ER as a membrane source for autophagosome 479
biogenesis (Zhuang et al., 2018) and as the entry point for fatty acids exported from the 480
plastid into the ER pathway of lipid biosynthesis (Bates et al., 2012). 481
Previous studies have shown that during autophagy-mediated chloroplast breakdown, 482
stromal proteins (Ishida et al., 2014) but not thylakoid components (Spitzer et al., 2015; 483
Wang et al., 2018) are delivered into vacuoles for degradation. In line with these 484
observations, our results showed that disruption of autophagy had no significant impact 485
on the dark-induced synthesis of TAG (Figure 12B), which is mainly derived from 486
thylakoid lipids (Kunz et al., 2009; Fan et al., 2017). Similarly, treatment with 3-MA did 487
not affect TAG content during the initial 2 d of dark treatment (Figure 12A), suggesting 488
that autophagy-independent breakdown of chloroplasts serves as a main source of fatty 489
acids for TAG synthesis. In addition, our microscopy analysis showed that the number 490
of chloroplasts per cell remained unaltered during dark treatment (Supplemental Figure 491
13), consistent with previous reports (Keech et al., 2007; Evans et al., 2010). These 492
results exclude the possibility of whole chloroplast autophagy as observed in plants 493
under photooxidative stress (Izumi et al., 2017; Nakamura et al., 2018) or in mutants 494
defective in plastid protein import (Niwa et al., 2004) as a means for thylakoid 495
degradation under dark-induced starvation. The autophagy-independent degradation of 496
thylakoids is also consistent with previous reports showing an internal dismantling of 497
thylakoid systems during senescence-induced chloroplast breakdown (Evans et al., 498
2010; van Doorn and Papini, 2013). 499
In addition to reduced organellar membrane turnover and TAG synthesis, disruption of 500
basal autophagy results in significant decreases in fatty acid synthesis in tgd1 or 501
PDAT1-OE lines (Figure 5A). Although the exact mechanistic basis as to how 502
autophagy impacts fatty acid synthesis remains unclear, it is possible that blocking 503
16
autophagy results in a build-up of fatty acids in the cytosol due to reduced cellular fatty 504
acid needs for organellar membrane lipid turnover, which act as feedback signals to 505
negatively regulate fatty acid synthesis in the chloroplast. On the other hand, 506
overexpression of PDAT1 or blocking the chloroplast lipid biosynthesis pathway in act1 507
accelerates autophagy-mediated membrane lipid turnover and hence increases the 508
cellular demand for fatty acids. This increased fatty acid demand may cause a decrease 509
in fatty acids in the cytosol thereby partially relieving feedback inhibition on plastid fatty 510
acid synthesis. In this context, it is worth noting that inefficient utilization of fatty acids 511
for glycerolipid biosynthesis in the ER has been shown to cause a feedback inhibition 512
on fatty acid synthesis by an unknown mechanism (Bates et al., 2014), and exogenous 513
fatty acid applications to cell cultures or isolated chloroplasts can also elicit feedback 514
inhibition on fatty acid synthesis (Ohlrogge and Jaworski, 1997), likely involving 18:1-515
acyl carrier protein as a signal molecule (Andre et al., 2012). 516
TAG and fatty acid synthesis are increased in tgd1 mutants (Fan et al., 2013b), and 517
disruption of SDP1 causes a significant increase (Fan et al., 2014), whereas blocking 518
autophagy caused a significant decrease (Figure 4) in TAG accumulation in tgd1. These 519
results suggest under normal growth conditions, autophagy functions in TAG synthesis, 520
whereas the cytosolic pathway mediated neutral lipases including SDP1 is the major 521
mechanism for TAG catabolism (Figure 13). 522
523
Inducible Autophagy Is Involved in LD Breakdown via Microlipophagy 524
Under extended darkness, TAG content decreases when autophagy is induced but 525
increases when autophagy is disabled in sdp1. In addition, disruption of SDP1 does not 526
impact autophagic flux under either normal growth or starvation conditions 527
(Supplemental Figure 10). These results suggest an important and general role of 528
lipophagy in mediating TAG hydrolysis under starvation conditions (Figure 13). TAG did 529
not accumulate in atg mutants under extended darkness (Supplemental Figure 11). This 530
result suggests that the SDP1-mediated cytosolic lipolytic pathway can functionally 531
compensate for the lack of lipophagy in TAG hydrolysis under starvation. 532
Previous studies showed that plant autophagic organelles contain hydrolytic enzymes, 533
including proteases and lipases, for cargo degradation at the onset of their formation 534
(Marty, 1978; Buvat and Robert, 1979; Marty, 1999) and are functionally sufficient to 535
17
break down the sequestered materials on their own (Rose et al., 2006; van Doorn and 536
Papini, 2013). In accordance with the autophagosome-autonomous hydrolysis, our 537
ultrastructural analysis showed that LDs and other cellular constituents were degraded 538
in AVs (Figures 7D and 10C), in addition to the central vacuole (Figure 10E). These 539
results point to the unique aspects of plant autophagy in comparison with this catabolic 540
process in yeast and mammals, where the autophagosome itself lacks degradative 541
enzymes and its cargo is broken down following fusion with lytic compartments such as 542
vacuoles and lysosomes, respectively (Eskelinen, 2005; Suzuki and Ohsumi, 2007; 543
Reggiori and Klionsky, 2013; Dikic, 2017; Galluzzi et al., 2017). 544
Our ultrastructural analysis showed that the autophagic degradation of LDs in 545
Arabidopsis occurs in a process resembling microlipophagy in yeast. Disruption of 546
autophagy genes increased TAG content in sdp1 under starvation conditions (Figure 547
12). These results suggest that microlipophagy in Arabidopsis depends on the core 548
machinery of macroautophagy, similar to the situation in yeast (van Zutphen et al., 549
2014). At present, the exact mechanism underlying microautophagy and the role of ATG 550
gene products in microlipophagy remain largely unknown (Noda and Inagaki, 2015; 551
Galluzzi et al., 2017; Oku and Sakai, 2018). Our results showed that microautophagy-552
like LD degradation occurs in AVs, key autophagic structures in macroautophagy 553
(Eskelinen, 2005). Therefore, it is possible that the observed dependence of starvation-554
induced TAG and LD accumulation on the macroautophagic machinery in Arabidopsis 555
may simply reflect the essential role of core ATG proteins in the formation of 556
autophagosomes and hence AVs. In support of this possibility, disruption of the core 557
ATG genes blocks the formation of both AVs and microlipophagy (Figure 11). Recently, 558
vacuolar membrane lipid rafts enriched in sterols have been shown to be necessary for 559
microlipophagy in yeast (Oku and Sakai, 2018). Further studies are needed to test 560
whether the sterol-enriched membrane rafts are involved in microlipophagy in plants, to 561
determine how TAG is hydrolyzed in vacuoles, and to establish the regulation and 562
physiological functions of lipophagy. 563
564
METHODS 565
Plant Materials and Growth Conditions 566
18
The Arabidopsis thaliana plants used in this study were of the Columbia ecotype. The 567
tgd1 mutant was previously described by Xu et al. (2003), act1 by Kunst et al. (1988) 568
and sdp1 mutants by Fan et al. (2017). The PDAT1 overexpressing lines 3 and 4 were 569
described in Fan et al. (2013a). The primers used for genotyping sdp1 were as 570
described previously (Fan et al., 2014). The atg2-1 (SALK_076727) and atg5-1 571
(SAIL_129_B07) mutant lines were ordered from the Arabidopsis Biological Research 572
Center at Ohio State University. The primers used for genotyping atg mutants are 5’-573
GTGGGGCTCATAGCTTAGACC-3’ and 5’- CACTTTCCATCAGCTACTCGC-3’ for 574
atg2-1; 5’-ATTTGCTATTTGTTTGGCACG -3’, and 5’-ATAATGGCAAACCAATTGCAG-575
3’ for atg5-1. Genotyping of tgd1 and act1 mutants was as described (Xu et al., 2005; 576
Fan et al., 2015). 577
For plant growth in soil, surface-sterilized seeds of Arabidopsis were germinated on 578
0.6% (w/v) agar-solidified half-strength Murashige and Skoog (MS) medium (Murashige 579
and Skoog, 1962) supplemented with 1% (w/v) Suc in an incubator with a photon flux 580
density of 50 to 80 μmol m–2 s–1 (cool white lamps), a light period of 16 h (22°C), and a 581
dark period of 8 h (18°C). After 10 d of growth, the seedlings were transferred to soil 582
and grown under a photosynthetic photon flux density of 80 to 150 μmol m−2 s−1 (a 583
combination of cool white fluorescent lamps and incandescent lamps) at 22/18°C 584
(day/night) with a 16-h-light/8-h-dark period, unless stated otherwise. For starvation 585
treatment, whole plants, unless stated otherwise in Figure 12A, were transferred to 586
continuous darkness at 24°C for the time indicated. 587
588
589
Plasmid Construction 590
591
To construct the DsRed-ATG8e expression vector, the coding region of ATG8e was 592
amplified with primers, 5’-agaggtaccAATAAAGGAAGCATCttt-3’ and 5’- 593
agaggatccTTAGATTGAAGAAGCAC-3’. To construct the tonoplast marker δTIP-594
DsRed, the coding sequence of At5g47450 was amplified using the primers: δTIP-fw-595
Sac1: 5’-agagagctcATGGTGAAGATCGAAGTTGG-3’ and δTIP-Rv-kpn1, 5’- 596
ctaggtaccCACTCGGATCTCACGGGTTT-3’. The PCR products were cloned into a 597
19
binary vector pPZP212 (Fan et al., 2015) with DsRed fused to the N-terminus of ATG8e 598
or to the C-terminus of δTIP. After confirming the integrity of the construct by 599
sequencing, plant stable transformation was carried out according to Clough and Bent 600
(1998). 601
602
603
Lipid and Fatty Acid Analyses 604
605
Lipids were extracted from leaves of 4-week-old plants grown in soil as described 606
previously (Fan et al., 2013b). Polar and neutral lipids were seperated on silica plates 607
(Silica Gel 60, EMD Millipore Corporation) by thin layer chromotagraphy (TLC) using 608
acetone-toluent-water (91:30:7, by volume) and/or hexane-diethyl ether-acetatic acid 609
(70:30:1, by volume), respectively. To quantitate low TAG levels in leaves of wild type 610
and atg mutants, total lipid extracts were first fractionated through silica columns 611
(Supelco Discovery SPE DSC-Si 6 mL) as described by James et al. (2010) and the 612
collected neutral lipid-containing fraction was separated by TLC predeveloped using 613
methanol/chloroform (1:1, by volume). Fatty acid methyl esters were prepared as 614
described by Li-Beisson et al. (2013). Separation and identification of the fatty 615
acid methyl esters were performed on an HP5975 gas chromatograph-mass 616
spectrometer (Hewlett-Packard) fitted with a 30 m × 250 μm DB-23 capillary column 617
(Agilent) with helium as the carrier gas as described (Fan et al., 2013b). Fatty acid 618
methyl esters were quantified using heptadecanoic acid as an internal stardard as 619
described (Fan et al., 2013b). 620
621
Immunoblot Analysis 622
Equal fresh weight of mature leaves of 4-week-old plants grown in soil was ground in 623
liquid nitrogen, homogenized with 2 X Laemmli sample buffer. The extracts were 624
incubated for 5 min in boiling water and clarified by centrifugation at 12,000g for 5 min 625
at 22°C. For ATG8 lipidation analysis, proteins were subjected to 15% SDS-PAGE with 626
6 M urea in the separating gel as previously described (Chung et al., 2010). For OLE1-627
GFP, proteins were subjected to 10% SDS-PAGE and blotted to a PVDF membrane. 628
20
Immunoblot analyses were carried out according to the ECL Western Blotting procedure 629
(Thermo Fisher, 32106) with antibodies against GFP (BioLegend; cat#902603; lot# 630
E11LF02512), ATG8a (Agrisera; cat# AS142811; lot# 1604) and actin (MyBioSource; 631
cat# MBS8500610; lot# M14L06). Targeted proteins were visualized using an 632
ImageQuant LAS 4000 biomolecular imager (GE Healthcare Life Sciences). 633
634
635
Assays for FA Synthesis and Degradation 636
637
In vivo labeling experiments with 14C-acetate or 3H2O were done as described (Fan et 638
al., 2013b, Yu et al., 2018). Developing seeds of 50 siliques were directly harvested into 639
labeling medium containing 20 mM MES pH5.5, one-tenth strength of MS salts and 640
0.01% Tween 20 on ice. For labeling, developing seeds or 4-d-old seedlings or 641
detached leaves were incubated in the light of 80 µmol m−2 s−1 (cool white fluorescent 642
lamps) at 22°C in 10 ml of the labeling medium. The assay was started by the addition 643
of 0.1 mCi of 14C-acetate or 0.2 mCi 3H2O (both radiochemicals were from American 644
Radiolabeled Chemicals, St. Louis). After incubation for 1 h, tissues were washed two 645
times with water and immediately used for lipid extraction. For pulse-chase labeling 646
experiments, leaves were labeled for 1 h with14C-acetate. After washing three times with 647
water, the leaves were incubated further with unlabeled solution under a 16-h-light/8-h-648
dark cycle for three days. Total lipids were extracted and separated as described above 649
and radioactivity associated with total lipids or different lipid classes was determined by 650
liquid scintillation counting or phosphor imaging. Radiolabel loss was calculated by 651
correcting for the dilution of radioactivity caused by tissue growth during the chase 652
period. 653
PDAT Activity Assays 654
655
Microsomal membranes were isolated from 3-week-old seedlings as described (Xu et 656
al., 2005). Radioactive PC for PDAT activity assays was prepared after incubating 2-657
week-old seedlings overnight in 20 M MES-KOH, pH 6.0, with 0.2 mCi 14C-acetate 658
(American Radiolabeled Chemicals, St. Louis). Lipids were extracted and separated by 659
21
TLC as described (Fan et al., 2013b). Radiolabeled PC was eluted from silica gel using 660
chloroform:methanol:formic acid (1:2:0.1, by volume) and re-dissolved in chloroform. 661
The reaction mixture contained 0.1 mg of microsomal proteins in 50 mM potassium 662
phosphate buffer, pH 7.8, 250 µM 14C-labeled PC (56 MBq/mmol) and 250 µM 18:1-663
DAG (Avanti Polar Lipids) in a final volume of 200 µL. The reaction solution was 664
thoroughly mixed and incubated at room temperature for 30 min. Lipid extraction and 665
TLC separation were as described above. Radioactivity in TAG was determined by 666
scintillation counting. 667
668
669
Treatment with 3-MA 670
671
Detached leaves of 4-week-old plants grown in soil were floated on water with or 672
without the addition of 5 mM 3-MA (Sigma) (dissolved in water) and 0.01% Tween-20 in 673
the dark at 24C. Samples were taken every 24 h over 4 d for lipid analysis as 674
described above. 675
676
677
Microscopy 678
679
For LD imaging, leaf tissues were stained with a neutral lipid specific fluorescent dye, 680
Nile red (Sigma-Aldrich) or BODIPY493/503 at a final concentration of 10 µg/mL or 5 681
µg/ml in PBS (PH 7.0), respectively, and observed under a Zeiss epifluorescence 682
microscope (Carl Zeiss, Axiovert 200M, Germany) with a green fluorescent protein filter 683
(Zeiss filter set 38). GFP was excited with a BP 470/40 nm and the emission was 684
captured at BP 525/50 nm. 685
686
For the co-localization study, leaf samples were mounted in water on slides and were 687
directly examined using a Leica TCS SP5 laser scan confocal microscope with 688
sequential scanning. GFP was excited with a wavelength of 488 nm and detected at 689
500–530 nm. DsRed was excited at 543 nm and detected at 560–630 nm. 690
22
691
For tonoplast imaging, transgenic plants coexpressing OLE1-GFP and δTIP-DsRed 692
were germinated on 0.6% (w/v) agar-solidified half-strength MS medium lacking 693
sucrose. Six-d-old seedlings were dark-treated for 1 d and then transferred to half-694
strength MS medium with or without 0.5 µM ConcA and incubated in the dark for 695
additional 20 h. The hypocotyls or cotyledons were observed under confocal 696
microscopy as described above. 697
698
For transmission electron microscopy, leaf tissues were fixed with 2.5% (v/v) 699
glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 2h, and then post-fixed 700
with 1% osmium tetroxide in the same buffer for 2 h at room temperature. After 701
dehydration in a graded series of ethanol, the tissues were embedded in EPON812 702
resin (Electron Microscopy Sciences, Hatfield, PA, USA), sectioned and stained with 2% 703
uranyl acetate and lead citrate before viewing under a JEOL JEM-1400 LaB6 120KeV 704
transmission electron microscope (JEOL Inc., Peabody, MA, USA). 705
706
For chloroplast counting, leaf tissues were fixed and embedded as described above. 707
Thin sections produced from the embedded leaf tissues were stained with 1% toluidine 708
blue and imaged using a Zeiss epifluorescence microscope (Carl Zeiss Axiovert 200M). 709
The number of chloroplasts was counted from at least 60 mesophyll cell cross-sections 710
for each time point of dark treatment. 711
712
713
Colocalization Analysis 714
715
Colocalization analysis of OLE1-GFP and ATG8e-DsRed signals was done with the 716
Coloc 2 plugin for Image J. Background subtraction from image pairs was carried out 717
using rolling ball subtraction with a 50-pixel ball size. Statistical significance of the 718
Pearson’s correlation coefficient of the image pairs was analyzed employing the Costes 719
image randomization test as described (Costes et al., 2004). ROIs were selected for 720
colocalization analysis with 100 Costes randomizations using a PSF of 3. 721
23
722
723
Immunogold Labeling 724
725
Five-d-old seedlings grown on 0.6% (w/v) agar-solidified half-strength MS medium with 726
1% Suc were dark-treated for 2 d. The seedlings were then transferred to half-strength 727
MS medium containing 0.5 µM of concA and incubated in the dark for 20 h. Hypocotyls 728
of concA-treated seedlings were harvested and fixed overnight at 4C in 4% 729
paraformaldehyde, 0.25% glutaraldehyde and 0.1 M cacodylate buffer, pH 7.4. The 730
fixed hypocotyls were washed twice with 0.1 M cacodylate buffer and once in distilled 731
water, each wash step for 30 min. The tissues were then dehydrated using a graded 732
ethanol series (50%, 70%, 80%, 95%, twice in 100%) for 30 min each. After 733
dehydration, the tissues were embedded in LR White resin (Electron Microscopy 734
Sciences, London Resin Company, 14381-CA) in gelatin capsules. Resin 735
polymerization was performed at 50–55C. 736
Ultrathin sections (70–90 nm) of LR White-embedded hypocotyls were collected with 737
formvar-coated 300 mesh nickel grids. The grids were first washed with 1xPBS 738
containing 0.2% glycine 2 times, 3 min each, then blocked with 1% BSA, 0.2% glycine 739
for 30 min. After blocking, the grids were incubated with the primary antibody:rabbit 740
polyclonal anti-ATG8a (Agrisera; cat# AS142811; lot# 1604) of Arabidopsis thaliana 741
(dilute 1:60, in blocking solution) at 4C overnight in a wet chamber. After rinsing with 742
blocking solution 5 times, 1 min each, the grids were then incubated in the secondary 743
antibody of goat anti-rabbit IgG conjugated with 10 nm gold particles (Sigma-Aldrich; 744
cat# G7402; lot# SLBW9500) (1:20 dilution in blocking solution) for 1 h at room 745
temperature. Following washing with 1x PBS, 0.2% glycine 5 times, 1 min each, the 746
grids were immersed in a drop of 2.5% glutaraldehyde solution for 30 min. After rinsing 747
with distilled water 2 times, 2 min each, the grids were stained with 2% uranyl acetate 748
for 15 min and lead citrate for 3 min, and then observed using electron microscopy as 749
described above. 750
751
24
752
Accession Numbers 753
754
Sequence data from this article can be found in the Arabidopsis Genome Initiative or 755
GenBank/EMBL databases under the following accession numbers: ACT1, At1g32200; 756
ATG2, At3g19190; ATG5, AT5g17290; ATG7, At5g45900; PDAT1, At5g13640; SDP1, 757
At5g04040; TGD1, At1g19800; δTIP, At5g47450. 758
759
760
Supplemental Data 761
762
Supplemental Figure 1. Time Course of the Incorporation of Radiolabel from 14C-763
Acetate or 3H2O into Total Fatty Acids in Wild-Type Developing Embryos. 764
765
Supplemental Figure 2. Rate of the Incorporation of Radiolabel from 14C-Acetate or 766 3H2O into TAG in Developing Embryos and Seedlings. 767
768
Supplemental Figure 3. Rate of the Incorporation of Radiolabel from 14C-Acetate or 769 3H2O into Total Fatty Acids in Leaves. 770
771
Supplemental Figure 4. Rate of the Incorporation of Radiolabel from 14C-Acetate into 772
Total Membrane Lipids in Leaves. 773
774
Supplemental Figure 5. PDAT Activity in Microsomal Membranes Isolated from 775
Seedlings. 776
777
Supplemental Figure 6. Disruption of Autophagy Reduces TAG Content in Mature 778
Leaves of 4-Week-Old PDAT1 Overexpressing Transgenic Plants. 779
780
Supplemental Figure 7. Increased Accumulation of DsRed-ATG8e-Labeled Structures 781
in Leaves of tgd1 Plants under Dark Treatment. 782 783 784 Supplemental Figure 8. Accumulation of Autophagosomes and Autophagic Vacuoles 785
in Mature Leaves of 4-Week-Old sdp1-4 Plants under Dark Treatment. 786
787
Supplemental Figure 9. The Appearance of LDs in the Central Vacuole in Wild-Type 788
Seedlings after Dark Treatment in the Presence of ConcA. 789
25
790
Supplemental Figure 10. Autophagic Activity in 4-Week-Old sdp1-4 Plants under Dark-791
Induced Starvation. 792
793
Supplemental Figure 11. TAG levels in Mature Leaves of 4-Week-Old Wild Type, atg2-794
1 and atg5-1 Plants under Dark-Induced Starvation. 795
796
Supplemental Figure 12. Membrane Lipid Levels in Mature Leaves of 4-Week-Old 797
sdp1-4, atg2-1 sdp1-4 and atg5-1 sdp1-4 Plants under Dark-Induced Starvation. 798
799
Supplemental Figure 13. Chloroplast Number in Mature Leaves of sdp1-4 Plants 800
under Dark-Induced Starvation. 801
802
Supplemental Dataset 1: The Results of Statistical Analyses 803
804
805
ACKNOWLEDGEMENTS 806
This work was supported by the U.S. Department of Energy, Office of Science, Office of 807
Basic Energy Sciences under contract number DE-SC0012704 - specifically through the 808
Physical Biosciences program of the Chemical Sciences, Geosciences and Biosciences 809
Division. Use of the transmission electron microscope and the confocal microscope at 810
the Center of Functional Nanomaterials was supported by the Office of Basic Energy 811
Sciences, U.S. Department of Energy, under Contract DE-SC0012704. 812
813
AUTHOR CONTRIBUTIONS 814
C.X. and J.F. designed the experiments. J.F., L.Y., and C.X. performed the research. 815
J.F. and C.X. participate in data analysis. C.X. wrote the article with contributions from 816
J.F. and L.Y. 817
818
REFERENCES 819
820
Anding, A.L., and Baehrecke, E.H. (2017). Cleaning House: Selective autophagy of 821
organelles. Dev. Cell 41: 10-22. 822
26
Andre, C., Haslam, R.P., and Shanklin, J. (2012). Feedback regulation of plastidic acetyl-CoA 823
carboxylase by 18:1-acyl carrier protein in Brassica napus. Proc. Natl. Acad. Sci.USA 109: 824
10107-10112. 825
Antonioli, M., Di Rienzo, M., Piacentini, M., and Fimia, G.M. (2017). Emerging mechanisms 826
in initiating and terminating autophagy. Trends Biochem. Sci. 42: 28-41. 827
Avin-Wittenberg, T., Bajdzienko, K., Wittenberg, G., Alseekh, S., Tohge, T., Bock, R., 828
Giavalisco, P., and Fernie, A.R. (2015). Global analysis of the role of autophagy in cellular 829
metabolism and energy homeostasis in Arabidopsis seedlings under carbon starvation. Plant 830
Cell 27: 306-322. 831
Bao, X., Focke, M., Pollard, M., and Ohlrogge, J. (2000). Understanding in vivo carbon 832
precursor supply for fatty acid synthesis in leaf tissue. Plant J. 22: 39-50. 833
Barros, J.A.S., Cavalcanti, J.H.F., Medeiros, D.B., Nunes-Nesi, A., Avin-Wittenberg, T., 834
Fernie, A.R., and Araujo, W.L. (2017). Autophagy deficiency compromises alternative 835
pathways of respiration following energy deprivation in Arabidopsis thaliana. Plant Physiol. 836
175: 62-76. 837
Bates, P.D., Fatihi, A., Snapp, A.R., Carlsson, A.S., Browse, J., and Lu, C.F. (2012). Acyl 838
editing and headgroup exchange are the major mechanisms that direct polyunsaturated fatty 839
acid flux into triacylglycerols. Plant Physiol. 160: 1530-1539. 840
Bates, P.D., Johnson, S.R., Cao, X., Li, J., Nam, J.W., Jaworski, J.G., Ohlrogge, J.B., and 841
Browse, J. (2014). Fatty acid synthesis is inhibited by inefficient utilization of unusual fatty 842
acids for glycerolipid assembly. Proc. Natl. Acad. Sci.USA 111: 1204-1209. 843
Blommaart, E.F.C., Krause, U., Schellens, J.P.M., Vreeling-Sindelárová, H., and Meijer, 844
A.J. (1997). The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit 845
autophagy in isolated rat hepatocytes. Eur. J. Biochem. 243: 240-246. 846
Breeze, E., Harrison, E., McHattie, S., Hughes, L., Hickman, R., Hill, C., Kiddle, S., Kim, 847
Y.S., Penfold, C.A., Jenkins, D., Zhang, C.J., Morris, K., Jenner, C., Jackson, S., 848
Thomas, B., Tabrett, A., Legaie, R., Moore, J.D., Wild, D.L., Ott, S., Rand, D., Beynon, J., 849
Denby, K., Mead, A., and Buchanan-Wollaston, V. (2011). High-Rresolution temporal 850
profiling of transcripts during Arabidopsis leaf senescence reveals a distinct chronology of 851
processes and regulation. Plant Cell 23: 873-894. 852
Browse, J., Roughan, P.G., and Slack, C.R. (1981). Light control of fatty-acid synthesis and 853
diurnal fluctuations of fatty-acid composition in leaves. Biochem. J. 196: 347-354. 854
Browse, J., and Somerville, C. (1991). Glycerolipid synthesis - Biochemistry and regulation. 855
Annu. Rev. Plant Physiol. Plant Mol. Biol. 42: 467-506. 856
27
Buvat, R., and Robert, G. (1979). Vacuole formation in the actively growing root meristem of 857
Barley (Hordeum sativum). Am. J. Bot. 66: 1219-1237. 858
Cabodevilla, A.G., Sanchez-Caballero, L., Nintou, E., Boiadjieva, V.G., Picatoste, F., 859
Gubern, A., and Claro, E. (2013). Cell survival during complete nutrient deprivation depends 860
on lipid droplet-fueled beta-oxidation of fatty acids. J.Biol. Chem. 288: 27777-27788. 861
Chanoca, A., Kovinich, N., Burkel, B., Stecha, S., Bohorquez-Restrepo, A., Ueda, T., 862
Eliceiri, K.W., Grotewold, E., and Otegui, M.S. (2015). Anthocyanin vacuolar inclusions 863
form by a microautophagy mechanism. Plant Cell 27: 2545-2559. 864
Chapman, K.D., and Ohlrogge, J.B. (2012). Compartmentation of triacylglycerol accumulation 865
in plants. J. Biol. Chem. 287: 2288-2294. 866
Chung, T., Suttangkakul, A., and Vierstra, R.D. (2009). The ATG autophagic conjugation 867
system in maize: ATG transcripts and abundance of the ATG8-lipid adduct are regulated by 868
development and nutrient availability. Plant Physiol. 149: 220-234. 869
Chung, T., Phillips, A.R., and Vierstra, R.D. (2010). ATG8 lipidation and ATG8-mediated 870
autophagy in Arabidopsis require ATG12 expressed from the differentially controlled 871
ATG12A and ATG12B loci. Plant J. 62: 483-493. 872
Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-873
mediated transformation of Arabidopsis thaliana. Plant J. 16: 735-743. 874
Costes, S.V., Daelemans, D., Cho, E.H., Dobbin, Z., Pavlakis, G., and Lockett, S. (2004). 875
Automatic and quantitative measurement of protein-protein colocalization in live cells. 876
Biophys. J. 86: 3993-4003. 877
Dikic, I. (2017). Proteasomal and autophagic degradation systems. Annu. Rev. Biochem. 86: 878
193-224. 879
Eastmond, P.J. (2006). SUGAR-DEPENDENT1 encodes a patatin domain triacylglycerol lipase 880
that initiates storage oil breakdown in germinating Arabidopsis seeds. Plant Cell 18: 665-675. 881
Elander, P.H., Minina, E.A., and Bozhkov, P.V. (2017). Autophagy in turnover of lipid stores: 882
trans-kingdom comparison. J. Exp. Bot. 69: 1301-1311. 883
Enrique Gomez, R., Joubes, J., Valentin, N., Batoko, H., Satiat-Jeunemaitre, B., and 884
Bernard, A. (2017). Lipids in membrane dynamics during autophagy in plants. J. Exp. Bot. 885
69: 1287-1200. 886
Eskelinen, E.L. (2005). Maturation of autophagic vacuoles in mammalian cells. Autophagy 1: 1-887
10. 888
28
Evans, I.M., Rus, A.M., Belanger, E.M., Kimoto, M., and Brusslan, J.A. (2010). Dismantling 889
of Arabidopsis thaliana mesophyll cell chloroplasts during natural leaf senescence. Plant 890
Biol. 12: 1-12. 891
Fan, J., Yan, C., Zhang, X., and Xu, C. (2013a). Dual role for phospholipid:diacylglycerol 892
acyltransferase: enhancing fatty acid synthesis and diverting fatty acids from membrane 893
lipids to triacylglycerol in Arabidopsis leaves. Plant Cell 25: 3506-3518. 894
Fan, J., Yan, C., Roston, R., Shanklin, J., and Xu, C. (2014). Arabidopsis lipins, PDAT1 895
acyltransferase, and SDP1 triacylglycerol lipase synergistically direct fatty acids toward β-896
oxidation, thereby maintaining membrane lipid homeostasis. Plant Cell 26: 4119-4134. 897
Fan, J.L., Yan, C.S., and Xu, C.C. (2013b). Phospholipid: diacylglycerol acyltransferase-898
mediated triacylglycerol biosynthesis is crucial for protection against fatty acid-induced cell 899
death in growing tissues of Arabidopsis. Plant J. 76: 930-942. 900
Fan, J.L., Yu, L.H., and Xu, C.C. (2017). A central role for triacylglycerol in membrane lipid 901
breakdown, fatty acid β-oxidation, and plant survival under extended darkness. Plant Physiol. 902
174: 1517-1530. 903
Fan, J.L., Zhai, Z.Y., Yan, C.S., and Xu, C.C. (2015). Arabidopsis 904
TRIGALACTOSYLDIACYLGLYCEROL5 interacts with TGD1, TGD2, and TGD4 to facilitate 905
lipid transfer from the endoplasmic reticulum to plastids. Plant Cell 27: 2941-2955. 906
Galluzzi, L., Baehrecke, E.H., Ballabio, A., Boya, P., Bravo-San Pedro, J.M., Cecconi, F., 907
Choi, A.M., Chu, C.T., Codogno, P., Colombo, M.I., Cuervo, A.M., Debnath, J., Deretic, 908
V., Dikic, I., Eskelinen, E.L., Fimia, G.M., Fulda, S., Gewirtz, D.A., Green, D.R., Hansen, 909
M., Harper, J.W., Jaattela, M., Johansen, T., Juhasz, G., Kimmelman, A.C., Kraft, C., 910
Ktistakis, N.T., Kumar, S., Levine, B., Lopez-Otin, C., Madeo, F., Martens, S., Martinez, 911
J., Melendez, A., Mizushima, N., Munz, C., Murphy, L.O., Penninger, J.M., Piacentini, M., 912
Reggiori, F., Rubinsztein, D.C., Ryan, K.M., Santambrogio, L., Scorrano, L., Simon, 913
A.K., Simon, H.U., Simonsen, A., Tavernarakis, N., Tooze, S.A., Yoshimori, T., Yuan, J., 914
Yue, Z., Zhong, Q., and Kroemer, G. (2017). Molecular definitions of autophagy and related 915
processes. EMBO J. 36: 1811-1836. 916
Graham, I.A. (2008). Seed storage oil mobilization. Annu. Rev. Plant Biol. 59: 115-142. 917
Guiboileau, A., Yoshimoto, K., Soulay, F., Bataille, M.P., Avice, J.C., and Masclaux-918
Daubresse, C. (2012). Autophagy machinery controls nitrogen remobilization at the whole-919
plant level under both limiting and ample nitrate conditions in Arabidopsis. New Phytol. 194: 920
732-740. 921
29
Huang, A.H.C. (1996). Oleosins and oil bodies in seeds and other organs. Plant Physiol. 110: 922
1055-1061. 923
Ishida, H., Izumi, M., Wada, S., and Makino, A. (2014). Roles of autophagy in chloroplast 924
recycling. Biochim. Biophys. Acta 1837: 512-521. 925
Izumi, M., Ishida, H., Nakamura, S., and Hidema, J. (2017). Entire photodamaged 926
chloroplasts are transported to the central vacuole by autophagy. Plant Cell 29: 377-394. 927
Jaishy, B., and Abel, E.D. (2016). Lipids, lysosomes, and autophagy. J. Lipid Res. 57: 1619-928
1635. 929
James, C.N., Horn, P.J., Case, C.R., Gidda, S.K., Zhang, D.Y., Mullen, R.T., Dyer, J.M., 930
Anderson, R.G.W., and Chapman, K.D. (2010). Disruption of the Arabidopsis CGI-58 931
homologue produces Chanarin-Dorfman-like lipid droplet accumulation in plants. Proc. Natl. 932
Acad. Sci.USA 107: 17833-17838. 933
Keech, O., Pesquet, E., Ahad, A., Askne, A., Nordvall, D., Vodnala, S.M., Tuominen, H., 934
Hurry, V., Dizengremel, P., and Gardestrom, P. (2007). The different fates of mitochondria 935
and chloroplasts during dark-induced senescence in Arabidopsis leaves. Plant Cell Environ. 936
30: 1523-1534. 937
Kelly, A.A., van Erp, H., Quettier, A.L., Shaw, E., Menard, G., Kurup, S., and Eastmond, 938
P.J. (2013). The SUGAR-DEPENDENT1 lipase limits triacylglycerol accumulation in 939
vegetative tissues of Arabidopsis. Plant Physiol. 162: 1282-1289. 940
Kim, J., Lee, H., Lee, H.N., Kim, S.H., Shin, K.D., and Chung, T. (2013). Autophagy-related 941
proteins are required for degradation of peroxisomes in Arabidopsis hypocotyls during 942
seedling growth. Plant Cell 25: 4956-4966. 943
Klionsky, D. (2016). Guidelines for the use and interpretation of assays for monitoring 944
autophagy. Autophagy 12: 443-443. 945
Kohlwein, S.D. (2010). Triacylglycerol homeostasis: Insights from yeast. J. Biol. Chem. 285: 946
15663-15667. 947
Kunst, L., Browse, J., and Somerville, C. (1988). Altered regulation of lipid biosynthesis in a 948
mutant of Arabidopsis deficient in chloroplast glycerol-3-phosphate acyltransferase activity. 949
Proc. Natl. Acad. Sci.USA 85, 4143-4147. 950
Kunz, H.H., Scharnewski, M., Feussner, K., Feussner, I., Flugge, U.I., Fulda, A., and 951
Gierth, M. (2009). The ABC transporter PXA1 and peroxisomal β-oxidation are vital for 952
metabolism in mature leaves of Arabidopsis during extended darkness. Plant Cell 21: 2733-953
2749. 954
30
Kurusu, T., Koyano, T., Hanamata, S., Kubo, T., Noguchi, Y., Yagi, C., Nagata, N., 955
Yamamoto, T., Ohnishi, T., Okazaki, Y., Kitahata, N., Ando, D., Ishikawa, M., Wada, S., 956
Miyao, A., Hirochika, H., Shimada, H., Makino, A., Saito, K., Ishida, H., Kinoshita, T., 957
Kurata, N., and Kuchitsu, K. (2014). OsATG7 is required for autophagy-dependent lipid 958
metabolism in rice postmeiotic anther development. Autophagy 10: 878-888. 959
Levine, B., and Kroemer, G. (2008). Autophagy in the pathogenesis of disease. Cell 132: 27-960
42. 961
Li-Beisson, Y., Shorrosh, B., Beisson, F., Andersson, M.X., Arondel, V., Bates, P.D., Baud, 962
S., Bird, D., Debono, A., Durrett, T.P., Franke, R.B., Graham, I.A., Katayama, K., Kelly, 963
A.A., Larson, T., Markham, J.E., Miquel, M., Molina, I., Nishida, I., Rowland, O., 964
Samuels, L., Schmid, K.M., Wada, H., Welti, R., Xu, C., Zallot, R., and Ohlrogge, J. 965
(2013). Acyl-lipid metabolism. Arabidopsis Book 11: e0161. 966
Li, F., Chung, T., and Vierstra, R.D. (2014). AUTOPHAGY-RELATED11 plays a critical role in 967
general autophagy- and senescence-induced mitophagy in Arabidopsis. Plant Cell 26: 788-968
807. 969
Liu, Y., Schiff, M., Czymmek, K., Talloczy, Z., Levine, B., and Dinesh-Kumar, S.P. (2005). 970
Autophagy regulates programmed cell death during the plant innate immune response. Cell 971
121: 567-577. 972
Liu, Y., Burgos, J.S., Deng, Y., Srivastava, R., Howell, S.H., and Bassham, D.C. (2012). 973
Degradation of the endoplasmic reticulum by autophagy during endoplasmic reticulum stress 974
in Arabidopsis. Plant Cell 24: 4635-4651. 975
Martinez-Lopez, N., Garcia-Macia, M., Sahu, S., Athonvarangkul, D., Liebling, E., Merlo, P., 976
Cecconi, F., Schwartz, G.J., and Singh, R. (2016). Autophagy in the CNS and periphery 977
coordinate lipophagy and lipolysis in the brown adipose tissue and liver. Cell Metab. 23: 113-978
127. 979
Marty, F. (1978). Cytochemical studies on gerl, provacuoles, and vacuoles in root meristematic 980
cells of Euphorbia. Proc. Natl. Acad. Sci.USA 75: 852-856. 981
Marty, F. (1999). Plant vacuoles. Plant cell 11: 587-599. 982
McLoughlin, F., Augustine, R.C., Marshall, R.S., Li, F., Kirkpatrick, L.D., Otegui, M.S., and 983
Vierstra, R.D. (2018). Maize multi-omics reveal roles for autophagic recycling in proteome 984
remodelling and lipid turnover. Nat. Plants 4: 1056-1070. 985
Merkulova, E.A., Guiboileau, A., Naya, L., Masclaux-Daubresse, C., and Yoshimoto, K. 986
(2014). Assessment and optimization of autophagy monitoring methods in Arabidopsis roots 987
indicate direct fusion of autophagosomes with vacuoles. Plant Cell Physiol. 55: 715-726. 988
31
Minina, E.A., Bozhkov, P.V., and Hofius, D. (2014). Autophagy as initiator or executioner of 989
cell death. Trends Plant Sci. 19: 692-697. 990
Murashige, T., and Skoog, F. (1962). A revised medium for rapid growth and bio assays with 991
tobacco tissue cultures. Physiol. Plant. 15: 473-497. 992
Nakamura, S., and Yoshimori, T. (2018). Autophagy and longevity. Mol. Cells 41: 65-72. 993
Nakamura, S., Hidema, J., Sakamoto, W., Ishida, H., and Izumi, M. (2018). Selective 994
elimination of membrane-damaged chloroplasts via microautophagy. Plant Physiol. 177: 995
1007-1026. 996
Nguyen, T.B., Louie, S.M., Daniele, J.R., Tran, Q., Dillin, A., Zoncu, R., Nomura, D.K., and 997
Olzmann, J.A. (2017). DGAT1-dependent lipid droplet biogenesis protects mitochondrial 998
function during starvation-induced autophagy. Dev. Cell 42: 9-21. 999
Niwa, Y., Kato, T., Tabata, S., Seki, M., Kobayashi, M., Shinozaki, K., and Moriyasu, Y. 1000
(2004). Disposal of chloroplasts with abnormal function into the vacuole in Arabidopsis 1001
thaliana cotyledon cells. Protoplasma 223: 229-232. 1002
Noda, N.N., and Inagaki, F. (2015). Mechanisms of autophagy. Annu. Rev. Biophys. 44: 101-1003
122. 1004
Ohlrogge, J., and Browse, J. (1995). Lipid biosynthesis. Plant Cell 7: 957-970. 1005
Ohlrogge, J.B., and Jaworski, J.G. (1997). Regulation of fatty acid synthesis. Annu. Rev. 1006
Plant Physiol. Plant Mol. Biol. 48: 109-136. 1007
Oku, M., and Sakai, Y. (2018). Three distinct types of microautophagy based on membrane 1008
dynamics and molecular machineries. Bioessays 40: e1800008. 1009
Oku, M., Maeda, Y., Kagohashi, Y., Kondo, T., Yamada, M., Fujimoto, T., and Sakai, Y. 1010
(2017). Evidence for ESCRT- and clathrin-dependent microautophagy. J. Cell Biol. 216: 1011
3263-3274. 1012
Peng, Y., Miao, H., Wu, S., Yang, W., Zhang, Y., Xie, G., Xie, X., Li, J., Shi, C., Ye, L., Sun, 1013
W., Wang, L., Liang, H., and Ou, J. (2016). ABHD5 interacts with BECN1 to regulate 1014
autophagy and tumorigenesis of colon cancer independent of PNPLA2. Autophagy 12: 2167-1015
2182. 1016
Rambold, A.S., Cohen, S., and Lippincott-Schwartz, J. (2015). Fatty acid trafficking in 1017
starved cells: regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion 1018
dynamics. Dev. Cell 32: 678-692. 1019
Reggiori, F., and Klionsky, D.J. (2013). Autophagic processes in yeast: mechanism, 1020
machinery and regulation. Genetics 194: 341-361. 1021
32
Rose, T.L., Bonneau, L., Der, C., Marty-Mazars, D., and Marty, F. (2006). Starvation-induced 1022
expression of autophagy-related genes in Arabidopsis. Biol. Cell 98: 53-67. 1023
Sathyanarayan, A., Mashek, M.T., and Mashek, D.G. (2017). ATGL promotes 1024
autophagy/lipophagy via SIRT1 to control hepatic lipid droplet catabolism. Cell Rep. 19: 1-9. 1025
Schwarz, V., Andosch, A., Geretschlager, A., Affenzeller, M., and Lutz-Meindl, U. (2017). 1026
Carbon starvation induces lipid degradation via autophagy in the model alga Micrasterias. J. 1027
Plant Physiol. 208: 115-127. 1028
Shatz, O., Holland, P., Elazar, Z., and Simonsen, A. (2016). Complex relations between 1029
phospholipids, autophagy, and neutral lipids. Trends Biochem. Sci. 41: 907-923. 1030
Shibata, M., Oikawa, K., Yoshimoto, K., Kondo, M., Mano, S., Yamada, K., Hayashi, M., 1031
Sakamoto, W., Ohsumi, Y., and Nishimura, M. (2013). Highly oxidized peroxisomes are 1032
selectively degraded via autophagy in Arabidopsis. Plant Cell 25: 4967-4983. 1033
Singh, R., Kaushik, S., Wang, Y., Xiang, Y., Novak, I., Komatsu, M., Tanaka, K., Cuervo, 1034
A.M., and Czaja, M.J. (2009). Autophagy regulates lipid metabolism. Nature 458: 1131-1035
1135. 1036
Soto-Burgos, J., Zhuang, X., Jiang, L., and Bassham, D.C. (2018). Dynamics of 1037
autophagosome formation. Plant Physiol. 176: 219-229. 1038
Spitzer, C., Li, F., Buono, R., Roschzttardtz, H., Chung, T., Zhang, M., Osteryoung, K.W., 1039
Vierstra, R.D., and Otegui, M.S. (2015). The endosomal protein CHARGED 1040
MULTIVESICULAR BODY PROTEIN1 regulates the autophagic turnover of plastids in 1041
Arabidopsis. Plant Cell 27: 391-402. 1042
Suzuki, K., and Ohsumi, Y. (2007). Molecular machinery of autophagosome formation in 1043
yeast, Saccharomyces cerevisiae. FEBS Lett. 581: 2156-2161. 1044
Toyooka, K., Okamoto, T., and Minamikawa, T. (2001). Cotyledon cells of Vigna mungo 1045
seedlings use at least two distinct autophagic machineries for degradation of starch granules 1046
and cellular components. J. Cell Biol. 154, 973-982. 1047
Ustun, S., Hafren, A., and Hofius, D. (2017). Autophagy as a mediator of life and death in 1048
plants. Curr. Opin. Plant Biol. 40: 122-130. 1049
van Doorn, W.G., and Papini, A. (2013). Ultrastructure of autophagy in plant cells: a review. 1050
Autophagy 9: 1922-1936. 1051
van Zutphen, T., Todde, V., de Boer, R., Kreim, M., Hofbauer, H.F., Wolinski, H., Veenhuis, 1052
M., van der Klei, I.J., and Kohlwein, S.D. (2014). Lipid droplet autophagy in the yeast 1053
Saccharomyces cerevisiae. Mol. Biol. Cell 25: 290-301. 1054
33
Wahlroos, T., Soukka, J., Denesyuk, A., Wahlroos, R., Korpela, T., and Kilby, N.J. (2003). 1055
Oleosin expression and trafficking during oil body biogenesis in tobacco leaf cells. Genesis 1056
35: 125-132. 1057
Wang, C.W. (2016). Lipid droplets, lipophagy, and beyond. Biochim. Biophys. Acta 1861: 793-1058
805. 1059
Wang, C.W., Miao, Y.H., and Chang, Y.S. (2014). A sterol-enriched vacuolar microdomain 1060
mediates stationary phase lipophagy in budding yeast. J. Cell Biol. 206: 357-366. 1061
Wang, P., Mugume, Y., and Bassham, D.C. (2018). New advances in autophagy in plants: 1062
Regulation, selectivity and function. Semin. Cell Dev. Biol. 80, 113-122. 1063
Wang, W.Y., Xu, M.Y., Wang, G.P., and Galili, G. (2017). Autophagy: an important biological 1064
process that protects plants from stressful environments. Front. Plant Sci. 7: 2030. 1065
Xu, C., Fan, J., Riekhof, W., Froehlich, J.E., and Benning, C. (2003). A permease-like protein 1066
involved in ER to thylakoid lipid transfer in Arabidopsis. EMBO J. 22: 2370-2379. 1067
Xu, C., Fan, J., Froehlich, J.E., Awai, K., and Benning, C. (2005). Mutation of the TGD1 1068
chloroplast envelope protein affects phosphatidate metabolism in Arabidopsis. Plant Cell 17: 1069
3094-3110. 1070
Xu, C., and Shanklin, J. (2016). Triacylglycerol metabolism, function, and accumulation in plant 1071
vegetative tissues. Annu. Rev. Plant Biol. 67: 179-206. 1072
Xu, C., Yu, B., Cornish, A.J., Froehlich, J.E., and Benning, C. (2006). Phosphatidylglycerol 1073
biosynthesis in chloroplasts of Arabidopsis mutants deficient in acyl-ACP glycerol-3- 1074
phosphate acyltransferase. Plant J. 47: 296-309. 1075
Yang, X., and Bassham, D.C. (2015). New insight into the mechanism and function of 1076
autophagy in plant cells. Int. Rev. Cell Mol. Biol. 320: 1-40. 1077
Yoshimoto, K. (2012). Beginning to understand autophagy, an intracellular self-degradation 1078
system in plants. Plant Cell Physiol. 53: 1355-1365. 1079
Yu, L.H., Fan, J.L., Yan, C.S., and Xu, C.C. (2018). Starch deficiency enhances 1080
lipidbiosynthesis and turnover in leaves. Plant Physiol. 178: 118-129. 1081
Zechner, R., Madeo, F., and Kratky, D. (2017). Cytosolic lipolysis and lipophagy: two sides of 1082
the same coin. Nat. Rev. Mol. Cell Biol. 18: 671-684. 1083
Zhang, M., Fan, J., Taylor, D.C., and Ohlrogge, J.B. (2009). DGAT1 and PDAT1 1084
acyltransferases have overlapping functions in Arabidopsis triacylglycerol biosynthesis and 1085
are essential for normal pollen and seed development. Plant Cell 21: 3885-3901. 1086
34
Zhao, L., Dai, J., and Wu, Q. (2014). Autophagy-like processes are involved in lipid droplet 1087
degradation in Auxenochlorella protothecoides during the heterotrophy-autotrophy transition. 1088
Front. Plant Sci. 5: 400. 1089
Zhuang, X., Chung, K.P., Luo, M., and Jiang, L. (2018). Autophagosome biogenesis and the 1090
endoplasmic reticulum: a plant perspective. Trends Plant Sci. 23: 677-692. 1091
1092
Figure legend 1093
Figure 1. Disruption of Autophagy Reduces TAG accumulation. 1094
TAG levels in dry seeds (A), 4-d-old seedlings (B) and leaves of 5-week-old plants (C). Data are means 1095 of three replicates with SD. FW, fresh weight. Asterisks indicate statistically significant differences from 1096 wild type based on Student’s t test (*P< 0.05, **P < 0.01; Supplemental Dataset 1) . 1097 1098 1099 Figure 2. Disruption of Autophagy Reduces TAG Synthesis in Mature and Senescing but not in Growing 1100 Leaves. 1101
Detached leaves of 5-week-old plants were incubated with 14
C-acetate (A) or 3H2O (B) for 1 h and total 1102
radioactivity in TAG was measured by scintillation counting following separation by thin layer 1103 chromatography. Data are means of three replicates with SD. FW, fresh weight. Asterisks indicate 1104 statistically significant differences from the wild type based on Student’s t test (P < 0.01). 1105 1106 Figure 3. Disruption of Autophagy Slows Down Membrane Lipid Turnover in Mature and Senescing but 1107 not in Growing Leaves. 1108
Radiolabel loss was calculated as percentage of loss of radioactivity in total membrane lipids during 3 d of 1109 chase following 1 h of
14C-acetate pulse of detached leaves of 5-week-old plants. Data are means of 1110
three replicates with SD. WT, wild type. Asterisks indicate statistically significant differences based on 1111 Student’s t test (P < 0.01). 1112 1113 1114 Figure 4. Disruption of Autophagy Reduces Leaf TAG Accumulation. 1115
TAG content in mature leaves of 4-week-old PDAT1 overexpressing line 4 (PDAT1-OE4) in the wild-type, 1116 act1, atg2-1, atg5-1, atg2-1 act1 or atg5-1 act1 background. Data are means of three replicates with SD. 1117 FW, fresh weight. Asterisks indicate statistically significant differences based on Student’s t test (*P< 1118 0.05, **P < 0.01). 1119 1120 1121 Figure 5. Disruption of Autophagy Reduces Fatty Acid Synthesis and Membrane Lipid Turnover in 1122 Growing Leaves of the tgd1 Mutant and PDAT1 Overexpressing Lines. 1123
(A) Rate of 14
C-acetate incorporation into total fatty acids in growing leaves of the 4-week-old PDAT1 1124 overexpressing line 4 (PDAT1-OE4) in the wild-type, act1, atg2-1, atg5-1, atg2-1 act1 or atg5-1 act1 1125 background. 1126 1127 (B) Radiolabel loss during the 3-d chase following 1 h incubation with
14C-acetate. 1128
1129
35
Data are means of three replicates with SD. FW, fresh weight; WT, wild type. Asterisks indicate 1130 statistically significant differences based on Student’s t test (*P< 0.05, **P < 0.01) . 1131 1132 1133 Figure 6. Colocalization of LDs with Autophagic Structures in Leaves under Dark-Induced Starvation. 1134
Confocal images of mature leaves of 4-week-old transgenic plants coexpressing OLE1-GFP (green) and 1135 DsRed-ATG8e (red) in tgd1 before and after 3 d of dark treatment. Boxed areas show colocalization of 1136 green and red signals under higher magnification. Quantification of colocalization is provided by the 1137 Pearson’s correlation coefficient (PCC) and the Costes P value below the images. Bars = 20 µm. 1138 1139 1140 Figure 7. Autophagy of Leaf LDs under Dark-Induced Starvation. 1141
(A) Confocal images of mature leaves of 4-week-old transgenic plants coexpressing OLE1-GFP and 1142 DsRed-ATG8e in tgd1 after 3 d of dark treatment. Bars = 1 µm. 1143 1144 (B) to (D) Electron micrographs of LD clusters in leaf cells of tgd1 overexpressing OLE1-GFP before (B) 1145 and after (C and D) 3 d of dark treatment. (D) Enlargement of the boxed area in (C). Arrows indicate LDs. 1146 AV, autophagic vacuole. LD, lipid droplet. Bars = 1 µm in (B and D) and 2 µm in (C). 1147 1148 1149 Figure 8. OLE1-GFP-Coated Leaf LDs Are Degraded in Vacuoles under Dark-Induced Starvation. 1150
(A) Accumulation of free GFP and ATG8-PE in mature leaves of the 4-week-old wild-type and tgd1 plants 1151 but not in mature leaves of atg5-1 tgd1 double mutant overexpressing OLE1-GFP following 3 d of 1152 darkness. 1153 1154 (B) Time course of free GFP and ATG8-PE accumulation in mature leaves of tgd1 overexpressing OLE1-1155 GFP under dark treatment. 1156 1157 Equal amounts of proteins were subjected to SDS-PAGE followed by immunoblot analysis with antibodies 1158 against GFP, ATG8 or the loading control actin. The dashed lines and asterisks locate free ATG8 proteins 1159 and ATG8-PE conjugates, respectively. WT, wild type. 1160 1161 1162 Figure 9. LD and Vacuole Interactions during Dark-Induced Starvation. 1163
(A) to (F) Confocal images of cotyledon cells of 7-d-old wild-type transgenic plants coexpressing the 1164 tonoplast marker δTIP-DsRed (red) and OLE1-GFP (green) after 2 d of darkness in the presence of 0.5 1165 µM concA. Overlay of red and green fluorescence showing the presence of LDs in vacuoles (A to D) or 1166 within tonoplast invagination (E and F). Bars = 10 µm. 1167 1168 (G) and (H) Three-dimensional images reconstructed from a series of confocal Z-stack images. Bars = 10 1169 µm. 1170 1171 1172 Figure 10. Vacuolar Uptake of LDs under Dark-Induced Starvation. 1173
(A) to (F) Electron micrographs of leaf cells of 4-week-old sdp1-4 plants dark-treated for 1 d (A) and 2 d 1174 (B to F). Arrows indicate lipid droplets. LD, lipid droplet. Bars = 0.5 µm in (A), 1 µm in (B), (C) and (D), 2 1175 µm in (E), 0.2 µm in (F) and 0.1 µm in the inset in (F). 1176 1177 (B) to (D) Various stages of LD internalization into autophagic vacuoles (AVs). Note that LD is partially 1178 degraded within invagination of the AV membrane in (C). 1179 1180 (E) The presence of LDs in the central vacuole (CV). 1181
36
1182 (F) Immunogold labeling of sdp1-4 seedlings dark-treated for 2 d in the presence of 0.5 µM concA using1183 the ATG8 antibody. Arrowheads indicate gold particles. The insert shows higher magnification of the1184 boxed region.1185
1186 1187
Figure 11. Disruption of Autophagy Blocks Autophagic Degradation of LDs. 1188
(A) to (D) Electron micrographs of leaf cells of 4-week-old atg2-1 sdp1-4 (A and B) and atg5-1 sdp1-4 (C1189 and D) plants dark-treated for 2 d. LD, lipid droplet. Bars = 1 µm in (A), (B) and (D) and 2 µm in (C).1190
1191 1192
Figure 12. Inhibition of Autophagy Enhances TAG Accumulation in sdp1-4 under Extended Darkness. 1193
(A) Changes in TAG levels in detached sdp1-4 mature leaves during dark treatment in the presence or1194 absence of 3-MA.1195
1196 (B) Changes in TAG levels in mature leaves of 4-week-old sdp1-4, atg2-1 sdp1-4 and atg5-1 sdp1-41197 plants during dark treatment.1198
1199 Data are means of three replicates with SD. FW, fresh weight. Asterisks indicate statistically significant 1200 differences from controls (A) or sdp1 (B) based on Student’s t test (*P< 0.05, **P < 0.01). 1201
1202 1203 1204 1205
Figure 13. A Proposed Model for the Role of Autophagy in Lipid Metabolism in Plants. 1206
De novo fatty acid (FA) synthesis in chloroplasts is mediated by a series of enzymatic reactions 1207 collectively referred to as fatty acid synthase (FAS). The resultant FAs feed into membrane lipid synthesis 1208 via two parallel pathways localized in the chloroplast or the endoplasmic reticulum. Autophagy-mediated 1209 degradation of cellular organelles other than chloroplasts provides a source of fatty acids for TAG 1210 synthesis under normal and starvation conditions. Thylakoid lipids are broken down by hydrolytic 1211 enzymes (HEs) inside the chloroplast and the released fatty acids are used for TAG synthesis. TAG is 1212 packaged in lipid droplets (LDs) in the cytosol. Under normal growth conditions, TAG stored in LDs is 1213 hydrolyzed by SDP1. Nutrient starvation triggers microlipophagy, which functions together with cytosolic 1214 lipolysis catalyzed by SDP1 to mediate LD breakdown into fatty acids for energy production through β-1215 oxidation. Black arrows represent processes occurring in both normal and starvation conditions. The red 1216 arrow is specific to starvation. 1217
1218 1219
Seeds
Seedlings
TAG
(µg/
10 s
eeds
)TA
G (µ
g/g
FW)
WT atg2-1 atg5-1
020406080
100120140160
WT atg2-1 atg5-1
Growing leaves
Mature leavesSenescing leaves
*
TAG
(µg/
g FW
)
0
20
40
60
80
100
0306090
120150180
WT atg2-1 atg5-1
A
B
C
** **
** **
********
**
020406080
100120140
WT atg2-1 atg5-1
Growing leavesMature leavesSenescing leaves
Labe
l inc
orpo
ratio
n in
to T
AG
(DP
M/m
g FW
/h)
010203040506070
C-acetate14
H3 2O
Labe
l inc
orpo
ratio
n in
to T
AG
(DP
M/m
g FW
/h)
WT atg2-1 atg5-1
A
B Growing leavesMature leavesSenescing leaves
**********
**** ****
OLE1 + ATG8e Bright field Bright field0 d darkness 3 d darkness
OLE1 + ATG8e
PPC = 0.51 ± 0.15 (n = 15); Costes P = 1.00 ± 0.01 (n = 15)
B
0 mM 3-MA5mM 3-MA
0 1 2 3 4Darkness (d)
Leaf
TA
G (µ
g/g
FW)
ALe
af T
AG
(µg/
g FW
) sdp1-4atg2-1 sdp1-4atg5-1 sdp1-4
0 1 2 3 Darkness (d)
** *
0
200
400
600
800
0100200300400500600700 ** **
****
****
FAs
HEs
FAS
Other organelles FAs TAG LD
FAs
SD
P1
β-oxidation
Micr
olipo
phag
y
Autophagy
FAs
Chloroplast
A
B
C
D
E
F
G
H
Brig
ht fi
eld
OLE
1 + δT
IP
A OLE1
B C DAV
AV
OLE1+ ATG8eATG8e Bright field
A B
C D
LDLD
LDLD
LD
LD
LD
Labe
l inc
orpo
ratio
n in
to to
tal
fatty
aci
ds (D
PM
/mg
FW/h
)
act1/
PDAT1-OE4
atg2-1
act1/
PDAT1-OE4
atg5-1
act1/
PDAT1-OE4
WT
act1
atg2-1
atg5-1
atg2-1
tgd1
atg5-1
tgd1tgd
1
PDAT1-OE4
atg2-1
/PDAT1-O
E4
atg5-1
/PDAT1-O
E4
Rad
iola
bel l
oss
durin
g ch
ase
(%
)
**
**
0
5
10
15
20
0
10
20
30
40
50
act1/
PDAT1-OE4
atg2-1
act1/
PDAT1-OE4
atg5-1
act1/
PDAT1-OE4
WT
act1
atg2-1
atg5-1
atg2-1
tgd1
atg5-1
tgd1tgd
1
PDAT1-OE4
atg2-1
/PDAT1-O
E4
atg5-1
/PDAT1-O
E4
A
B
*
*
*
****
****
****
****
1um
A B
D
LD
AV
E
AV
LD
LD
C
AVLD
LD
E
LD
LD
CV
LD
FLD
LD
LD
WT atg2-1 atg5-1
Growing leavesMature leavesSenescing leaves
05
1015202530
Rad
iola
bel l
oss
durin
g ch
ase
(%
)
****
****
Leaf
TA
G (µ
g/g
FW)
0
200
400
600
800
1000
act1/
PDAT1-OE4
atg2-1
act1/
PDAT1-OE4
atg5-1
act1/
PDAT1-OE4
WT
act1
atg2-1
atg5-1
atg2-1
tgd1
atg5-1
tgd1tgd
1
PDAT1-OE4
atg2-1
/PDAT1-O
E4
atg5-1
/PDAT1-O
E4
****
***
*
****
**
ATG8ATG8-PE
Actin
GFP
OLE1-GFP
GFP
OLE1-GFP
WT/O
LE1
tgd1/O
LE1
tgd1/OLE1
0 1 2 3 4 5
atg5-1
tgd1
/OLE
1
*
Actin
ATG8-PEATG8
GFP
*
OLE1-GFP
Darkness (d)
A
B
atg5-1
/OLE
1
WT/O
LE1
tgd1/O
LE1
atg5-1
tgd1
/OLE
1
atg5-1
/OLE
1
0 d darkness 3 d darkness
ATG8ATG8-PE
Actin
* * * *
Parsed CitationsAnding, A.L., and Baehrecke, E.H. (2017). Cleaning House: Selective autophagy of organelles. Dev. Cell 41: 10-22.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Andre, C., Haslam, R.P., and Shanklin, J. (2012). Feedback regulation of plastidic acetyl-CoA carboxylase by 18:1-acyl carrier protein inBrassica napus. Proc. Natl. Acad. Sci.USA 109: 10107-10112.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Antonioli, M., Di Rienzo, M., Piacentini, M., and Fimia, G.M. (2017). Emerging mechanisms in initiating and terminating autophagy.Trends Biochem. Sci. 42: 28-41.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Avin-Wittenberg, T., Bajdzienko, K., Wittenberg, G., Alseekh, S., Tohge, T., Bock, R., Giavalisco, P., and Fernie, A.R. (2015). Globalanalysis of the role of autophagy in cellular metabolism and energy homeostasis in Arabidopsis seedlings under carbon starvation.Plant Cell 27: 306-322.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Bao, X., Focke, M., Pollard, M., and Ohlrogge, J. (2000). Understanding in vivo carbon precursor supply for fatty acid synthesis in leaftissue. Plant J. 22: 39-50.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Barros, J.A.S., Cavalcanti, J.H.F., Medeiros, D.B., Nunes-Nesi, A., Avin-Wittenberg, T., Fernie, A.R., and Araujo, W.L. (2017). Autophagydeficiency compromises alternative pathways of respiration following energy deprivation in Arabidopsis thaliana. Plant Physiol. 175: 62-76.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Bates, P.D., Fatihi, A., Snapp, A.R., Carlsson, A.S., Browse, J., and Lu, C.F. (2012). Acyl editing and headgroup exchange are the majormechanisms that direct polyunsaturated fatty acid flux into triacylglycerols. Plant Physiol. 160: 1530-1539.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Bates, P.D., Johnson, S.R., Cao, X., Li, J., Nam, J.W., Jaworski, J.G., Ohlrogge, J.B., and Browse, J. (2014). Fatty acid synthesis isinhibited by inefficient utilization of unusual fatty acids for glycerolipid assembly. Proc. Natl. Acad. Sci.USA 111: 1204-1209.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Blommaart, E.F.C., Krause, U., Schellens, J.P.M., Vreeling-Sindelárová, H., and Meijer, A.J. (1997). The phosphatidylinositol 3-kinaseinhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur. J. Biochem. 243: 240-246.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Breeze, E., Harrison, E., McHattie, S., Hughes, L., Hickman, R., Hill, C., Kiddle, S., Kim, Y.S., Penfold, C.A., Jenkins, D., Zhang, C.J.,Morris, K., Jenner, C., Jackson, S., Thomas, B., Tabrett, A., Legaie, R., Moore, J.D., Wild, D.L., Ott, S., Rand, D., Beynon, J., Denby, K.,Mead, A., and Buchanan-Wollaston, V. (2011). High-Rresolution temporal profiling of transcripts during Arabidopsis leaf senescencereveals a distinct chronology of processes and regulation. Plant Cell 23: 873-894.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Browse, J., Roughan, P.G., and Slack, C.R. (1981). Light control of fatty-acid synthesis and diurnal fluctuations of fatty-acid compositionin leaves. Biochem. J. 196: 347-354.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Browse, J., and Somerville, C. (1991). Glycerolipid synthesis - Biochemistry and regulation. Annu. Rev. Plant Physiol. Plant Mol. Biol.42: 467-506.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Buvat, R., and Robert, G. (1979). Vacuole formation in the actively growing root meristem of Barley (Hordeum sativum). Am. J. Bot. 66:1219-1237.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Cabodevilla, A.G., Sanchez-Caballero, L., Nintou, E., Boiadjieva, V.G., Picatoste, F., Gubern, A., and Claro, E. (2013). Cell survivalduring complete nutrient deprivation depends on lipid droplet-fueled beta-oxidation of fatty acids. J.Biol. Chem. 288: 27777-27788.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Google Scholar: Author Only Title Only Author and Title
Chanoca, A., Kovinich, N., Burkel, B., Stecha, S., Bohorquez-Restrepo, A., Ueda, T., Eliceiri, K.W., Grotewold, E., and Otegui, M.S.(2015). Anthocyanin vacuolar inclusions form by a microautophagy mechanism. Plant Cell 27: 2545-2559.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Chapman, K.D., and Ohlrogge, J.B. (2012). Compartmentation of triacylglycerol accumulation in plants. J. Biol. Chem. 287: 2288-2294.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Chung, T., Suttangkakul, A., and Vierstra, R.D. (2009). The ATG autophagic conjugation system in maize: ATG transcripts andabundance of the ATG8-lipid adduct are regulated by development and nutrient availability. Plant Physiol. 149: 220-234.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Chung, T., Phillips, A.R., and Vierstra, R.D. (2010). ATG8 lipidation and ATG8-mediated autophagy in Arabidopsis require ATG12expressed from the differentially controlled ATG12A and ATG12B loci. Plant J. 62: 483-493.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.Plant J. 16: 735-743.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Costes, S.V., Daelemans, D., Cho, E.H., Dobbin, Z., Pavlakis, G., and Lockett, S. (2004). Automatic and quantitative measurement ofprotein-protein colocalization in live cells. Biophys. J. 86: 3993-4003.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Dikic, I. (2017). Proteasomal and autophagic degradation systems. Annu. Rev. Biochem. 86: 193-224.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Eastmond, P.J. (2006). SUGAR-DEPENDENT1 encodes a patatin domain triacylglycerol lipase that initiates storage oil breakdown ingerminating Arabidopsis seeds. Plant Cell 18: 665-675.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Elander, P.H., Minina, E.A., and Bozhkov, P.V. (2017). Autophagy in turnover of lipid stores: trans-kingdom comparison. J. Exp. Bot. 69:1301-1311.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Enrique Gomez, R., Joubes, J., Valentin, N., Batoko, H., Satiat-Jeunemaitre, B., and Bernard, A. (2017). Lipids in membrane dynamicsduring autophagy in plants. J. Exp. Bot. 69: 1287-1200.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Eskelinen, E.L. (2005). Maturation of autophagic vacuoles in mammalian cells. Autophagy 1: 1-10.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Evans, I.M., Rus, A.M., Belanger, E.M., Kimoto, M., and Brusslan, J.A. (2010). Dismantling of Arabidopsis thaliana mesophyll cellchloroplasts during natural leaf senescence. Plant Biol. 12: 1-12.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Fan, J., Yan, C., Zhang, X., and Xu, C. (2013a). Dual role for phospholipid:diacylglycerol acyltransferase: enhancing fatty acid synthesisand diverting fatty acids from membrane lipids to triacylglycerol in Arabidopsis leaves. Plant Cell 25: 3506-3518.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Fan, J., Yan, C., Roston, R., Shanklin, J., and Xu, C. (2014). Arabidopsis lipins, PDAT1 acyltransferase, and SDP1 triacylglycerol lipasesynergistically direct fatty acids toward β-oxidation, thereby maintaining membrane lipid homeostasis. Plant Cell 26: 4119-4134.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Fan, J.L., Yan, C.S., and Xu, C.C. (2013b). Phospholipid: diacylglycerol acyltransferase-mediated triacylglycerol biosynthesis is crucialfor protection against fatty acid-induced cell death in growing tissues of Arabidopsis. Plant J. 76: 930-942.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Fan, J.L., Yu, L.H., and Xu, C.C. (2017). A central role for triacylglycerol in membrane lipid breakdown, fatty acid β-oxidation, and plantsurvival under extended darkness. Plant Physiol. 174: 1517-1530.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Fan, J.L., Zhai, Z.Y., Yan, C.S., and Xu, C.C. (2015). Arabidopsis TRIGALACTOSYLDIACYLGLYCEROL5 interacts with TGD1, TGD2, andTGD4 to facilitate lipid transfer from the endoplasmic reticulum to plastids. Plant Cell 27: 2941-2955.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Galluzzi, L., Baehrecke, E.H., Ballabio, A., Boya, P., Bravo-San Pedro, J.M., Cecconi, F., Choi, A.M., Chu, C.T., Codogno, P., Colombo,M.I., Cuervo, A.M., Debnath, J., Deretic, V., Dikic, I., Eskelinen, E.L., Fimia, G.M., Fulda, S., Gewirtz, D.A., Green, D.R., Hansen, M.,Harper, J.W., Jaattela, M., Johansen, T., Juhasz, G., Kimmelman, A.C., Kraft, C., Ktistakis, N.T., Kumar, S., Levine, B., Lopez-Otin, C.,Madeo, F., Martens, S., Martinez, J., Melendez, A., Mizushima, N., Munz, C., Murphy, L.O., Penninger, J.M., Piacentini, M., Reggiori, F.,Rubinsztein, D.C., Ryan, K.M., Santambrogio, L., Scorrano, L., Simon, A.K., Simon, H.U., Simonsen, A., Tavernarakis, N., Tooze, S.A.,Yoshimori, T., Yuan, J., Yue, Z., Zhong, Q., and Kroemer, G. (2017). Molecular definitions of autophagy and related processes. EMBO J.36: 1811-1836.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Graham, I.A. (2008). Seed storage oil mobilization. Annu. Rev. Plant Biol. 59: 115-142.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Guiboileau, A., Yoshimoto, K., Soulay, F., Bataille, M.P., Avice, J.C., and Masclaux-Daubresse, C. (2012). Autophagy machinery controlsnitrogen remobilization at the whole-plant level under both limiting and ample nitrate conditions in Arabidopsis. New Phytol. 194: 732-740.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Huang, A.H.C. (1996). Oleosins and oil bodies in seeds and other organs. Plant Physiol. 110: 1055-1061.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Ishida, H., Izumi, M., Wada, S., and Makino, A. (2014). Roles of autophagy in chloroplast recycling. Biochim. Biophys. Acta 1837: 512-521.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Izumi, M., Ishida, H., Nakamura, S., and Hidema, J. (2017). Entire photodamaged chloroplasts are transported to the central vacuole byautophagy. Plant Cell 29: 377-394.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Jaishy, B., and Abel, E.D. (2016). Lipids, lysosomes, and autophagy. J. Lipid Res. 57: 1619-1635.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
James, C.N., Horn, P.J., Case, C.R., Gidda, S.K., Zhang, D.Y., Mullen, R.T., Dyer, J.M., Anderson, R.G.W., and Chapman, K.D. (2010).Disruption of the Arabidopsis CGI-58 homologue produces Chanarin-Dorfman-like lipid droplet accumulation in plants. Proc. Natl. Acad.Sci.USA 107: 17833-17838.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Keech, O., Pesquet, E., Ahad, A., Askne, A., Nordvall, D., Vodnala, S.M., Tuominen, H., Hurry, V., Dizengremel, P., and Gardestrom, P.(2007). The different fates of mitochondria and chloroplasts during dark-induced senescence in Arabidopsis leaves. Plant CellEnviron. 30: 1523-1534.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kelly, A.A., van Erp, H., Quettier, A.L., Shaw, E., Menard, G., Kurup, S., and Eastmond, P.J. (2013). The SUGAR-DEPENDENT1 lipaselimits triacylglycerol accumulation in vegetative tissues of Arabidopsis. Plant Physiol. 162: 1282-1289.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kim, J., Lee, H., Lee, H.N., Kim, S.H., Shin, K.D., and Chung, T. (2013). Autophagy-related proteins are required for degradation ofperoxisomes in Arabidopsis hypocotyls during seedling growth. Plant Cell 25: 4956-4966.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Klionsky, D. (2016). Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 12: 443-443.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kohlwein, S.D. (2010). Triacylglycerol homeostasis: Insights from yeast. J. Biol. Chem. 285: 15663-15667.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kunst, L., Browse, J., and Somerville, C. (1988). Altered regulation of lipid biosynthesis in a mutant of Arabidopsis deficient inchloroplast glycerol-3-phosphate acyltransferase activity. Proc. Natl. Acad. Sci.USA 85, 4143-4147.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kunz, H.H., Scharnewski, M., Feussner, K., Feussner, I., Flugge, U.I., Fulda, A., and Gierth, M. (2009). The ABC transporter PXA1 andperoxisomal β-oxidation are vital for metabolism in mature leaves of Arabidopsis during extended darkness. Plant Cell 21: 2733-2749.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kurusu, T., Koyano, T., Hanamata, S., Kubo, T., Noguchi, Y., Yagi, C., Nagata, N., Yamamoto, T., Ohnishi, T., Okazaki, Y., Kitahata, N.,Ando, D., Ishikawa, M., Wada, S., Miyao, A., Hirochika, H., Shimada, H., Makino, A., Saito, K., Ishida, H., Kinoshita, T., Kurata, N., andKuchitsu, K. (2014). OsATG7 is required for autophagy-dependent lipid metabolism in rice postmeiotic anther development. Autophagy10: 878-888.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Levine, B., and Kroemer, G. (2008). Autophagy in the pathogenesis of disease. Cell 132: 27-42.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Li-Beisson, Y., Shorrosh, B., Beisson, F., Andersson, M.X., Arondel, V., Bates, P.D., Baud, S., Bird, D., Debono, A., Durrett, T.P., Franke,R.B., Graham, I.A., Katayama, K., Kelly, A.A., Larson, T., Markham, J.E., Miquel, M., Molina, I., Nishida, I., Rowland, O., Samuels, L.,Schmid, K.M., Wada, H., Welti, R., Xu, C., Zallot, R., and Ohlrogge, J. (2013). Acyl-lipid metabolism. Arabidopsis Book 11: e0161.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Li, F., Chung, T., and Vierstra, R.D. (2014). AUTOPHAGY-RELATED11 plays a critical role in general autophagy- and senescence-induced mitophagy in Arabidopsis. Plant Cell 26: 788-807.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Liu, Y., Schiff, M., Czymmek, K., Talloczy, Z., Levine, B., and Dinesh-Kumar, S.P. (2005). Autophagy regulates programmed cell deathduring the plant innate immune response. Cell 121: 567-577.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Liu, Y., Burgos, J.S., Deng, Y., Srivastava, R., Howell, S.H., and Bassham, D.C. (2012). Degradation of the endoplasmic reticulum byautophagy during endoplasmic reticulum stress in Arabidopsis. Plant Cell 24: 4635-4651.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Martinez-Lopez, N., Garcia-Macia, M., Sahu, S., Athonvarangkul, D., Liebling, E., Merlo, P., Cecconi, F., Schwartz, G.J., and Singh, R.(2016). Autophagy in the CNS and periphery coordinate lipophagy and lipolysis in the brown adipose tissue and liver. Cell Metab. 23:113-127.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Marty, F. (1978). Cytochemical studies on gerl, provacuoles, and vacuoles in root meristematic cells of Euphorbia. Proc. Natl. Acad.Sci.USA 75: 852-856.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Marty, F. (1999). Plant vacuoles. Plant cell 11: 587-599.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
McLoughlin, F., Augustine, R.C., Marshall, R.S., Li, F., Kirkpatrick, L.D., Otegui, M.S., and Vierstra, R.D. (2018). Maize multi-omics revealroles for autophagic recycling in proteome remodelling and lipid turnover. Nat. Plants 4: 1056-1070.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Merkulova, E.A., Guiboileau, A., Naya, L., Masclaux-Daubresse, C., and Yoshimoto, K. (2014). Assessment and optimization of autophagymonitoring methods in Arabidopsis roots indicate direct fusion of autophagosomes with vacuoles. Plant Cell Physiol. 55: 715-726.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Minina, E.A., Bozhkov, P.V., and Hofius, D. (2014). Autophagy as initiator or executioner of cell death. Trends Plant Sci. 19: 692-697.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Murashige, T., and Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15:473-497.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Google Scholar: Author Only Title Only Author and Title
Nakamura, S., and Yoshimori, T. (2018). Autophagy and longevity. Mol. Cells 41: 65-72.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Nakamura, S., Hidema, J., Sakamoto, W., Ishida, H., and Izumi, M. (2018). Selective elimination of membrane-damaged chloroplasts viamicroautophagy. Plant Physiol. 177: 1007-1026.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Nguyen, T.B., Louie, S.M., Daniele, J.R., Tran, Q., Dillin, A., Zoncu, R., Nomura, D.K., and Olzmann, J.A. (2017). DGAT1-dependent lipiddroplet biogenesis protects mitochondrial function during starvation-induced autophagy. Dev. Cell 42: 9-21.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Niwa, Y., Kato, T., Tabata, S., Seki, M., Kobayashi, M., Shinozaki, K., and Moriyasu, Y. (2004). Disposal of chloroplasts with abnormalfunction into the vacuole in Arabidopsis thaliana cotyledon cells. Protoplasma 223: 229-232.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Noda, N.N., and Inagaki, F. (2015). Mechanisms of autophagy. Annu. Rev. Biophys. 44: 101-122.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Ohlrogge, J., and Browse, J. (1995). Lipid biosynthesis. Plant Cell 7: 957-970.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Ohlrogge, J.B., and Jaworski, J.G. (1997). Regulation of fatty acid synthesis. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48: 109-136.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Oku, M., and Sakai, Y. (2018). Three distinct types of microautophagy based on membrane dynamics and molecular machineries.Bioessays 40: e1800008.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Oku, M., Maeda, Y., Kagohashi, Y., Kondo, T., Yamada, M., Fujimoto, T., and Sakai, Y. (2017). Evidence for ESCRT- and clathrin-dependent microautophagy. J. Cell Biol. 216: 3263-3274.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Peng, Y., Miao, H., Wu, S., Yang, W., Zhang, Y., Xie, G., Xie, X., Li, J., Shi, C., Ye, L., Sun, W., Wang, L., Liang, H., and Ou, J. (2016).ABHD5 interacts with BECN1 to regulate autophagy and tumorigenesis of colon cancer independent of PNPLA2. Autophagy 12: 2167-2182.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Rambold, A.S., Cohen, S., and Lippincott-Schwartz, J. (2015). Fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis,autophagy, and mitochondrial fusion dynamics. Dev. Cell 32: 678-692.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Reggiori, F., and Klionsky, D.J. (2013). Autophagic processes in yeast: mechanism, machinery and regulation. Genetics 194: 341-361.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Rose, T.L., Bonneau, L., Der, C., Marty-Mazars, D., and Marty, F. (2006). Starvation-induced expression of autophagy-related genes inArabidopsis. Biol. Cell 98: 53-67.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Sathyanarayan, A., Mashek, M.T., and Mashek, D.G. (2017). ATGL promotes autophagy/lipophagy via SIRT1 to control hepatic lipiddroplet catabolism. Cell Rep. 19: 1-9.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Schwarz, V., Andosch, A., Geretschlager, A., Affenzeller, M., and Lutz-Meindl, U. (2017). Carbon starvation induces lipid degradation viaautophagy in the model alga Micrasterias. J. Plant Physiol. 208: 115-127.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Shatz, O., Holland, P., Elazar, Z., and Simonsen, A. (2016). Complex relations between phospholipids, autophagy, and neutral lipids.Trends Biochem. Sci. 41: 907-923.
Pubmed: Author and Title
Google Scholar: Author Only Title Only Author and Title
Shibata, M., Oikawa, K., Yoshimoto, K., Kondo, M., Mano, S., Yamada, K., Hayashi, M., Sakamoto, W., Ohsumi, Y., and Nishimura, M.(2013). Highly oxidized peroxisomes are selectively degraded via autophagy in Arabidopsis. Plant Cell 25: 4967-4983.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Singh, R., Kaushik, S., Wang, Y., Xiang, Y., Novak, I., Komatsu, M., Tanaka, K., Cuervo, A.M., and Czaja, M.J. (2009). Autophagy regulateslipid metabolism. Nature 458: 1131-1135.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Soto-Burgos, J., Zhuang, X., Jiang, L., and Bassham, D.C. (2018). Dynamics of autophagosome formation. Plant Physiol. 176: 219-229.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Spitzer, C., Li, F., Buono, R., Roschzttardtz, H., Chung, T., Zhang, M., Osteryoung, K.W., Vierstra, R.D., and Otegui, M.S. (2015). Theendosomal protein CHARGED MULTIVESICULAR BODY PROTEIN1 regulates the autophagic turnover of plastids in Arabidopsis. PlantCell 27: 391-402.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Suzuki, K., and Ohsumi, Y. (2007). Molecular machinery of autophagosome formation in yeast, Saccharomyces cerevisiae. FEBS Lett.581: 2156-2161.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Toyooka, K., Okamoto, T., and Minamikawa, T. (2001). Cotyledon cells of Vigna mungo seedlings use at least two distinct autophagicmachineries for degradation of starch granules and cellular components. J. Cell Biol. 154, 973-982.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Ustun, S., Hafren, A., and Hofius, D. (2017). Autophagy as a mediator of life and death in plants. Curr. Opin. Plant Biol. 40: 122-130.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
van Doorn, W.G., and Papini, A. (2013). Ultrastructure of autophagy in plant cells: a review. Autophagy 9: 1922-1936.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
van Zutphen, T., Todde, V., de Boer, R., Kreim, M., Hofbauer, H.F., Wolinski, H., Veenhuis, M., van der Klei, I.J., and Kohlwein, S.D.(2014). Lipid droplet autophagy in the yeast Saccharomyces cerevisiae. Mol. Biol. Cell 25: 290-301.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Wahlroos, T., Soukka, J., Denesyuk, A., Wahlroos, R., Korpela, T., and Kilby, N.J. (2003). Oleosin expression and trafficking during oilbody biogenesis in tobacco leaf cells. Genesis 35: 125-132.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Wang, C.W. (2016). Lipid droplets, lipophagy, and beyond. Biochim. Biophys. Acta 1861: 793-805.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Wang, C.W., Miao, Y.H., and Chang, Y.S. (2014). A sterol-enriched vacuolar microdomain mediates stationary phase lipophagy inbudding yeast. J. Cell Biol. 206: 357-366.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Wang, P., Mugume, Y., and Bassham, D.C. (2018). New advances in autophagy in plants: Regulation, selectivity and function. Semin.Cell Dev. Biol. 80, 113-122.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Wang, W.Y., Xu, M.Y., Wang, G.P., and Galili, G. (2017). Autophagy: an important biological process that protects plants from stressfulenvironments. Front. Plant Sci. 7: 2030.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Xu, C., Fan, J., Riekhof, W., Froehlich, J.E., and Benning, C. (2003). A permease-like protein involved in ER to thylakoid lipid transfer inArabidopsis. EMBO J. 22: 2370-2379.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Xu, C., Fan, J., Froehlich, J.E., Awai, K., and Benning, C. (2005). Mutation of the TGD1 chloroplast envelope protein affects
phosphatidate metabolism in Arabidopsis. Plant Cell 17: 3094-3110.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Xu, C., and Shanklin, J. (2016). Triacylglycerol metabolism, function, and accumulation in plant vegetative tissues. Annu. Rev. PlantBiol. 67: 179-206.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Xu, C., Yu, B., Cornish, A.J., Froehlich, J.E., and Benning, C. (2006). Phosphatidylglycerol biosynthesis in chloroplasts of Arabidopsismutants deficient in acyl-ACP glycerol-3- phosphate acyltransferase. Plant J. 47: 296-309.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yang, X., and Bassham, D.C. (2015). New insight into the mechanism and function of autophagy in plant cells. Int. Rev. Cell Mol. Biol.320: 1-40.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yoshimoto, K. (2012). Beginning to understand autophagy, an intracellular self-degradation system in plants. Plant Cell Physiol. 53:1355-1365.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yu, L.H., Fan, J.L., Yan, C.S., and Xu, C.C. (2018). Starch deficiency enhances lipidbiosynthesis and turnover in leaves. Plant Physiol.178: 118-129.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Zechner, R., Madeo, F., and Kratky, D. (2017). Cytosolic lipolysis and lipophagy: two sides of the same coin. Nat. Rev. Mol. Cell Biol. 18:671-684.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Zhang, M., Fan, J., Taylor, D.C., and Ohlrogge, J.B. (2009). DGAT1 and PDAT1 acyltransferases have overlapping functions inArabidopsis triacylglycerol biosynthesis and are essential for normal pollen and seed development. Plant Cell 21: 3885-3901.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Zhao, L., Dai, J., and Wu, Q. (2014). Autophagy-like processes are involved in lipid droplet degradation in Auxenochlorellaprotothecoides during the heterotrophy-autotrophy transition. Front. Plant Sci. 5: 400.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Zhuang, X., Chung, K.P., Luo, M., and Jiang, L. (2018). Autophagosome biogenesis and the endoplasmic reticulum: a plant perspective.Trends Plant Sci. 23: 677-692.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
DOI 10.1105/tpc.19.00170; originally published online April 29, 2019;Plant Cell
Jilian Fan, Linhui Yu and Changcheng XuDual Role for Autophagy in Lipid Metabolism in Arabidopsis
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