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RESEARCH ARTICLE 1 2 Orosomucoid Proteins Interact with the Small Subunit of Serine 3 Palmitoyltransferase and Contribute to Sphingolipid Homeostasis and Stress 4 Responses in Arabidopsis 5 6 Jian Li*, Jian Yin*, Chan Rong*, Kai-En Li*, Jian-Xin Wu*, Li-Qun Huang, Hong-Yun Zeng, Sunil 7 Kumar Sahu, Nan Yao† 8 9 State Key Laboratory of Biocontrol, Guangdong Provincial Key Laboratory of Plant Resources, School of 10 Life Sciences, Sun Yat-sen University, Guangzhou 510275, P. R. China 11 *These authors contributed equally to this work. 12 †Corresponding Author: [email protected] 13 14 Short title: ORMs play a vital role in sphingolipid regulation 15 16 One-sentence summary: Loss-of-function of the orosomucoid proteins stimulate the de novo 17 biosynthesis of sphingolipids, cause an early senescence phenotype, and enhance resistance to oxidative 18 stress and pathogen infection. 19 20 The author responsible for distribution of materials integral to the findings presented in this article in 21 accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Nan Yao 22 ([email protected]) 23 24 ABSTRACT 25 Serine palmitoyltransferase (SPT), a pyridoxyl-5′-phosphate-dependent enzyme, catalyzes the first and 26 rate-limiting step in sphingolipid biosynthesis. In humans and yeast, orosomucoid proteins (ORMs) 27 negatively regulate SPT and thus play an important role in maintaining sphingolipid levels. Despite the 28 importance of sphingoid intermediates as bioactive molecules, the regulation of sphingolipid biosynthesis 29 through SPT is not well understood in plants. Here, we identified and characterized the Arabidopsis 30 thaliana ORMs, ORM1 and ORM2. Loss-of-function of both ORM1 and ORM2 (orm1 amiR-ORM2) 31 stimulated de novo sphingolipid biosynthesis, leading to strong sphingolipid accumulation, especially of 32 long-chain bases and ceramides. Yeast two-hybrid, bimolecular fluorescence complementation, and 33 coimmunoprecipitation assays confirmed that ORM1 and ORM2 physically interact with the small 34 subunit of SPT (ssSPT), indicating that ORMs inhibit ssSPT function. We found that orm1 amiR-ORM2 35 plants exhibited an early-senescence phenotype accompanied by H2O2 production at the cell wall and in 36 mitochondria, active vesicular trafficking, and formation of cell wall appositions. Strikingly, the orm1 37 amiR-ORM2 plants showed increased expression of genes related to endoplasmic reticulum stress and 38 defenses, and also had enhanced resistance to oxidative stress and pathogen infection. Taken together, our 39 findings indicate that ORMs interact with SPT to regulate sphingolipid homeostasis and play a pivotal 40 role in environmental stress tolerance in plants. 41
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Plant Cell Advance Publication. Published on December 6, 2016, doi:10.1105/tpc.16.00574
©2016 American Society of Plant Biologists. All Rights Reserved
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INTRODUCTION 43
In eukaryotes, sphingolipids make up ~40% of the lipids of the plasma membrane and are 44
also abundant in other endomembranes. The functions of these key lipids have been intensively 45
investigated in mammals and yeast for decades (Hannun and Obeid, 2008) and recent work has 46
begun to explore sphingolipid biochemistry in plants. Sphingolipids play pivotal roles as 47
membrane structural components, as bioactive molecules involved in signal transduction and cell 48
regulation, and in a wide range of other biological processes including secretion, programmed 49
cell death, autophagy, stress responses, and cell-cell interactions (Liang et al., 2003; Markham et 50
al., 2011; Sentelle et al., 2012; Bi et al., 2014; Li et al., 2015; Wu et al., 2015). On the outer 51
leaflet of the membrane, sphingolipids form membrane microdomains with cholesterol to 52
provide conformational support for membrane proteins and serve as a platform for recruitment of 53
signaling molecules (Lingwood and Simons, 2010). 54
In humans, changes in sphingolipid contents have been closely linked to diabetes 55
(Summers and Nelson, 2005), cancer (Modrak et al., 2006), Alzheimer’s disease (Mizuno et al., 56
2016), cardiovascular disease, and respiratory disease (Park et al., 2006). For example, 57
sphingosine-1-phosphate can bind to the G protein-coupled receptor EDG5 to inhibit the activity 58
of Rac protein, which could prevent the metastasis of tumor cells (Okamoto et al., 2000). The 59
sphingosine-1-phosphate analog fingolimod (FTY720) has a similar activity and can be used for 60
cancer treatment (Brinkmann et al., 2010). Sphingolipids also function in plant development and 61
responses to biotic or abiotic stresses (Chen et al., 2006; Dietrich et al., 2008; Teng et al., 2008; 62
Chen et al., 2009; Markham et al., 2011; Ternes et al., 2011; König et al., 2012; Zhang et al., 63
2013; Bi et al., 2014). 64
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Regulation of the levels of sphingolipids involves the modulation of key enzymes such as 65
ceramide synthases, ceramidases, ceramide kinase, glucosylceramidase, and 66
inositolphosphorylceramidase (Liang et al., 2003; Wang et al., 2008; Ternes et al., 2011; Bi et al., 67
2014 ; Li et al., 2015; Msanne et al., 2015; Wu et al., 2015). Serine palmitoyltransferase (SPT) is 68
a pyridoxyl-5′-phosphate-dependent enzyme that catalyzes the first and rate-limiting step in 69
sphingolipid biosynthesis, the condensation between L-serine and a long-chain acyl thioester 70
such as palmitoyl-CoA (C16-CoA) to generate long-chain bases (LCBs) (Chen et al., 2006). In 71
Arabidopsis, the SPT enzyme has three subunits, LCB1, LCB2a, and LCB2b. The fbr11-2/lcb1-1 72
mutant, a loss-of-function mutation of LCB1, shows abnormal development, initiating apoptotic 73
cell death in binucleate microspores, and the LCB2 loss-function mutant displays gametophytic 74
lethality (Chen et al., 2006; Dietrich et al., 2008; Teng et al., 2008). 75
Work in animals and yeast showed that orosomucoid (ORM) proteins can modulate SPT 76
activity (Breslow et al., 2010; Han et al., 2010; Gururaj et al., 2013). The ORM family proteins 77
are endoplasmic reticulum (ER)-resident membrane proteins encoded by ORM or ORMDL genes, 78
which are conserved from yeast to humans (Hjelmqvist et al., 2002; Moffatt et al., 2007). 79
Depletion of the mammalian ORMDL1-3 eliminates the feedback of exogenous ceramide on 80
ceramide biosynthesis, indicating that ORMDL proteins function as the primary regulators of 81
ceramide biosynthesis in mammalian cells (Siow and Wattenberg, 2012). In yeast, genetic studies 82
established a link between ORM1 and ORM2 and sphingolipid metabolism, as deletion of ORM1 83
and ORM2 leads to toxic accumulation of sphingolipids, whereas overexpression of ORM1 or 84
ORM2 leads to reduced sphingolipid levels (Breslow et al., 2010). Thus, ORM1 and ORM2 85
negatively regulate sphingolipid synthesis. 86
Yeast (Saccharomyces cerevisiae) Orm proteins regulate sphingolipid metabolism by 87
4
forming a multiprotein complex with SPT, termed the SPOTS complex, which also contains the 88
SPT accessory subunit Tsc3 and the phosphoinositide phosphatase Sac1. Phosphorylation of 89
Orm proteins, mediated by both branches of the TOR signaling pathway, regulates SPT activity 90
to maintain sphingolipid homeostasis (Breslow et al., 2010; Han et al., 2010; Breslow et al., 91
2013). The Ypk1 protein kinase, downstream of rapamycin complex TORC, regulates Orm1 and 92
Orm2 phosphorylation (Roelants et al., 2011; Berchtold et al., 2012; Niles et al., 2012; Sun et al., 93
2012; Shimobayashi et al., 2013). Orm proteins’ SPT-inhibitory activity is subject to feedback 94
regulation by multiple sphingolipid intermediates, including LCBs, ceramide, and complex 95
sphingolipids. When sphingolipid biosynthesis is disrupted, phosphorylation of their amino 96
termini activates Orm1 and Orm2, thus enabling a compensatory increase in SPT activity 97
(Breslow et al., 2010; Liu et al., 2012). Orm1 activity is adjusted in response to manipulation of 98
Orm2 expression levels, with increased Orm2 expression causing a corresponding increase in 99
Orm1 phosphorylation and vice versa. Phosphoregulation of Orm proteins controls sphingolipid 100
biosynthesis in response to various stresses, including heat stress, ER stress, iron stress, and cell 101
wall stress (Sun et al., 2012; Lee et al., 2012; Gururaj et al., 2013). 102
In addition to ORM, eukaryotes have a class of polypeptides, the SPT small subunits 103
(ssSPT), which can regulate SPT activity. For example, yeast has an 80-amino acid polypeptide 104
called Tsc3, which combines with the LCB1 and LCB2 subunits to form a trimer, thereby 105
activating SPT (Gable et al., 2000). Humans have two ssSPT: ssSPTa (68 amino acids) and 106
ssSPTb (76 amino acids). Co-expression of human ssSPTa or ssSPTb and LCB1 or LCB2a, 107
LCB2b can activate SPT in yeast lcb1 lcb2△ mutants (Han et al., 2009). Recent work reported 108
that Arabidopsis has two ssSPTs, encoded by ssSPTa (At1g06515) and ssSPTb (At2g30942). 109
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These 56-amino acid ssSPTs can activate SPT and play an important role in the formation of 110
mature pollen (Kimberlin et al., 2013). 111
Considerable progress has been made in advancing our knowledge of plant sphingolipid 112
metabolism in the past 10-15 years. However, our understanding of ORMs, particularly in plants, 113
remains in its infancy. Here we reported functional characterization of the ORM genes (ORM1, 114
AT1G01230; ORM2, AT5G42000) in Arabidopsis thaliana. We discovered that ORM can 115
interact with and inhibit ssSPT, thus affecting sphingolipid levels. Our studies strongly suggest 116
that ORM plays a key role in the plant response to biotic and abiotic stress. 117
118
RESULTS 119
120
Loss of ORM1 and ORM2 Function Causes an Early-Senescence Phenotype and High 121
Sphingolipid Accumulation 122
ORM family genes encode conserved ER membrane proteins in eukaryotic cells. 123
Arabidopsis thaliana has two genes encoding homologs of S. cerevisiae Orm, AT1G01230 and 124
AT5G42000, designated ORM1 and ORM2 (Figure 1A), which encode 157- and 154-amino-acid 125
polypeptides with 39% and 35% identity to Orm1 in yeast, respectively (Supplemental Figure 126
1A). Using the TMHMM protein structure analysis tool 127
(http://www.cbs.dtu.dk/services/TMHMM/), we found that ORM1 and ORM2 have three 128
predicted transmembrane domains (Supplemental Figure 2). The N-terminal extension found in 129
yeast was absent from Arabidopsis ORMs, but phosphorylation site analysis (using NetPhos and 130
the plant-specific algorithm PhosPhAt), showed ORM1 and ORM2 to have possible 131
phosphorylation sites (Supplemental Figure 1A). ORM1 and ORM2 were expressed at higher 132
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levels in siliques than in other tissues (Supplemental Figure 1B). Our confocal microscopy 133
observations confirmed that ORM1 and ORM2 localized to the ER of Arabidopsis (Figure 1D). 134
To test the function of the Arabidopsis ORMs, we characterized the available T-DNA 135
mutant lines for these genes: SALK_046054 (predicted T-DNA insertion in the first intron of 136
ORM1), and SAIL_1286_D09 (predicted T-DNA insertion in the 5′ UTR of ORM2). 137
Homozygous lines were identified for each of these mutants. We did not detect the full-length 138
transcript of ORM1 in SALK_046054, indicating that the line is a null mutant (Supplemental 139
Figures 1C and 1E). In Arabidopsis, according to TAIR (http://www.arabidopsis.org), ORM2 has 140
two splice isoforms, AT5G42000.1 and AT5G42000.2. However, our RT-PCR experiments 141
detected only the transcripts of AT5G42000.1 (ORM2.1) in plants (Supplemental Figure 1D). 142
Since the full-length transcript of ORM2 was still detected in SAIL_1286_D09 (Supplemental 143
Figure 1D), we also generated an artificial micro RNA (amiR) line targeting ORM2 (amiR-144
ORM2) (Supplemental Figure 1E). For subsequent analysis, we crossed orm1 with amiR-ORM2 145
plants and obtained the orm1 amiR-ORM2 homozygous lines. In addition, we placed ORM1 or 146
ORM2 cDNAs under control of the cauliflower mosaic virus 35S (CaMV35S) promoter to create 147
the overexpression lines ORM1-OX and ORM2-OX in Arabidopsis (Supplemental Figures 1C 148
and 1D). The orm1 amiR-ORM2 plants initially underwent the same development as wild type 149
(Supplemental Figure 3A, top panel, orm1 amiR-ORM2-6 and orm1 amiR-ORM2-5). However, 150
in late development, the orm1 amiR-ORM2 plants showed accelerated senescence, characterized 151
by early chlorosis of rosette leaf tips (Figure 1B and 1C). By contrast, the development of the 152
orm1 single mutant and amiR-ORM2 plants was similar to that of wild-type plants 153
(Supplemental Figure 3A). 154
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In yeast, Orm proteins act as negative mediators of sphingolipid biosynthesis by 155
inhibiting SPT activity (Han et al., 2010). To determine the role of ORM1 and ORM2 in the 156
regulation of sphingolipid biosynthesis in Arabidopsis, we comprehensively analyzed the major 157
classes of sphingolipids in orm1 and amiR-ORM2 plants. We observed no obvious changes in 158
sphingolipids in orm1 and amiR-ORM2 plants compared with wild type (Supplemental Figure 159
3B). However, the sphingolipid profile of 28-day-old orm1 amiR-ORM2 plants showed a 160
dramatic increase in the total sphingolipid contents, especially in LCBs (~15-fold) and ceramides 161
(8-fold), compared with the wild type. By contrast, the amount of glucosylceramides did not 162
change relative to wild type (Figure 1E). Among LCBs, we noticed that d18:0 LCB showed the 163
largest difference in orm1 amiR-ORM2 (Supplemental Figure 4). Remarkably, the ceramide 164
backbones containing long-chain fatty acids (C16) exhibited a more dramatic increase than 165
ceramide containing very-long-chain fatty acids (VLCFA) (C20, C24 and C26) (Supplemental 166
Figure 4). 167
We also measured the sphingolipid contents of orm1 amiR-ORM2 and wild-type rosettes 168
at different developmental stages, and the results showed a gradual accumulation of 169
sphingolipids in orm1 amiR-ORM2 plants (Supplemental Figure 5). The sphingolipid profile of 170
21-day-old plants revealed that the total amounts of LCB, ceramides, and hydroxyceramides 171
were higher in orm1 amiR-ORM2 plants, compared to wild-type plants. However, no visible 172
early-senescence phenotype was observed in the orm1 amiR-ORM2 plants at this stage 173
(Supplemental Figure 3). In other words, orm1 amiR-ORM2 plants accumulated sphingolipids 174
prior to the early senescence. There were no significant changes in sphingolipids in orm1 175
mutants, amiR-ORM2 plants, or overexpression transgenic plants with respect to wild-type plants 176
at different developmental stages (Supplemental Figures 3 and 5). These results indicated that 177
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ORM1 and ORM2 negatively co-regulate sphingolipid biosynthesis in plants. In addition, the 178
early-senescence phenotype of orm1 amiR-ORM2 plants was associated with over-accumulation 179
of sphingolipids. 180
181
ORMs Suppress the de novo Biosynthesis of Sphingolipids in Arabidopsis 182
To decipher the possible cause of the over-accumulation of sphingolipids, we used 15N-183
labeling and metabolic turnover analysis to directly measure in vivo sphingolipid changes, as we 184
reported previously (Shi et al., 2015). Seven-day-old orm1 amiR-ORM2 and wild-type seedlings 185
were transferred to N-deficient ½x MS liquid medium containing 5 mM 15N- serine in a time 186
course. Isotope-labeled LCBs and ceramides of orm1 amiR-ORM2 plants accumulated to 187
significant levels after 24 h of incubation with 15N-serine, relative to wild-type and orm1 or 188
amiR-ORM2 plants (Figure 2). It is noteworthy that the increase of 15N-labeled d18:0 LCB was 189
greater than that of t18:0 LCB (Figure 2). In addition to quantitative changes in ceramides, d18:0 190
ceramides showed a more dramatic increase than t18:0 ceramides (Figure 2). The initial LCB 191
produced in plants is dihydrosphingosine (d18:0 LCB) (Chen et al., 2009). Hence, these 192
measurements confirmed our hypothesis that the loss of ORM1 and ORM2 function in the orm1 193
amiR-ORM2 plants promotes the de novo biosynthesis of sphingolipids, and ultimately leads to 194
higher accumulation of sphingolipids. Moreover, the higher levels of 15N-labeled ceramides 195
containing C16 fatty acid in orm1 amiR-ORM2 plants (Figure 2) were consistent with 196
accumulation of C16 ceramides in 28-day-old orm1 amiR-ORM2 plants (Supplemental Figure 4). 197
198
ORM1 and ORM2 Interact with ssSPT 199
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Previous studies in yeast and mammals showed that ORM proteins physically interact with 200
Lcb1 and Lcb2 (Han et al., 2010). To test the interactions of ORM proteins in Arabidopsis, we 201
first applied yeast two-hybrid assays. Surprisingly, our yeast two-hybrid assays revealed that the 202
ORM proteins physically interact with the small subunit of SPT (ssSPT) (Figure 3A). The 203
interaction was further confirmed by co-immunoprecipitation (co-IP) assays. Anti-Flag resins 204
could precipitate not only ORM-Flag but also GFP-ssSPT (Figure 3B). When free GFP was used 205
as a control, no GFP signals were detected in the eluate (Figure 3B). We also used bimolecular 206
fluorescence complementation (BiFC) assays to confirm the ORM-ssSPT interaction. Clear YFP 207
fluorescence was observed in protoplasts co-transformed with pSATN-nEYFP-ORM1 and 208
pSATN-cEYFP-ssSPTa or pSATN-cEYFP-ssSPTb, pSATN-nEYFP-ORM2, and pSATN-cEYFP-209
ssSPTa or pSATN-cEYFP-ssSPTb constructs (Figure 3C). However, no YFP signal was detected 210
in the protoplasts co-transformed with one construct in combination with an empty vector 211
(Figure 3C). In addition, our confocal microscopy observations confirmed that ssSTPa and 212
ssSTPb also localized to the ER where they interact with ORMs (Supplemental Figure 6). These 213
results are consistent with the physical interaction of ORM1 and ORM2 with ssSPTa and ssSPTb 214
at the ER. We also found that SPTs localized to ER in the orm1 amiR-ORM2 plants, as 215
confirmed by transient co-expression with 35Spro:EGFP-LCB1 or 35Spro:EGFP-LCB2a/b and an 216
ER marker, suggesting that loss of function of ORMs did not affect subcellular localization of 217
the SPT complex (Supplemental Figure 7). 218
219
ORM Proteins Decrease the Biosynthesis of Sphingolipids 220
Reduced SPT activity may decrease sensitivity to fumonisin B1 (FB1) by decreasing levels 221
of cytotoxic LCBs, whereas increased SPT activity may increase sensitivity to FB1 by increasing 222
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the levels of LCBs (Shi et al., 2007; Saucedo-Garcia et al., 2011). ssSPTa/ssSPTb can strongly 223
stimulate SPT activity and increase the production of LCBs in Arabidopsis (Kimberlin et al., 224
2013). To explore the function of Arabidopsis ORMs, we grew plants on agar plates with FB1 to 225
test the significance of the interaction between ORM and ssSPT. Both ORM1-OX and ORM2-OX 226
overexpression lines showed resistance to 0.5 μM FB1, whereas the orm1 amiR-ORM2 plants 227
were highly sensitive to FB1 (Figure 4). Consistent with a previous human ORM study (Breslow 228
et al., 2010), ORM proteins thus appear to negatively regulate sphingolipid biosynthesis. 229
230
Loss of ORM Proteins Increases ER Stress-Related Responses 231
To understand whether the loss of ORM proteins affects cellular ultrastructure, we fixed 232
the orm1 amiR-ORM2 leaves before the appearance of the senescence phenotype. As shown in 233
Figure 5, compared with the normal nuclei observed in the wild-type cells (Figure 5A), the orm1 234
amiR-ORM2 cells showed condensed chromatin aggregated in the perinuclear membrane, which 235
indicates dying cells (Figure 5B; Liang et al., 2003), and a large amount of tiny membrane sacs 236
close to the plasma membrane and the cell wall (Figure 5C and 5D). Interestingly, we found a 237
large cell wall apposition, a clear marker of the defense response, in the orm1 amiR-ORM2 leaf 238
cells (Figure 5E). When we used the cerium chloride method (Bi et al., 2014) to detect reactive 239
oxygen species (ROS), we observed H2O2 production on the cell wall (Figure 5G and 5H), the 240
cytosolic area close to the apoplast (Figure 5I) and in the mitochondria (Figure 5J). No cerium 241
deposits were observed in the wild-type control (Figure 5F). In the dying cells, irregular ER and 242
vacuolization were frequently observed (Figure 5K). These observations suggest that loss of 243
ORMs may induce active vesicular trafficking around the plasma membrane, and this ER stress 244
phenomenon is associated with defense responses. 245
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To support the TEM observations, we investigated the expression of genes related to ER 246
stress and defenses. Strikingly, the expression of ER stress marker genes (bZIP28, bZIP60, 247
IRE1a, and TBF1) significantly increased in orm1 amiR-ORM2 plants, compared with wild type 248
(Figure 6A). To determine whether the transcription of defense-related genes was affected in 249
plants with decreased ORM function, we analyzed the expression of salicylic acid-related and 250
pathogenesis-related (PR) genes in wild-type and orm1 amiR-ORM2 plants using RT-qPCR. The 251
orm1 amiR-ORM2 plants showed high transcript levels of these genes, such as PAD4, SID2, and 252
NPR1, compared with wild type (Figure 6B). PR1 showed especially dramatic enhancement, 253
which is highly linked to the formation of cell wall appositions. No obvious changes of those 254
genes were detected in orm1 and amiR-ORM2 plants (Figure 6). 255
256
Loss of ORM Proteins Enhances Plant Resistance to Abiotic and Biotic Responses 257
Recent studies have shown that sphingolipids are involved in plant stress responses (Wang 258
et al., 2008; Peer et al., 2010; Bi et al., 2014; Li et al., 2015). The orm1 amiR-ORM2 plants 259
exhibit highly induced expression of ER stress and salicylic acid-related genes, which suggested 260
to us that we should explore the role of ORMs during abiotic and biotic stress. We first treated 261
the orm1 amiR-ORM2 plants with the oxidative stress agent methyl viologen (MV), which 262
produces reactive oxygen species (ROS). Surprisingly, orm1 amiR-ORM2 plants proved to be 263
tolerant to ROS and exhibited dramatically higher survival rates than wild type (Figure 7A and 264
7B). 265
We further inoculated orm1 amiR-ORM2 plants with the bacterial pathogen Pseudomonas 266
syringae strain DG3. The orm1 amiR-ORM2 plants showed subtle symptoms after infection and 267
proved to be relatively resistant to DG3 bacteria when compared with wild type (Figure 7C). 268
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This enhanced disease resistance was consistent with the detection of high transcript levels of 269
resistance genes in orm1 amiR-ORM2 plants. These results indicate that loss of ORM function in 270
Arabidopsis affects the plant’s response to abiotic and biotic stress. 271
272
273
DISCUSSION 274
The orosomucoid (ORM) proteins, encoded by ORMDL (ORM) genes, are highly 275
conserved eukaryotic transmembrane proteins located in the ER (Hjelmqvist et al., 2002; Han et 276
al., 2010). The three ORMDL proteins are highly conserved in humans and yeast has two 277
homologous ORM genes (ORM1 and ORM2) with redundant functions. Compared with other 278
organisms, the extent of conservation of ORM function in plants remains unclear. In this report, 279
we characterized the two ORM genes found in Arabidopsis thaliana. Our results indicate that 280
Arabidopsis ORMs function to regulate sphingolipid biosynthesis in maintaining sphingolipid 281
homeostasis and play a role in response to abiotic and biotic stresses. 282
ORM proteins have few conserved regions, mainly located in the middle of the amino 283
acid sequence, and are generally predicted to form two to four transmembrane domains. The 284
amino acid sequences of the Arabidopsis ORMs share 81% identity and also share 35-39% 285
identity with S. cerevisiae Orm1 and Orm2 (Supplemental Figure 1), but interestingly, the 286
Arabidopsis isoforms are N-terminally truncated relative to the yeast proteins and therefore lack 287
the three serine residues that are phosphorylated by Ypk1 in the yeast proteins (Roelants et al., 288
2011). Although we found some possible phosphorylation sites in Arabidopsis ORM1 and 289
ORM2, we still do not know whether the ORMs in Arabidopsis are regulated by phosphorylation, 290
similar to yeast Orm proteins. Another possibility is that Arabidopsis ORMs may be regulated by 291
13
an allosteric effect on the ORM proteins themselves, where sphingolipids trigger a change in 292
conformation within a pre-existing ORM/SPT complex rather than enhancing formation of the 293
complex (Kiefer et al., 2015). 294
Orm1 and 2 negatively regulate SPT, activating or inhibiting its activity through 295
phosphorylation and dephosphorylation (Tafesse et al., 2010). Regulation of sphingolipids and 296
Orm proteins involves a feedback mechanism; Orms regulate SPT and sphingolipid metabolites 297
such as LCBs, ceramide, and accumulated sphingoid intermediates then trigger Orm 298
dephosphorylation, which in turn downregulates sphingolipid biosynthesis (Sun et al., 2012). In 299
yeast, Orm1 and Orm2 are mainly phosphorylated by Npr1 and Ypk1, respectively (Roelants et 300
al., 2011; Sun et al., 2012; Shimobayashi et al., 2013). Besides ORMs, other polypeptides can 301
also regulate the activity of SPT, namely Tsc3 in yeast (Gable et al., 2000) and ssSPTa (68 302
amino acids) and ssSPTb (76 amino acids) in human (Han et al., 2009). The 56-amino-acid 303
ssSPTs, ssSPTa, and ssSPTb strongly stimulate Arabidopsis SPT activity when coexpressed with 304
Arabidopsis LCB1 and LCB2a or 2b in a yeast spt null mutant (Kimberlin et al., 2013). Also, 305
ssSPTa suppression lines increase the resistance to FB1, and the ssSPTa overexpression lines 306
display strong sensitivity to FB1 by increasing or decreasing SPT activity (Kimberlin et al., 307
2013). In the present study, the de novo biosynthesis of sphingolipids was activated due to loss of 308
Arabidopsis ORM function. The isotope-labeled LCBs and ceramides of orm1 amiR-ORM2 309
accumulated significantly after 24 h of incubation in 15N-labeled serine, relative to wild type, 310
which showed that ORM was indeed a negative regulator of SPT in Arabidopsis, like in human 311
and yeast. Through yeast two-hybrid system, BiFC experiments, and co-IP assays, we confirmed 312
that Arabidopsis ORMs can directly bind with the Arabidopsis ssSPTs. Based on our data, we 313
put forward two possible mechanisms for the function of ORMs (Figure 8). On the one hand, 314
14
ORM protein might bind to the SPT complex through the physical interaction with ssSPT and 315
inhibit the enzyme activity of SPT, rather than changing its localization; on the other hand, the 316
interaction between ORM and ssSPT might reduce the available ssSPTs that can interact with 317
core LCB1/LCB2. We favor the first mechanism, in which ORM binds to the SPT complex with 318
the ssSPT interaction, based on a recent study by Kimberlin et al (2016). In addition, we 319
observed that the ORMs and ssSPT localize in the ER, which could be the site where they 320
interact (Figure 1 and Supplemental Figure 6). Moreover, during FB1 treatment, ORM-321
overexpression lines displayed strong resistance to FB1, the opposite effect to that of ssSPTa. 322
Taking these results together, we speculate that ORMs could modulate sphingolipid levels by 323
inhibiting the function of ssSPTs in Arabidopsis. When the ORMs are knocked out, SPT could 324
be activated without changing its location, resulting in substantial accumulation of sphingolipids 325
(Figure 8). 326
Like in humans and yeast, ORM proteins in Arabidopsis play an important role in 327
maintaining the level of sphingolipids (Tafesse et al., 2010; Breslow et al., 2010; Liu et al., 2012). 328
In this study, we analyzed the ORM1 T-DNA insertion mutant orm1 and the ORM2 silencing line 329
(amiR-ORM2). Under normal growth conditions, these plants showed no obvious differences 330
compared to wild type. However, the orm1 amiR-ORM2 plants exhibited premature senescence 331
and considerable changes in sphingolipid contents, including multiple-fold increases in overall 332
sphingolipids, especially in LCBs and ceramides. Among these, d18:0 and t18:0 LCB, the 333
ceramide backbones containing long-chain fatty acids (C16) exhibited the largest changes. 334
Measurements of the sphingolipid contents of orm1 amiR-ORM2 and wild-type rosettes at 335
different developmental stages showed that prior to the onset of the early-senescence phenotype, 336
the total LCB, ceramides, and hydroxyceramides, mainly ceramide backbones containing 337
15
dihydroxy LCBs and C16 fatty acids, increased in orm1 amiR-ORM2 plants compared with wild-338
type plants. These observations suggest that LCBs or ceramides may induce plant senescence, 339
and accumulate to threshold levels due to loss of ORM function. 340
Sphingolipids are involved in the regulation of plant responses to biotic and abiotic stresses. 341
Our previous studies showed that the Arabidopsis neutral ceramidase mutant ncer1 accumulates 342
hydroxyceramides and is sensitive to oxidative stress (Li et al., 2015) and the ceramide kinase 343
mutant acd5 accumulates ceramides and is sensitive to Pseudomonas syringae and Botrytis 344
cinerea (Liang et al., 2003; Bi et al., 2014). In addition, the fatty acid α hydroxylase mutant fah1 345
fah2 has a stronger resistance to Diplodia powdery mildew and Verticillium fungi (Verticillium 346
longisporum) compared to wild type (Konig et al., 2012). In this study, we found that plants with 347
loss of ORM function are more resistant to MV and pathogen infection. This phenomenon could 348
be attributed to the higher accumulation of LCBs in the ORMs mutants compared with the acd5 349
and fah1 fah2 mutants. Several previous studies reported that LCBs play an important role 350
during abiotic and biotic stress (Peer et al., 2010; Saucedo-Garcia et al., 2011; Li et al., 2015). 351
LCBs can induce MPK6 expression and programmed cell death by an MPK6-mediated signal 352
transduction pathway (Saucedo-Garcia et al., 2011). The expression of MPK6 can be induced 353
rapidly by Flg22, which mediates the pathogen-associated pattern-induced basal resistance 354
(Galletti et al., 2011). Phytosphingosine content has been reported to rapidly escalate at two 355
hours after Pseudomonas inoculation, and is also involved in plant resistance (Peer et al., 2010). 356
Our sphingolipid data also showed an increase in the phytosphingosine contents in plants with 357
loss of ORM function, which leads to increased resistance against P. syringae infection. In 358
addition, our TEM observation gives us another indication of the defense response in plants with 359
decreased ORM1 or ORM2 (Figure 8), showing that active vesicular transport may be caused by 360
16
perturbing the ORM-mediated sphingolipid homeostasis, and this may strengthen cell surface 361
defenses (including formation of cell wall appositions and ROS production on the cell wall). 362
Further, based on the high transcript levels of resistance-related genes (PR1) in plants with loss 363
of ORM function, and especially the up-regulated ER stress-related genes (Liu et al., 2010) and 364
expression of PAD4, SID2, and NPR1 genes related to the SA pathway, we propose that loss of 365
ORM function may affect SA biosynthesis, thereby affecting the plant’s resistance to pathogens. 366
367
368
METHODS 369
Plant Materials and Growth Conditions 370
Wild-type Arabidopsis thaliana (Columbia, Col-0) and T-DNA insertion orm mutants 371
(SALK_046054 and SAIL_1286_D09) from ABRC (http://abrc.osu.edu/) were sown on soil after 372
3 days of stratification at 4°C, followed by cultivation in the greenhouse at 22°C and 50% 373
relative humidity, 16 h light/ 8 h dark with 4800-6000 lux light intensity (PAK bulb, 374
PAK090311). 375
To construct the ORM1 and ORM2 RNAi suppression vectors, the RS300 plasmid was 376
employed to clone an artificial microRNA, as previously described (Schwab et al., 2006). The 377
artificial microRNA was inserted into pCAMBIA1300 and fused with the 35S promoter and 378
NOS terminator. The ORM1 and ORM2 over-expression constructs were generated using 379
pCAMBIA1300 by inserting the open reading frame of ORM1 or ORM2, the 35S promoter, and 380
NOS terminator sequences. Finally, these constructs were transformed into wild type using an 381
Agrobacterium tumefaciens (EHA105)-mediated method. Transformed progenies were screened 382
on 1/2x MS medium containing 0.25 mg/l hygromycin. Homozygous transgenic lines were 383
17
isolated from the T3 generation for further study. All primers used for cloning are shown in 384
Supplemental Table 1. 385
386
Phosphorylation Site Analysis of ORMs 387
For the analysis of potential phosphorylation sites presented in ORM proteins, the amino acid 388
sequences of Arabidopsis ORM1 and ORM2 were submitted to the NetPhos 3.1 389
(http://www.cbs.dtu.dk/services/NetPhos/) and PhosPhAt 4.0 (http://phosphat.uni-390
hohenheim.de/phosphat.html) online tools, respectively (Blom et al., 2004; Durek et al., 2010). 391
392
Quantitative RT-PCR Analysis 393
Total RNA was extracted using the E.Z.N.A. plant RNA kit (R6827-01, Omega Bio-tek). 394
For each sample, 1 μg RNA was reverse transcribed into cDNA using the Primescript RT reagent 395
kit (Takara, DRR047A). Real-time PCR was performed with the SYBR Premix ExTaq kit 396
(Takara, RR820L) according to the manufacturer’s instructions, and quantitatively analyzed with 397
a Step One Plus real-time PCR system (ABI). The 2−△△CT method (Livak and Schmittgen, 2001) 398
was used to determine the relative transcript level of target genes according to the expression 399
level of ACT2 (the internal control). All the experiments were performed in triplicate. The 400
primers used in the present study are listed in Supplemental Table 1. Unless otherwise mentioned, 401
all chemicals were purchased from Sigma (Shanghai, China). 402
403
Subcellular Protein Localization 404
For subcellular protein localization, the 35Spro:GFP:ORM1 and 35Spro:GFP:ORM2 gene 405
expression cassettes were constructed and inserted into pCAMBIA1300. Mesophyll protoplasts 406
were isolated by the tape-Arabidopsis sandwich method and transformed by PEG-calcium 407
18
mediated transfection (Wu et al., 2009). The transfected protoplasts were cultured under dim 408
light (about 300 lux) for 16–24 h at room temperature and observed by confocal microscopy 409
(LSM-780, Carl Zeiss). The excitation/emission wavelengths were: 488 nm/500–530 nm for 410
green fluorescent protein (GFP), 561 nm/580–630 nm for mCherry, and 488 nm/650–750 nm for 411
chlorophyll. 412
413
Sphingolipid Analysis 414
Measurement of sphingolipids was performed and the data were analyzed by Shimadzu 415
UFLC-XR (Shimadzu, Japan) coupled with a hybrid quadrupole time-of-flight mass 416
spectrometer (AB SCIEX Triple TOF 5600+, Foster City, CA, USA) using Phenomenex Luna 417
C8 column (150 mm × 2.0 mm, 3 μm). Briefly, 30 mg of lyophilized sample was homogenized. 418
The internal standards (C17 base D-erythro-sphingosine and d18:1 C12:0-ceramide) were added 419
and extracted with the isopropanol/hexane/water (55:20:25 v/v/v) and incubated at 60°C for 15 420
min. After centrifugation, the supernatants were dried and de-esterified in methylamine in 421
ethanol/water (70:30 v/v) as described previously (Bi et al., 2014; Li et al., 2015; Wu et al., 422
2015). The sphingolipid species were analyzed using the software Multiquant (AB SCIEX). 423
424
Yeast Two-Hybrid Assay 425
Yeast two-hybrid analysis was conducted following the Matchmaker Gold Yeast Two-426
Hybrid System User Manual (Clontech). The full-length open reading frames of ssSPTa and 427
ssSPTb were fused to the bait vector pGBKT7, and the full-length open reading frames of ORM1 428
and ORM2 were cloned into the prey vector pGADT7. Prey and bait vectors were transformed 429
into the yeast strain Y2H Gold (Clontech), and yeast was grown on SD/-Trp-Leu medium for 3 430
19
days. Transformants were incubated at 30°C in a shaking incubator with SD/-Trp-Leu broth until 431
OD600 = 1.0 was obtained, and then tested on selective SD medium at 30°C for 5 days. Empty 432
vectors were used as the negative control. 433
434
Bimolecular Fluorescence Complementation Assay 435
The full-length coding sequences of ORM1 and ORM2 were cloned into pSATN-nEYFP-C1, and 436
full-length coding sequences of ssSPTa and ssSPTb were cloned into pSATN-cYFP-C1 437
(Citovsky et al., 2006). Protoplast isolation and transient expression were performed as described 438
previously (Wu et al., 2009). Empty vectors were co-transformed as negative controls. 439
440
Co-Immunoprecipitation Assay 441
Mesophyll protoplast isolation from 3 to 4-week-old Arabidopsis leaves and DNA transfection 442
were performed as described previously (Wu et al., 2009). For the protocol, 100 μg of prey 443
plasmids and 100 μg of bait plasmids were co-transfected into 1 ml protoplasts (5×105 cells). 444
After expression of proteins for 12 h, protoplasts were pelleted and lysed in 200 μl of 445
immunoprecipitation buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% 446
glycerol, 1% Triton X-100, 13 Roche EDTA-free protease inhibitor cocktail) by vigorous 447
vortexing. For each sample, 20 μl of lysate was saved as the input fraction. The remaining of the 448
lysate was mixed with 300 μl immunoprecipitation buffer and vigorously vortexed. The clear 449
lysate was centrifuged at 16,000 g for 10 min at 4°C, and the supernatant was incubated with 20 450
μl of anti-Flag agarose resins (Sigma) for 4 h at 4°C. The resin was washed three times with 451
immunoprecipitation buffer. The resins were boiled in 40 μl of SDS-PAGE loading buffer to 452
obtain the eluate and prey proteins was detected by immunoblotting analysis using anti-Flag 453
20
antibody and anti-GFP antibody (Cell Signal) at 1:2000 dilution. The immunoblot signal was 454
visualized with the Clarity Western ECL Substrate kit (Bio-Rad). 455
456
H2O2 Detection by CeCl3 Staining and TEM Observation 457
For electron microscopy samples, ~25-day-old Arabidopsis rosette leaves from soil-grown plants 458
were used. The histochemical cerium chloride method was used to detect H2O2 based on 459
generation of cerium hydroxide, as described previously (Bi et al., 2014). The leaves were cut 460
and incubated in 10 mM CeCl3 dissolved in 50 mM MOPS buffer (pH 7.2) for 1 h. Control 461
samples were incubated in MOPS buffer only. Samples were fixed in 2.5% (v/v) glutaraldehyde 462
and 2% (v/v) paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4). Samples were embedded in 463
SPI-PON812 resin (SPI Supplies). Ultrathin sections were obtained on a microtome (Leica EM 464
UC6, Vienna, Austria) and examined without staining. The images were photographed using a 465
transmission electron microscope (JEM-1400, JEOL, Tokyo, Japan) at an accelerating voltage of 466
120 kV. 467
468
Abiotic and Biotic Stress Treatments 469
The seeds of each line were sown on 1/2x MS medium supplemented with FB1 (0.5 μM) or MV 470
(0.5 μM). After 2-3 days at 4°C in the dark, plates were transferred into an incubator with a 16 h 471
light / 8 h dark light regimen. Phenotypes were characterized and scored. For bacterial infection, 472
leaves from 3-week-old plants were injected with the virulent P. syringae strain DG3 at OD600 473
= 0.001 or with 10 mM MgSO4 as a mock-inoculated control. Leaf discs were harvested for 474
21
bacterial quantification at indicated days after inoculation as previous reports (Bi et al., 2014; Wu 475
et al., 2015). 476
477
Accession Numbers 478
Sequence data from this article can be found in the Arabidopsis Genome Initiative or 479
GenBank/EMBL databases under the following accession numbers: ORM1 (At1G01230), ORM2 480
(At5G42000), ssSPTa (At1g06515), ssSPTb (At2g30942), LCB1 (At4g36480), LCB2a 481
(At5g23670), LCB2b (At3g48780), ACT2 (At3g18780), TUBULIN (At5G62690), IRE1a 482
(At2G17520), bZIP28 (At3G10800), bZIP60 (At1G42990), TBF1 (At4G36990), PAD4 483
(At3g52430), SID2 (At1g74710), NPR1 (At1g64280), PR1 (At2g14610), orm1 (SALK_046054), 484
and orm2 (SAIL_1286_D09). 485
486
Supplemental Data 487
Supplemental Figure 1. Sequence alignment of the two Arabidopsis ORM proteins and 488
expression of Arabidopsis ORMs. 489
Supplemental Figure 2. Transmembrane helices predicted in ORM1 and ORM 2. 490
Supplemental Figure 3. Phenotypes and sphingolipid profiles in orm1 and ORM transgenic 491
plants. 492
Supplemental Figure 4. Comparison of major LCB and ceramide components between orm1 493
amiR-ORM2 and wild type. 494
Supplemental Figure 5. Sphingolipid contents in wild type and orm1 amiR-ORM2 plants at 495
different developmental stages. 496
22
Supplemental Figure 6. Subcellular localization of ssSPT and biomolecular fluorescence 497
complementation assay between ORM1 and ssSPTa. 498
Supplemental Figure 7. Subcellular localization of SPT in orm1 amiR-ORM2 plants. 499
Supplemental Table 1. List of primers used in this study. 500
501
ACKNOWLEDGMENTS 502
We thank Edgar Cahoon (University of Nebraska-Lincoln) for helpful discussion and Jian-503
Feng Li (Sun Yat-sen University) for assistance with co-IP experiments. This work was funded 504
by the National Natural Science Foundation of China (31570255), National Key Basic Science 505
973 program (2012CB114006), the Foundation of Guangzhou Science and Technology Project 506
(201504010021) and the Fundamental Research Funds for the Central Universities (16lgjc74) to 507
NY, and China Postdoctoral Science Foundation (2016M592575) to SKS. 508
509
510
Author contributions 511
NY, JL and JXW conceived and designed experiments. JL, JY, CR, KEL, LQH, and HYZ 512
performed the experiments. JL, JY, KEL, JXW, and NY analyzed the data. NY, JL, and SKS 513
wrote the article. 514
515
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Figure legends 666
667
Figure 1. Loss-of-function of ORM1 and ORM2 causes an early-senescence phenotype and 668
sphingolipid accumulation. 669
(A) The structures of Arabidopsis ORM1 and ORM2 and the locations of T-DNA insertions. 670
(B and C) The phenotype of wild-type and ORM loss-of-function plants at 26 (B) and 35 (C) 671
days old. The white arrows indicate leaf senescence in orm1 amiR-ORM2 plants. 672
(D) Subcellular localization of ORM proteins. The fusion constructs 35Spro:GFP-ORM1 and 673
35Spro:GFP-ORM2 were co-expressed with the ER mCherry marker (TAIR, CD3-960) by 674
transient expression in protoplasts. The 35Spro:GFP construct and CD3-960 were co-transformed 675
as controls. The images were obtained by confocal microscopy after 16 h incubation. The 676
experiments were repeated at least 3 times with similar results. Bars = 5 μm. 677
(E) Sphingolipids accumulated in orm1 amiR-ORM2 plants. Sphingolipids were extracted from 678
28-day-old plants following the steps described in Methods. The main sphingolipids were 679
separated and identified by HPLC-ESI-MS. The amount of total long-chain bases (LCB), 680
ceramides (Cer), hydroxyceramides (hCer) and glucosylceramides (gCer) were quantified (see 681
Supplemental Figure 4 for major LCB and ceramide species). The experiment was repeated three 682
times with similar results using independent samples. Values are means ±SE from three technical 683
replicates. Asterisks show a significant difference from the wild type using Student’s t-test (*** 684
p < 0.001). 685
686
Figure 2. ORM suppresses the de novo biosynthesis of sphingolipids in Arabidopsis. 687
29
Seven-day-old wild-type (WT), orm1, amiR-ORM2, and orm1 amiR-ORM2 seedlings were 688
transferred to 5 mM 15N-serine labeled N-deficient 1/2x MS liquid medium for 9-24 h. 689
Sphingolipids were then extracted and measured as described in Methods. Note that except total 690
LCB and total ceramides (top panels), major LCB and ceramide species significantly increased at 691
24 h in orm1 amiR-ORM2 seedlings. Error bars represent the means ±SE from triplicate 692
biological repeats. Asterisks show a significant difference from the wild type using Student’s t-693
test (*p < 0.05, **p < 0.01, *** p < 0.001). 694
695
Figure 3. Physical interaction between ORM and ssSPT. 696
(A) Yeast two-hybrid assay showing that ssSPTa and ssSPTb interact with ORM1 and ORM2. 697
Vectors pGADT7 (AD) and pGBKT7 (BD) were used as negative controls. 698
(B) Coimmunoprecipitation assays of ORM1, ORM2, and ssSPTa, ssSPTb in protoplasts. Flag-699
tagged ORM1 and ORM2 were immunoprecipitated with an anti-Flag antibody, and 700
coimmunoprecipitated ssSPTa-GFP and ssSPTb-GFP were detected by an anti-GFP antibody. 701
“+” or “-”denote the presence or absence of the protein in each sample. Free GFP was used as a 702
negative control. 703
(C) BiFC interaction of ssSPTa and ORM1, ssSPTb and ORM2, ssSPTa and ORM2, ssSPTb and 704
ORM2 in Arabidopsis leaf protoplasts. Overlaid images show signals for YFP (yellow) and 705
chloroplasts (blue). Empty vectors were used as negative controls. Scale bars = 5 μm. 706
707
Figure 4. Phenotypes of wild-type and ORM transgenic seedlings after FB1 treatments. 708
Seedlings of wild-type, orm1, amiR-ORM2, and orm1 amiR-ORM2 plants and overexpression 709
lines (ORM1-OX, ORM2-OX) were grown on 1/2x MS with or without 0.5 μM FB1 for 9 days 710
30
and then photographed. Percentage of seedling mortality after 0.5 μM FB1 treatment is shown in 711
the right panel. Asterisks show a significant difference from the wild type using Student’s t-test 712
(*p < 0.05, **p < 0.01, *** p < 0.001). 713
714
Figure 5. Ultrastructural features of orm1 amiR-ORM2 leaves. 715
(A-E) Representative TEM images of ultrastructure of 25-day-old wild-type (A) and orm1 amiR-716
ORM2 (B-E) leaves. Note an aggregate of condensed chromatin (B, a white arrow), abnormal ER 717
with loose structure (C), many small bubbles (C and D, white stars) around the plasma 718
membrane and cell wall and a large cell wall apposition in the orm1 amiR-ORM2 leaf cell (E). 719
(F-K) H2O2 production in wild-type (F) and orm1 amiR-ORM2 (G-K) leaves observed by TEM 720
using the histochemical cerium chloride method. (F) No cerium deposits were observed in the 721
representative wild-type control cell. (G and H) Cerium deposits in the plasma membrane and 722
cell wall (black arrows). Note vesicular bodies with double membrane (black arrowheads). (I) 723
Cerium deposits around the ER close to the cell wall. (J and K) H2O2 inside mitochondria (J, 724
white arrowheads) and irregular vacuolization (K). 725
Ch, chloroplast; CW, cell wall; CWA, cell wall apposition; ER, endoplasmic reticulum; G, Golgi; 726
M, mitochondrion; N, nucleus; S, starch grain. Bars = 500 nm. 727
728
Figure 6. Expression of genes related to ER stress and defense in orm1 amiR-ORM2 plants. 729
Approximately 26-day-old wild-type, orm1, amiR-ORM2, and orm1 amiR-ORM2 plants were 730
used to monitor the expression of indicated genes by RT-qPCR. IRE1a (At2G17520), bZIP28 731
(At3G10800), bZIP60 (At1G42990), TBF1 (At4G36990), PR1 (At2g14610), PAD4 (At3g52430), 732
SID2 (At1g74710), and NPR1 (At1g64280) were used for analysis. ACT2 transcript levels were 733
31
used as the internal control. (A) ER stress-related genes. (B) Salicylic acid pathway-related genes. 734
Gene expression values are presented relative to average wild-type levels (set to 1). Samples 735
were analyzed in triplicate biological repeats with similar results. Bars indicate ±SE from three 736
technical replicates. Different letters represent a significant difference from the wild type using a 737
post hoc multiple t-test (p<0.001). 738
739
Figure 7. Loss of ORM protein enhances the resistance to abiotic stress and biotic stress. 740
(A) The phenotype of wild-type and mutant seedlings after MV treatments. Seedlings were 741
grown on 1/2x MS with or without 0.5 μM MV for 7 days and then photographed. The white 742
square indicates one seedling with a severe phenotype. 743
(B) Percentage of seedlings with a severe phenotype after 0.5 μM MV treatments. At least 350 744
seedlings were counted in each genotype. Values represent means ± SE from two independent 745
experiments. 746
(C) Cell death symptom after infection by P. syringae virulent strain DG3 at OD600 = 0.005 for 747
3 days. MgSO4 as a mock-inoculated control was used. 748
(D) Growth of P. syringae virulent strain DG3 in wild-type and orm1 amiR-ORM2 plants after 749
infection at OD600 = 0.001. Lesion-free plants were inoculated with bacteria at 3 weeks of age. 750
The mean value of the growth of bacteria in nine leaves is indicated in each case. Bars indicate 751
standard deviations. This experiment was repeated three times with similar results. Asterisks 752
show a significant difference from the wild type using Student’s t-test (*p < 0.05). 753
754
Figure 8. Model of ORM1 and ORM2-mediated sphingolipid homeostasis and plant resistance. 755
32
The proposed model for ORM1 and ORM2 functions as negative regulators of de novo 756
sphingolipid synthesis in Arabidopsis based on the current study. Serine palmitoyltransferase 757
(SPT), localized on the ER, consists of LCB1, LCB2a/b, and ssSPTa/b and catalyzes the first 758
step in sphingolipid biosynthesis. SPT demonstrates extremely low enzyme activity without 759
ssSPTa/b (Kimberlin et al., 2013). We found that ORM1 and ORM2 inhibit the biosynthesis of 760
LCB through physically interacting with ssSPTa/b. Moreover, in plants lacking ORM1 or ORM2, 761
the profile of sphingolipids was enhanced, accompanied by a senescence phenotype and 762
increased disease resistance. A possible mechanism related to defense in ORM1 and ORM2 loss-763
of-function plants is proposed. LCB, long chain base; ER, endoplasmic reticulum; CW, cell wall; 764
SA, salicylic acid; ROS, reactive oxygen species; CWA, cell wall apposition. Red letters and 765
arrows represent data in this study. The black dotted arrow represents our speculation. 766
767
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DOI 10.1105/tpc.16.00574; originally published online December 6, 2016;Plant Cell
Sahu and Nan YaoJian Li, Jian Yin, Chan Rong, Kai-En Li, Jian-Xin Wu, Li-Qun Huang, Hong-Yun Zeng, Sunil Kumar
Contribute to Sphingolipid Homeostasis and Stress Responses in ArabidopsisOrosomucoid Proteins Interact with the Small Subunit of Serine Palmitoyltransferase and
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Supplemental Data /content/suppl/2016/12/06/tpc.16.00574.DC1.html
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