1 Short Title: ORM Mediation of Sphingolipid Homeostasis 2 · PDF file59 SPT activity when...

1

Short Title: ORM Mediation of Sphingolipid Homeostasis 1 2 3 Corresponding authors: 4 5 Edgar B. Cahoon 6

Department of Biochemistry 7

University of Nebraska-Lincoln 8

E318 Beadle Center 9

Lincoln, NE 68588 10

Email: [email protected] 11

Telephone: +1 402 472 5611 12

13

Teresa M. Dunn 14

Uniformed Services University of the Health Sciences 15

Department of Biochemistry and Molecular Biology 16

4301 Jones Bridge Road, C1094 17

Bethesda, MD 20814-4799 18

Email: [email protected] 19

Phone +1 301 295 3592 20

21

22

Plant Physiology Preview. Published on August 9, 2016, as DOI:10.1104/pp.16.00965

Copyright 2016 by the American Society of Plant Biologists

www.plantphysiol.orgon May 21, 2018 - Published by Downloaded from Copyright © 2016 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

2

Orosomucoid-like protein Expression Alters Sphingolipid Homeostasis & Affects 23

Ceramide Synthase Activity 24

Athen N. Kimberlin1†, Gongshe Han2†, Kyle D. Luttgeharm1, Ming Chen1, 25

Rebecca E. Cahoon1, Julie M. Stone1, Jonathan E. Markham1, 26

Teresa M. Dunn2, and Edgar B. Cahoon1 27

28 1Center for Plant Science Innovation and Department of Biochemistry, E318 Beadle 29

Center, 1901 Vine Street, University of Nebraska-Lincoln, Lincoln, NE 68588, USA 30 2Department of Biochemistry and Molecular Biology, Uniformed Services University of 31

the Health Sciences, 4301 Jones Bridge Road, Bethesda, Maryland 20814 USA 32

†Contributed equally. 33

34

Summary statement: AtORM1 and AtORM2 negatively regulate serine 35

palmitoyltransferase and altered expression differentially affects functionally distinct 36

ceramide synthase activities to maintain sphingolipid homeostasis. 37

38

Footnotes 39

List of author contributions: ANK, GH, JMS, TMD, and EBC contributed to the 40

conception of the research; ANK and GH conducted most of the research; ANK, GH, 41

TMD, and EBC analyzed data and wrote the manuscript; KDL and JEM contributed to 42

the research conception and studies on ceramide synthases; MC initiated the research; 43

REC conducted mass spectrometry-based sphingolipid analyses; TMD and EBC 44

supervised the project. 45

Funding: The research was supported by a National Science Foundation grant MCB-46

1158500 to JS, TMD, and EBC. 47

Corresponding authors email addresses: Edgar B. Cahoon, [email protected]; 48

Teresa M. Dunn, [email protected]. 49

50



3

ABSTRACT 51

Sphingolipid synthesis is tightly regulated in eukaryotes. This regulation in plants 52

ensures sufficient sphingolipids to support growth, while limiting accumulation of 53

sphingolipid metabolites that induce programmed cell death (PCD). Serine 54

palmitoyltransfersase (SPT) catalyzes the first step in sphingolipid biosynthesis and is 55

considered the primary sphingolipid homeostatic regulatory point. In this report, 56

Arabidopsis putative SPT regulatory proteins, orosomucoid-like proteins AtORM1 and 57

AtORM2 were found to physically interact with the Arabidopsis SPT and to suppress 58

SPT activity when co-expressed with Arabidopsis SPT subunits LCB1 and LCB2 and 59

the small subunit of SPT in a yeast SPT-deficient mutant. Consistent with a role in SPT 60

suppression, AtORM1 and AtORM2 overexpression lines displayed increased 61

resistance to the PCD-inducing mycotoxin fumonisin B1 (FB1), with an accompanying 62

reduced accumulation of long-chain bases (LCBs) and C16-fatty acid-containing 63

ceramides relative to wild type plants. Conversely, RNAi suppression lines of AtORM1 64

and AtORM2 displayed increased sensitivity to FB1 and an accompanying strong 65

increase in LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. 66

Overexpression lines were also found to have reduced activity of the Class I ceramide 67

synthase that uses C16-fatty acid acyl-CoA and dihydroxy LCB substrates, but 68

increased activity of Class II ceramide synthases that use very long-chain fatty acyl-CoA 69

and trihydroxy LCB substrates. RNAi suppression lines, in contrast, displayed 70

increased Class I ceramide synthase activity, but reduced Class II ceramide synthase 71

activity. These findings indicate that ORM-mediation of SPT activity differentially 72

regulates functionally distinct ceramide synthase activities as part of a broader 73

sphingolipid homeostatic regulatory network. 74

75

76

77



4

INTRODUCTION 78

Sphingolipids play critical roles in plant growth and development as essential 79

components of endomembranes, including the plasma membrane where they comprise 80

more than 40% of the total lipid (Sperling et al., 2005; Cacas et al., 2016). Sphingolipids 81

are also highly enriched in detergent insoluble membrane fractions of the plasma 82

membrane that form microdomains for proteins with important cell surface activities, 83

including cell wall biosynthesis and hormone transport (Cacas et al., 2012; Perraki et 84

al., 2012; Bayer et al., 2014; Cacas et al., 2016). In addition, sphingolipids, particularly 85

those with very long-chain fatty acids (VLCFAs), are integrally-associated with Golgi-86

mediated protein trafficking that underlies processes related to the growth of plant cells 87

(Bach et al., 2008; Bach et al., 2011; Markham et al., 2011; Melser et al., 2011). 88

Furthermore, sphingolipids function through their bioactive long-chain base (LCB) and 89

ceramide metabolites to initiate programmed-cell death (PCD), important for mediating 90

plant pathogen resistance through the hypersensitive response (HR) (Greenberg et al., 91

2000; Liang et al., 2003; Shi et al., 2007; Bi et al., 2014; Simanshu et al., 2014). 92

Sphingolipid biosynthesis is highly regulated in all eukaryotes. In plants, 93

maintenance of sphingolipid homeostasis is vital to ensure sufficient sphingolipids for 94

growth (Chen et al., 2006; Kimberlin et al., 2013) while restricting the accumulation of 95

PCD-inducing ceramides and long-chain bases (LCBs), until required for processes 96

such as pathogen-triggered HR. Serine palmitoyltransferase (SPT), which catalyzes the 97

first step in LCB synthesis, is generally believed to be the primary control point for 98

sphingolipid homeostasis (Hanada, 2003). SPT synthesizes LCBs, unique components 99

of sphingolipids, by catalyzing a pyridoxal phosphate-dependent condensation of serine 100

and palmitoyl (16:0)-CoA in plants (Markham et al., 2013). Similar to other eukaryotes, 101

the Arabidopsis SPT is a heterodimer consisting of LCB1 and LCB2 subunits (Chen et 102

al., 2006; Dietrich et al., 2008; Teng et al., 2008). Research to date has shown that SPT 103

is regulated primarily by post-translational mechanisms involving physical interactions 104

with non-catalytic, membrane-associated proteins that confer positive and negative 105

regulation of SPT activity (Han et al., 2009; Breslow et al., 2010; Han et al., 2010). 106

These proteins include a 56-amino acid small subunit of SPT (ssSPT) in Arabidopsis, 107

which was recently shown to stimulate SPT activity and to be essential for generating 108



5

sufficient amounts of sphingolipids for pollen and sporophytic cell viability (Kimberlin et 109

al., 2013). 110

Evidence from yeast and mammalian research points to a more critical role for 111

proteins termed ORMs in sphingolipid homeostatic regulation (Breslow et al., 2010; Han 112

et al., 2010). The Saccharomyces cerevisiae Orm1p and Orm2p negatively regulate 113

SPT through reversible phosphorylation of these polypeptides in response to 114

intracellular sphingolipid levels (Breslow et al., 2010; Han et al., 2010; Roelants et al., 115

2011; Gururaj et al., 2013; Muir et al., 2014). Phosphorylation/de-phosphorylation of 116

ORMs in S. cerevisiae presumably affects the higher order assembly of SPT to mediate 117

flux through this enzyme for LCB synthesis (Breslow, 2013). In this sphingolipid 118

homeostatic regulatory mechanism, the S. cerevisiae Orm1p and Orm2p are 119

phosphorylated at their N-termini by Ypk1, a TORC2-dependent protein kinase (Han et 120

al., 2010; Roelants et al., 2011). The absence of this phosphorylation domain in 121

mammalian and plant ORM homologs brings into question the nature of SPT reversible 122

regulation by ORMs in other eukaryotic systems (Hjelmqvist et al., 2002). 123

Sphingolipid synthesis is also mediated by N-acylation of LCBs by ceramide 124

synthases to form ceramides, the hydrophobic backbone of the major plant 125

glycosphingolipids, glucosylceramides (GlcCer) and glycosyl inositolphosphoceramides 126

(GIPCs). Two functionally distinct classes of ceramide synthases occur in Arabidopsis, 127

designated Class I and II (Chen et al., 2008). Class I ceramide synthase activity 128

resulting from the Longevity Assurance Gene One Homolog2 (LOH2)-encoded 129

ceramide synthase acylates, almost exclusively, LCBs containing two hydroxyl groups 130

(“dihydroxy” LCBs) with 16:0-CoA, to form C16-ceramides, which are primarily used for 131

GlcCer synthesis (Markham et al., 2011; Ternes et al., 2011; Luttgeharm et al., 2016). 132

Class II ceramide synthase activity resulting from the LOH1- and LOH3-encoded 133

ceramide synthases are most active in the acylation of LCBs containing three hydroxyl 134

groups (“trihydroxy” LCBs) with very long-chain fatty acyl (VLCFA)-CoAs, including 135

primarily C24 and C26 acyl-CoAs (Markham et al., 2011; Ternes et al., 2011; Luttgeharm 136

et al., 2016). Class II (LOH1 and LOH3) ceramide synthase activity is essential for 137

producing VLCFA-containing glycosphingolipids to support growth of plant cells, 138

whereas Class I (LOH2) ceramide synthase activity is non-essential under normal 139



6

growth conditions (Markham et al., 2011; Luttgeharm et al., 2015). It was recently 140

speculated that LOH2 ceramide synthase functions, in part, as a “safety valve” to 141

acylate excess LCBs for glycosylation, resulting in a less cytotoxic form (Luttgeharm et 142

al., 2015; Msanne et al., 2015). Recent studies have shown that the Lag1/Lac1 143

components of the S. cerevisiae ceramide synthase are phosphorylated by Ypk1, and 144

this phosphorylation stimulates ceramide synthase activity in response to heat and 145

reduced intracellular sphingolipid levels (Muir et al., 2014). This finding points to 146

possible coordinate regulation of ORM-mediated SPT and ceramide synthase activities 147

to regulate sphingolipid homeostasis, which is likely more complicated in plants and 148

mammals due to the occurrence of functionally distinct ceramide synthases in these 149

systems (Stiban et al., 2010; Markham et al., 2011; Ternes et al., 2011; Luttgeharm et 150

al., 2016). 151

RNAi suppression of ORM genes in rice has been shown to affect pollen viability 152

(Chueasiri et al., 2014), but no mechanistic characterization of ORM proteins in plants 153

has yet to be reported. Here we describe, two Arabidopsis ORMs, AtORM1 and 154

AtORM2, that suppress SPT activity through direct interaction with the LCB1/LCB2 155

heterodimer. We also show that strong upregulation of AtORM expression impairs 156

growth. In addition, up- or down-regulation of ORMs is shown to differentially affect 157

sensitivity of Arabidopsis to the PCD-inducing mycotoxin fumonisin B1, a ceramide 158

synthase inhibitor, and to also differentially affect activities of Class I and II ceramide 159

synthases as a possible additional mechanism for regulating sphingolipid homeostasis. 160

161

RESULTS 162

Two Functional Homologs of Mammalian ORMDLs Physically Interact with and 163

Inhibit Arabidopsis SPT 164

Two genes, designated AtORM1 (At1g01230) and AtORM2 (At5g42000), encoding 157 165

and 154 amino acid polypeptides respectively, were identified in homology searches 166

using human ORMDLs as query (Hjelmqvist et al., 2002). The amino acid sequences of 167

the Arabidopsis polypeptides share 81% identity and have predicted homologs 168

throughout the plant kingdom. Arabidopsis ORM1 (AtORM1) and ORM2 (AtORM2) 169

share 38 to 43% identity with human ORMDL1, ORMDL2, and ORMDL3. AtORM1 and 170



7

AtORM2 also share 35-39% identity with S. cerevisiae ORM1 and ORM2, but 171

interestingly lack the N-terminal phosphorylation domain found in the yeast ORMs 172

(Figure 1A). Notably, this N-terminal domain is also absent from ORMDL proteins from 173

human and other mammals. Similar to the mammalian ORMDL proteins, AtORM1 and 174

AtORM2 are predicted to have two, three or four transmembrane domains based on in 175

silico analyses using the TopPred II, SOSUI, TMPred, MEMSAT, and DAS-TMfilter 176

programs (von Heijne, 1992; Hirokawa et al., 1998). However, glycosylation cassettes 177

(GCs) inserted at key sites along the yeast ORM2 protein support the presence of four 178

transmembrane domains with the N- and C-termini residing in the cytosol (Figure 1B). 179

Given the significant homology between the yeast, plant and human ORM proteins, it is 180

likely that all members of the ORM family of proteins have the topology depicted in 181

Figure 1C. 182

To test whether the AtORM polypeptides physically interact with AtSPT, FLAG-183

tagged AtLCB1 was expressed along with Myc-AtLCB2a, HA-AtssSPTa, and HA-tagged 184

AtORM1 or -AtORM2 in a yeast mutant that lacks endogenous SPT due to knockout of 185

the yeast LCB1 and TSC3 gene. Pull-down assays using anti-FLAG antibodies with 186

solubilized microsomes of cells expressing these polypeptides resulted in the detection 187

of not only AtLCB1, but also AtLCB2a, and AtssSPTa, AtORM1 or AtORM2, but no 188

detection of ELO3, an ER polypeptide not known to associate with SPT (Figure 1D). 189

These results are consistent with the physical interaction of AtORM1 and AtORM2 with 190

the core SPT complex. 191

To examine their ability to function as suppressors of SPT activity, Arabidopsis 192

ORM1 and ORM2 were expressed in the S. cerevisiae orm2Δ mutant. The orm2Δ 193

mutant in yeast has a strong sensitivity to excess LCB, and the inclusion of 15 µM 194

phytosphingosine (PHS) in media is toxic to this mutant (Han et al., 2010). Expression 195

of AtORM1 or AtORM2 in the orm2Δ mutant rescued the sensitivity of the mutant to 196

exogenous LCB, consistent with the ability of the Arabidopsis proteins to suppress SPT 197

activity (Figure 1E). In addition, Arabidopsis ORM1 and ORM2 were co-expressed 198

along with AtLCB1C144W, AtLCB2a, and AtssSPTa in the yeast lcb1Δtsc3Δ mutant strain 199

lacking endogenous SPT (Figure 2C). The AtLCB1C144W contains a single amino acid 200

substitution analogous to the HSAN1-causing mutation in human LCB1 (Gable et al., 201



8

2010). SPT containing the AtLCB1C144W subunit condenses both serine and alanine 202

with palmitoyl-CoA to form normal LCB (d18:0) and deoxy-LCB (deoxy-sphinganine, 203

DoxSA; m18:0), respectively (Figure 2A). Deoxy-LCBs cannot be degraded due to the 204

missing hydroxyl group and serve as an in situ read-out of SPT activity. When 205

expressed in this background, Arabidopsis ORM1 and ORM2 markedly decreased the 206

amount of deoxy-LCB produced indicating that they act as SPT inhibitors (Figure 2B, 207

C). The inhibitory effect of the AtORMs was also observed using native AtSPT 208

expressed in a yeast mutant lacking endogenous SPT (Figure 2D). 209

AtORM1 and AtORM2 Polypeptides are ER-Associated and AtORM1 and AtORM2 210

are Constitutively Expressed 211

The subcellular localization of ORM1 and ORM2 polypeptides was visualized using N-212

terminal and C-terminal yellow fluorescent protein (YFP) tags with transient expression 213

in Nicotiana benthamiana (Figure 3A-F). Tagged-ORM1 and -ORM2 co-localized with 214

the ER marker mCherry-HDEL (Figure 3C, F), consistent with the known ER localization 215

of AtLCB1, AtLCB2a/2b, and AtssSPTa/b (Chen et al., 2006; Dietrich et al., 2008; Teng 216

et al., 2008; Kimberlin et al., 2013), but were also detected in other subcellular 217

locations, including the cytosol (Figure 3A, C, D, F). 218

To assess the in planta contributions of each gene to SPT inhibition, transcript 219

levels of AtORM1 and AtORM2 were measured in different organs of Arabidopsis. Our 220

analyses revealed that AtORM1 transcript was approximately 3-4 fold more abundant in 221

all tissues tested except pollen, where AtORM2 transcript was approximately 4-fold 222

more abundant (Figure 3G). Our results are broadly consistent with those for these 223

genes in the AtGenExpress public microarray database (Figure S1). 224

Using AtORM1 and AtORM2 promoter::GUS fusion constructs, the location of in 225

planta expression was examined. Promoters for both genes conferred expression in 226

vegetative tissues, with GUS staining most pronounced in vascular tissues. Differences 227

in promoter activity for AtORM1 and AtORM2 were observed in floral tissues and roots. 228

Based on the intensity of GUS staining, AtORM1 promoter conferred higher expression 229

in anthers and developing embryos than the AtORM2 promoter (Figure 3H-J), while 230

AtORM2 promoter conferred greater expression than the AtORM1 promoter in 231

filaments, petals, sepals, pistil and siliques (Figure 3M-O). Both AtORM1 and AtORM2 232



9

promoters yielded GUS expression in mature pollen grains and in roots. However, the 233

AtORM1 promoter, but not the AtORM2 promoter, conferred detectable GUS 234

expression in lateral root buds (Figure 3K, L, P, Q). 235

Overexpression of AtORM1 and AtORM2 Results in Dwarfed Growth 236

To examine the in planta functions of AtORM1 and AtORM2, three available T-DNA 237

mutant lines for these genes were initially characterized: SALK_046054 (predicted T-238

DNA insertion in the first intron of AtORM1), GK-143A01 (predicted T-DNA insertion in 239

the first exon of AtORM1), and SAIL_1286_D09 (predicted T-DNA insertion in the 240

5’UTR of AtORM2). Homozygous lines were identified for each of these mutants, but 241

full length transcripts were still detected, indicating that these lines are not null mutants 242

for the AtORM1 and AtORM2 genes (Figure S2). As an alternative approach for 243

characterization of the in planta functions of AtORM1 and AtORM2, overexpression and 244

RNAi suppression lines for these genes were created in Arabidopsis Col-0. 245

Overexpression lines were prepared by placing the AtORM1 or AtORM2 cDNA under 246

control of the cauliflower mosaic virus 35S (CaMV35S) promoter. Homozygous lines 247

were screened by qPCR to identify those with increased expression. A portion of the 248

lines with confirmed overexpression of AtORM1 or AtORM2 were visually 249

indistinguishable from Col-0 plants, including those with ~5-fold overexpression of 250

AtORM1 and ~35-fold overexpression of AtORM2 in leaves (Figure 4B, E). However, a 251

second class of overexpression lines for both genes was observed with strong dwarfing. 252

These lines included those with >80-fold overexpression of AtORM1 and ~800-fold 253

overexpression of AtORM2 in leaves relative to expression levels of these genes in Col-254

0 leaves (Figure 4C, F). These results suggest that a threshold of AtORM1 and 255

AtORM2 protein levels can be reached to suppress SPT activity sufficiently to reduce 256

growth, as previously observed for AtLCB1 RNAi suppression (Chen et al., 2006) and 257

reduced AtssSPTa expression (Kimberlin et al., 2013). Sphingolipidomic profiling 258

revealed little quantitative differences between amounts of sphingolipids in Col-0 and 259

AtORM overexpression lines and virtually no difference in amounts of sphingolipids 260

between AtORM overexpression lines with or without a growth phenotype, especially in 261

the GIPC and GlcCer classes (Figures S3-S6), similar to previous findings with AtssSPT 262

overexpression or RNAi suppression (Kimberlin et al., 2013). It was notable that 263



10

concentrations of the LCB t18:0 were elevated in ceramide and GIPC molecular species 264

in dwarfed AtORM1 and AtORM2 plants compared to Col-0 control plants (Figure S3). 265

AtORM RNAi suppression lines showed significant suppression of both AtORM1 and 266

AtORM2 transcript (Figure S7). Consistent with a role of AtORMs as SPT repressors, 267

SPT activity assayed from root microsomes of RNAi suppression lines showed 268

increased SPT activity (Figure S8). No reductions in growth and no significant 269

quantitative differences were seen in the sphingolipidome, particularly in GIPC and 270

GlcCer amounts, in AtORM RNAi lines relative to the Col-0 controls (Figures S4-S6). 271

Modulation of AtORM Expression Affects Sensitivity to FB1 272

The PCD-inducing mycotoxin FB1, a ceramide synthase inhibitor, has been routinely 273

used as a means of perturbing sphingolipid homeostasis for study of SPT activity. From 274

these studies, reduced SPT activity, such as that achieved by suppression of AtssSPTa, 275

results in enhanced resistance to FB1 due presumably to reduced accumulation of 276

cytotoxic LCBs (Kimberlin et al., 2013). Conversely, increased SPT activity, such as that 277

obtained by overexpression of AtssSPTa, results in enhanced sensitivity to FB1 due 278

presumably to accumulation of cytotoxic LCBs (Kimberlin et al., 2013). Consistent with 279

suppression of SPT activity, AtORM1 or AtORM2 overexpression lines displayed 280

increased resistance to FB1, along with a decrease in accumulation of free LCBs and 281

LCB-phosphates (LCBPs) (Figure 5A, 5B). These lines were viable at 0.5 μM FB1, a 282

concentration that was toxic to wild-type Arabidopsis (Figure 5A). This phenotype was 283

observed in a range of overexpression lines, including those with and without the 284

dwarfing phenotype described above (Figure S9). Conversely, AtORM RNAi 285

suppression lines were not viable on media containing 0.3 μM FB1, but no loss of 286

viability was observed in wild type plants at this concentration (Figure 5A). RNAi 287

suppression lines of AtORM were observed to have a large increase in free LCBs and 288

LCBPs when grown on FB1, which was particularly accentuated relative to AtORM 289

overexpression lines and wild type Col-0 on media containing 0.5 µM FB1 (Figure 5B). 290

Mirroring alterations in LCB concentrations in transgenic and wild type plants in 291

response to FB1, concentrations of ceramides containing C16 fatty acids were lower in 292

lines with overexpression of ORM and higher in RNAi suppression lines (Figure 5C). 293



11

These results indicate that altered ORM expression is an effective way of modulating 294

plant sensitivity to FB1 and indicates that AtORMs act as inhibitors of SPT. 295

Modulation of AtORM Expression Impacts Ceramide Synthase Activity 296

Recent studies in S. cerevisiae have suggested a role for ceramide synthase in 297

maintenance of sphingolipid homeostasis through a phosphorylation/de-phosphorylation 298

mechanism similar to that used for reversible modulation of ORMs (Muir et al., 2014). In 299

contrast to S. cerevisiae, Arabidopsis has two functionally distinct ceramide synthase 300

classes: Class I and Class II. This difference likely results in more complexity of 301

potential ceramide synthase regulation of sphingolipid homeostasis in Arabidopsis. To 302

gain insights into coordinate regulation of ORMs and ceramide synthases, Class I 303

ceramide synthase was assayed in microsomes from roots of AtORM1 and AtORM2 304

overexpression lines and RNAi lines using 16:0-CoA and d18:0, the preferred 305

substrates of this enzyme class. Class II ceramide synthase activity was assayed using 306

24:0-CoA and t18:0, preferred substrates of this enzyme class. Lower Class I ceramide 307

synthase activity was detected in microsomes of AtORM1 and AtORM2 overexpression 308

lines and increased activity was detected in microsomes from AtORM RNAi suppression 309

lines (Figure 6A). Conversely, Class II ceramide synthase activity was decreased in 310

microsomes from AtORM RNAi suppression lines, but increased in AtORM1 and 311

AtORM2 overexpression lines (Figure 6B). These results suggest preferential metabolic 312

flux through Class I ceramide synthase for the synthesis of C16 fatty acid-containing 313

ceramides with increased SPT activity resulting from disrupted ORM-mediation of SPT 314

in AtORM RNAi lines, and preferential flux through Class II ceramide synthases for 315

production of VLCFA-containing ceramides when SPT activity is limited by suppression 316

from increased ORMs in AtORM1 and AtORM2 overexpression lines (Figure 7). These 317

findings are consistent with the increased concentrations of C16 fatty acid-containing 318

ceramides detected in AtORM RNAi lines in response to FB1 treatment (Figure 5C). 319

320

DISCUSSION 321

Findings reported here demonstrate the occurrence of two ORM proteins in Arabidopsis 322

that share significant identity with human and yeast ORM polypeptides. We show that 323

these polypeptides physically interact with a complex that includes the AtLCB1/AtLCB2a 324



12

and AtssSPTa. Consistent with a function as suppressors of SPT activity, expression of 325

AtORM1 and AtORM2 rescued the sensitivity of the yeast orm2Δ mutant to exogenous 326

LCBs. Also consistent with a function as a suppressor of SPT activity, AtORM1 and 327

AtORM2 inhibited SPT activity when overexpressed with the core Arabidopsis SPT 328

components in a yeast mutant deficient in the native SPT and ORMs. Furthermore, 329

overexpression of AtORM1 and AtORM2 resulted in enhanced resistance to FB1, a 330

phenotype also observed with reduced SPT activity associated with suppressed 331

expression of AtLCB1 (Shi et al., 2007), AtLCB2 (Saucedo-Garcia et al., 2011), or 332

AtssSPT (Kimberlin et al., 2013). Moreover, RNAi-mediated downregulation of AtORM 333

expression resulted in increased sensitivity to FB1, a phenotype also observed with 334

increased SPT activity associated with upregulation of AtssSPT expression (Kimberlin 335

et al., 2013). Altered AtORM expression was also accompanied by either enhanced 336

accumulation of LCBs (AtORM RNAi lines) or reduced accumulation of LCBs (AtORM 337

overexpression lines) relative to Col-0 controls in response to FB1 treatment. These 338

findings are consistent with altered SPT regulation in the ORM transgenic lines and with 339

a function of AtORM1 and AtORM2 as negative regulators of SPT activity in 340

Arabidopsis. 341

Also observed in parallel with these changes was enhanced accumulation of 342

ceramides containing C16 fatty acids (AtORM RNAi lines) or reduced accumulation of 343

ceramides containing C16 fatty acids (AtORM overexpression lines). These findings 344

are consistent with enhanced sequestration of LCBs as ceramides via activity of 345

ceramide synthase I (LOH2), which uses primarily C16 fatty acyl-CoAs as substrates, in 346

response to de-regulated SPT in AtORM RNAi lines. Also consistent with this, LOH2 347

activity was found to be significantly increased in microsomes from AtORM RNAi lines, 348

but decreased by >2-fold in AtORM overexpression lines compared to the Col-0 control. 349

Unexpectedly, the opposite effect was observed in microsomes of these plants for Class 350

II ceramide synthase activity, which uses primarily trihydroxy LCBs and very long-chain 351

fatty acyl-CoAs as substrates: microsomes from AtORM RNAi lines had >3-fold less 352

Class II ceramide synthase activity, while microsomes for ORM overexpression lines 353

had >2-fold more activity of this enzyme class relative to the control. The findings point 354

to coordinate control of the activities of the two functionally distinct ceramide synthase 355



13

classes in response to altered ORM-mediated flux through SPT. As shown in Figure 7, 356

we propose that the LOH2 ceramide synthase, a non-essential enzyme under normal 357

growth conditions, provides a “safety-valve” for excess LCB production, which is 358

particularly accentuated in response to FB1. As we have previously proposed, 359

increased flux through LOH2 ceramide synthase may be coupled with GlcCer synthase 360

activity to glucosylate C16 fatty acid-containing ceramides to a less cytotoxic form. 361

Conversely, downregulation of SPT activity by AtORM overexpression may result in the 362

directing of LCB flux toward the synthesis of ceramides containing trihydroxy LCBs and 363

VLCFAs, via Class II ceramide synthases, to support growth. One notable observation 364

of this and similar studies is that perturbations in sphingolipid homeostasis are 365

accentuated in the presence of FB1. One possibility is that the C16 fatty acid-containing 366

ceramides resulting from LOH2 activity in FB1-treated plants deregulate SPT or dilute 367

possible sphingolipid signals (e.g., ceramides containing VLCFAs) that typically provide 368

homeostatic regulation of SPT. 369

How this apparent coordination of ORM-mediation of SPT and ceramide 370

synthase activities is regulated is currently under investigation. One possibility is that 371

Class I and II ceramide synthases are differentially regulated by phosphorylation. It is 372

known, for example, that activity of the yeast Lag1/Lac1 ceramide synthase is subject to 373

regulation by phosphorylation at residues Ser23 and Ser24 in Lag1 and Lac1 by Ypk1 374

(Muir et al., 2014) and at residues Ser393, Ser395, and Ser397 near their C-termini by 375

casein kinase 2 (CK2) (Fresques et al., 2015). In the latter case, phosphorylation of 376

Lag1 and Lac1 affects the localization of these polypeptides by disrupting their ER 377

retrieval (Fresques et al., 2015). Similar to the yeast ceramide synthase, the AtLOH1 378

ceramide synthase has four experimentally confirmed phosphorylated serines (Ser295, 379

Ser300, Ser302, Ser304) (Nühse et al., 2004), and the AtLOH2 has two experimentally 380

confirmed phosphorylated serines (Ser289, Ser291) (Nühse et al., 2004; Sugiyama et 381

al., 2008). Like the yeast Lag1 and Lac1 polypeptides, these phosphorylated serine 382

residues are in the vicinity of the C-termini of AtLOH1 and AtLOH2, near presumptive 383

ER retrieval sequences. Although not experimentally verified, AtLOH3 also has serine 384

residues analogous to those in AtLOH1 near its C-termini. The influence of this 385

phosphorylation on ceramide synthase activity and possible changes in phosphorylation 386



14

of these polypeptides in response to altered ORM regulation of SPT are currently under 387

study. Overall, our findings indicate that regulation of sphingolipid homeostasis extends 388

beyond SPT and is more complex in eukaryotic organisms with multiple, functionally 389

distinct ceramide synthases, than that found in S. cerevisiae that has only one ceramide 390

synthase type (Mullen et al., 2012). 391

Although our results clearly show that AtORM1 and AtORM2 function to 392

suppress SPT activity, still unclear in plants and mammals is how ORMs reversibly 393

regulate SPT in response to changes in intracellular sphingolipid levels. Absent from 394

plant and mammalian ORMs is the N-terminal serine-rich domain found in the yeast 395

ScORM1 and ScORM2 polypeptides. Phosphorylation of serine residues in this domain 396

by Ypk1 relieves SPT inhibition in response to low intracellular sphingolipid levels 397

(Breslow et al., 2010; Han et al., 2010; Roelants et al., 2011), and dephosphorylation of 398

these residues by Cdc55-protein phosphatase 2A results in ORM suppression of SPT in 399

response to excess intracellular sphingolipid levels (Sun et al., 2012).. The lack of the 400

regulatory domain in plant and mammalian ORMs does not preclude that other residues 401

are subject to reversible post-translational modifications (e.g., 402

phosphorylation/dephosphorylation). Other possibilities include changes in ORM 403

turnover rates or ORM localization in response to intracellular sphingolipid levels. With 404

regard to the latter possibility, our localization studies showed that AtORM polypeptides 405

are present primarily, but not exclusively in the ER. As described above, changes in ER 406

localization have been proposed as a component of yeast ceramide synthase activity 407

(Fresques et al., 2015). More extensive studies, beyond the scope of those in this 408

report, are currently underway to address this central question of how SPT is regulated 409

to maintain intracellular homeostasis in plants and animals. 410

411

MATERIALS AND METHODS 412

Yeast Growth and Expression Plasmids. Yeast (Saccharomyces cerevisiae) strain 413

TDY9113 (Mata tsc3Δ:NAT lcb1Δ:KAN ura3 leu2 lys2 trp1Δ) lacking endogenous SPT 414

was used for expression and characterization of the Arabidopsis thaliana SPT subunits 415

and ORM proteins. The mutant was cultured in medium containing 15 μM 416

phytosphingosine and 0.2% (w/v) Tergitol NP-40. The AtORM1 (At1g01230) and 417



15

AtORM2 (At5g42000) open reading frames were amplified by PCR and inserted into 418

pPR3-N (Dualsystems Biotech) for expression with N-terminal HA tags. The pAL2-URA 419

was constructed for divergent constitutive expression of AtLCB1-FLAG (or 420

AtLCB1C144W-FLAG) and Myc-At LCB2a by replacing the GAL1 and GAL10 promoters 421

of pESC-URA (Stratagene) with the yeast LCB2 and ADH promoters, respectively. The 422

AtssSPTa cDNA open reading frame was inserted after the 3xHA tag in pADH1 423

(Kohlwein et al., 2001; Kimberlin et al., 2013). 424

Immunoprecipitation. Immunoprecipitation was conducted as described (Kimberlin et 425

al., 2013) with minor modifications. Microsomal membrane proteins were prepared from 426

yeast cells expressing FLAG-tagged AtLCB1 (or AtLCB1C144W), Myc-tagged AtLCB2a, 427

HA-tagged AtssSPTa and HA-tagged AtORM1 or AtORM2. Microsomal membrane 428

proteins were solubilized in 1.5% digitonin at 4°C for 2.5 h and incubated with Flag-429

beads (Sigma-Aldrich) overnight. The bound proteins were eluted in 430

immunoprecipitation buffer (50 mM HEPES-KOH, pH 6.8, 150 mM potassium acetate, 2 431

mM magnesium acetate, 1 mM calcium chloride, and 15% glycerol) containing 0.25% 432

digitonin and 200 μg/mL of FLAG peptide, resolved on a 4 to 12% Bis-Tris NuPAGE gel 433

(Invitrogen), and detected by immunoblotting with antibodies, anti-HA (Covance; 1:5000 434

dilution), anti-Myc (Sigma-Aldrich; 1:3000 dilution), and anti-FLAG (GenScript; 1:5000 435

dilution). 436

LCB Extraction and Analysis. LCBs were extracted, derivatized, and analyzed by 437

reverse-phase HPLC as described previously {Gable, 2010 #46}. For measurement of 438

LCBs and DoxSA formation, the areas under HPLC peaks, measured in luminescence 439

units, were normalized to the internal C17 sphingosine standard and the amounts of 440

LCBs, and 1-deoxysphinganine (1-DoxSA) were determined using standard curves for 441

each of the LCBs and are reported as nmol/O.D.cells at 600 nm (Gable et al., 2010). 442

SPT Assay. Plant microsomes were prepared and SPT activity was assayed as 443

described (Kimberlin et al., 2013) except that 50 μM palmitoyl-CoA and 20 µM BSA 444

were used for the Arabidopsis microsomal SPT assays. SPT activity was measured 445

using the substrates [3H] Ser and 16:0-CoA as described (Han et al., 2009). 446

Yeast Complementation. Synthetic complete (SC) media was used to grow yeast 447

(BY4741). SC media was supplemented with 15 μM phytosphingosine (PHS) and 0.1% 448



16

tergitol. Yeast knockout mutants, orm1Δ and orm2Δ, were obtained from the S. 449

cerevisiae knockout library kindly provided by Professor Jaekwon Lee (University of 450

Nebraska-Lincoln). AtORM1 and AtORM2 cDNAs were cloned into the centromeric 451

plasmid pSH15 using the XhoI and PstI restriction sites using primers P1-P4 (Table S1). 452

The plasmid, pSH15, containing native S. cerevisiae ORM2 was received as a gift from 453

Dr. Amy Chang (University of Michigan). Cells were grown at 30°C and then normalized 454

to OD600=0.1 before being serially diluted and plated. 455

Plant Material and Growth Conditions. All Arabidopsis thaliana Col-0 lines used in 456

this study were stratified at 4°C for four days and were maintained at 22°C with a 16 h 457

light (100 µmol/m-2/s-1)/8 h dark cycle. Plants sown on Linsmaier and Skoog (LS) agar 458

plates were surface sterilized before stratification. Plants that were grown 459

hydroponically were maintained on modified Hoagland’s solution as described 460

previously, in custom made hydroponics tanks (Conn et al., 2013). 461

Arabidopsis Transformation and Selection. Binary vectors were transformed into 462

Agrobacterium tumefaciens GV3101 by electroporation. The floral dip method was used 463

to create transgenic plants in Arabidopsis thaliana (Col-0) (Clough and Bent, 1998). A 464

green LED flashlight and a Red 2 camera filter were used to identify transformed seeds 465

by fluorescence of the DsRed marker protein. 466

RNA Isolation and qPCR. For expression analyses of AtORM1 and AtORM2, RNA 467

extraction was done using the RNeasy Plant Kit (Qiagen) according to the 468

manufacturer’s protocol. RNA (1 µg) was treated with DNaseI (Invitrogen) according to 469

the manufacturer’s protocol. Treated RNA was then reverse transcribed to cDNA with 470

the iScript cDNA synthesis kit (BioRad) according to the manufacturer’s protocol. For 471

tissue-specific expression analysis, 6- to 8-week old Col-0 plants were used as sources 472

of plant material. qPCR was performed on cDNA using the BioRad MyiQ iCycler qPCR 473

instrument. Values shown are the average of three independent measurements ± SD. 474

SYBR green was used as the fluorophore in a qPCR supermix (Qiagen). QuantiTect 475

(Qiagen) primer sets P5-P7 (Table S1) were used for relative quantification. PP2AA3 476

(At1g13320) was used as an internal reference gene. RT-PCR analysis of homozygous 477

T-DNA mutant lines was performed on cDNA using primers P8-P11 (Table S1). 478



17

Analysis of Promoter GUS Expressing Plants. To generate the AtORM1 479

promoter::GUS and AtORM2 promoter::GUS constructs, a ~1 kb region upstream of the 480

start codon was PCR amplified from genomic DNA (oligonucleotides P12-P15; Table 481

S1), and cloned into a pBinGlyRed2 vector containing the GUS gene using the BamHI 482

and EcoRI restriction sites. This vector was then transformed into Agrobacterium 483

tumefaciens C58, and cells harboring the binary vector were used to transform wild-type 484

Arabidopsis as described previously (Clough and Bent, 1998). GUS staining solution 485

was comprised of 20 mM sodium phosphate (monobasic), 30 mM sodium phosphate 486

(dibasic), 2 mM potassium ferricyanide and 2 mM potassium ferrocyanide, along with 487

1μl/ml Triton X-100 and 1mg/ml 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-GLUC). 488

Tissues were pre-incubated in chilled 90% acetone for 10 minutes then vacuum 489

infiltrated with chilled GUS staining solution for 10 min. The tissues were then incubated 490

overnight at 37°C and then cleared with 100% ethanol followed by 70% ethanol. Images 491

of GUS analyzed tissue were taken with an Olympus AX70 optical microscope. 492

Subcellular Localization of ORM1 and ORM2. YFP fusion proteins with AtORM1 and 493

AtORM2 were prepared by amplification of the AtORM1 and AtORM2 open reading 494

frames using gene-specific primers (P16-P19; Table S1). PCR products were cloned 495

into the 35S-pFAST-eYFP vector using the SacI and KpnI restriction sites generating C-496

terminal YFP fusion constructs. Agrobacterium tumefaciens-mediated infiltration of 497

Nicotiana benthamiana leaves was performed with ORM1-YFP and ORM2-YFP 498

constructs separately and in conjunction with the ER marker CD3-959 (HDEL-mCherry). 499

Sequential imaging was performed using a Nikon A1 confocal imaging system mounted 500

on a Nikon Eclipse 90i microscope. Excitation/emission wavelengths for YFP and 501

mCherry were 488 nm/500-550 nm and 561.6 nm/570-620 nm, respectively as 502

described previously (Kimberlin et al., 2013). 503

Arabidopsis Mutant Genotyping. T-DNA insertion mutants were acquired from the 504

Arabidopsis Biological Resource Center and the GABI-Kat collections. The REDextract-505

N-Amp Tissue PCR kit (Sigma) was used to extract genomic DNA from leaf tissue. 506

Genotyping was performed by PCR using gene-specific and T-DNA-specific primer sets 507

P20-P28 (Table S1). 508



18

Membrane Topology Mapping of ScORM2. The topology of ScORM2 protein was 509

mapped based on the previous described methodology (Han et al., 2004). In brief, NheI 510

restriction sites were created before codons for amino acids 2, 100, 135 and 169, and 511

an XbaI site before the stop codon (217) by use of QuikChange mutagenesis kit (Agilent 512

Technology, Santa Clara, CA). A glycosylation cassette (GC) encoding the 53-amino 513

acid domain of invertase (Suc2p) was inserted into NheI or XbaI sites. The microsomal 514

proteins were isolated from the strains transformed with the GC topology reporter 515

plasmids and treated with Endoglycosidase H (Endo H). The proteins were resolved 516

using a 4–12% BisTris NuPAGE gel system (Invitrogen). 517

Fumonisin B1 Screening of Arabidopsis ORM1 and ORM2 Overexpression and 518

RNAi lines. ORM overexpressing plants were generated by transforming Col-0 with the 519

CaMV35S promoter:ORM cDNA constructs. AtORM1 and AtORM2 cDNAs were cloned 520

into the binary vector pBinGlyRed3-35S using the EcoRI/XbaI restriction sites (P29-P32; 521

Table S1). ORM RNAi lines were generated by overexpressing a hairpin composed of 522

an ORM1 or ORM2 gene fragment. The ORM gene fragments were cloned into the 523

pINTRON vector using the XbaI, XhoI, SpeI, and HindIII restriction sites forming a 524

hairpin using primers P33-P36 (Table S1). The pINTRON fragment containing the 525

hairpin was amplified using primers P37 and P38 (Table S1) and cloned into 526

pBinGlyRed3-35S using the EcoRI/XbaI restriction sites. Arabidopsis (Col-0) plants 527

were transformed with these constructs, and the resulting transformants were selected 528

and screened by qPCR for ORM1 and ORM2 expression. Sensitivity screening relative 529

to a wild type control was done at 0 µM, 0.3 µM, and 0.5 µM FB1 (Sigma) 530

concentrations in LS media. Sensitivity was determined by plant growth rate and 531

germination at the varying FB1 concentrations (Kimberlin et al., 2013). Plants were 532

grown for two weeks on FB1 before analysis. 533

Plant Microsomal Membrane Isolation. Microsomal membrane isolation from 534

hydroponically grown 4-week old Arabidopsis roots was performed as described 535

previously (Lynch and Fairfield, 1993) and protein concentration was measured using 536

the BCA method (Smith et al., 1985; Lynch and Fairfield, 1993). 537

Generation of AtLCB1C144W Mutant. The C144W mutation was introduced into the 538

AtLCB1 by changing codon 144 TGT to TGG by QuikChange mutagenesis (Stratagene, 539



19

La Jolla, CA), using oligonucleotide primers P39 and P40 (Table S1). The mutation was 540

confirmed by sequencing. 541

Ceramide Synthase Assays. Ceramide synthase assays were performed on 542

microsomal extracts (10 μg protein) derived from hydroponically grown root using 543

previously described methodology (Luttgeharm et al., 2015, 2016). Reaction substrates 544

for class I ceramide synthase assays were d18:0 LCB and 16:0-CoA, while substrates 545

for class II ceramide synthase assays were t18:0 LCB and 24:0-CoA. After incubation 546

and extraction, sphingolipids produced in the assay were analyzed by mass 547

spectrometry as described (Luttgeharm et al., 2015, 2016). 548

Sphingolipid Analysis. Sphingolipids were extracted from 2-15 mg of lyophilized plant 549

material and analyzed by LC-MS/MS as described previously (Markham and Jaworski, 550

2007; Kimberlin et al., 2013). 551

552

ACKNOWLEDGEMENTS 553

Financial support was provided by the National Science Foundation (MCB-1158500 to 554

JS, TMD, and EBC). We thank Professor Amy Chang (University of Michigan) for 555

providing plasmid pSH15 containing S. cerevisiae ORM2, and Professor Jaekwon Lee 556

(University of Nebraska-Lincoln) for providing S. cerevisiae knockout lines for ORM1 557

and ORM2. 558

559

SUPPLEMENTAL DATA 560

Table S1. Oligonucleotide primers used in the reported studies. 561

Figure S1. Gene expression levels for AtORM1 (At1g01230) and AtORM2 (At5g42000) 562 in different tissue types. 563

Figure S2. Genotyping of T-DNA mutants for AtORM1 and AtORM2. 564

Figure S3. Sphingolipid profile of AtORM1 and AtORM2 overexpression lines that 565 display the dwarf phenotype. 566

Figure S4. Ceramide profile for Col-0 and AtORM overexpression and RNAi lines 567 grown on LS media ±FB1. 568

Figure S5. Glucosylceramide (GlcCer) profile for Col-0 and AtORM overexpression and 569 RNAi lines 570 grown on LS ±FB1. 571



20

Figure S6. Glycosylinositolphosphorylceramide (GIPC) profile for Col-0 and AtORM 572 overexpression and RNAi 573 lines grown on LS ±FB1. n=3 independent analyses of seedlings ± SD. 574

Figure S7. qPCR analysis of ORM RNAi lines. RNA was extracted from leaf tissue of 3 575 week old plants. 576

Figure S8. SPT activity from root microsomes of wild-type and transgenic plants. 577

578

579

FIGURE LEGENDS 580

Figure 1. Arabidopsis ORMs physically interact with the Arabidopsis core serine 581

palmitoyltransferase (SPT) components and complement a S. cerevisiae ORM2 582

knockout mutant. 583

(A) Amino acid sequence alignment for ORM polypeptides from Saccharomyces 584

cerevisiae (ScORM1, ScORM2), Arabidopsis thaliana (AtORM1, AtORM2), and Homo 585

sapiens (ORMDL1, ORMDL2, ORMDL3). The alignments shows the N-terminal 586

extension, found only in yeast, responsible for reversible phosphorylation (at residues 587

marked with asterisks) that modulates SPT activity (Breslow et al., 2010). The dotted 588

lines mark the positions of 4 potential transmembrane domains identified by hydropathy 589

analyses. 590

(B) Topology mapping of ScORM2. Glycosylation cassettes (GC) were inserted after 591

the indicated amino acid and the GC-tagged proteins were expressed in yeast. 592

Increased mobility following treatment of microsomes with Endoglycosidase H (EndoH) 593

revealed that the GCs at residue 100 and 169 are glycosylated and therefore reside in 594

the lumen of the ER. 595

(C) Model of ORM protein topology. The figure shows the experimentally determined 596

membrane topology of ScORM2 as shown in (B). 597

(D) Co-immunoprecipitation of FLAG-tagged AtLCB1 in yeast expressing AtLCB1-598

FLAG, AtLCB2a-Myc, AtssSPTa-HA, and either AtORM1-HA or AtORM2-HA. 599

Solubilized yeast microsomes were incubated with anti-FLAG beads and protein was 600

eluted with FLAG peptide. Solubilized microsomes (Input) and the eluent (IP-FLAG) 601

were analyzed by SDS-PAGE and the polypeptides were detected by immunoblotting. 602

Elo3p was used as a negative control. 603



21

(E) AtORM1 and AtORM2 complement the sensitivity of the S. cerevisiae orm2Δ mutant 604

to exogenous long-chain bases. Shown are plates containing synthetic complete media 605

lacking or supplemented with the long-chain base phytosphingosine at a concentration 606

of 15 µM. As shown, plates contain dilutions of the parental cell line BY4741, S. 607

cerevisiae orm1Δ mutant, S. cerevisiae orm2Δ mutant, and the S. cerevisiae 608

orm2Δ complemented with either wild-type copy of the S. cerevisiae ORM2 (+ScORM2), 609

AtORM1 (+AtORM1), or AtORM2 (+AtORM2). 610

611

Figure 2. AtORM1 and AtORM2 are negative regulators of SPT activity. 612

(A) Schematic representation of the core SPT complex consisting of AtLCB1, 613

AtLCB2a/b, AtssSPT, and AtORM. The complex resides in the ER membrane and 614

catalyzes a condensation reaction between serine and palmitoyl-CoA to produce LCB. 615

The C144W (HSAN1) mutation in AtLCB1 allows SPT to use alanine as well as serine 616

as a substrate. The deoxy-LCB produced with alanine is referred to as deoxy-617

sphinganine (DoxSA) and lacks the hydroxyl group that is needed for LCB degradation 618

and for conversion to the glycosphingolipids glucosylceramides in the ER or 619

glycosylinositolphosphoceramides in the Golgi. 620

(B) In vivo AtSPT activity was measured in a yeast mutant that lacks endogenous SPT. 621

Cells expressing AtLCB1C144W, AtLCB2a, AtssSPTa with or without AtORM1 or AtORM2 622

were used to demonstrate an inhibitory effect on SPT activity. The activity is measured 623

by accumulation of DoxSA, produced by the AtLCB1C144W-containing mutant SPT 624

enzyme. The DoxSA product is not naturally produced and is not degraded. Values 625

shown are the average of three independent assays ± SD. **P<0.01. 626

(C) Immunoblot of yeast microsomes expressing AtLCB1C144W-FLAG, AtLCB2a-Myc, 627

AtORM1/2-HA, and AtssSPTa-HA. Anti-FLAG, anti-Myc, and anti-HA antibodies were 628

used for detection. The results show that the C144W mutation in AtLCB1 does not 629

affect the interaction of SPT components and interacting proteins. 630

(D) In vivo AtSPT activity measured in cells of yeast lacking endogenous SPT but 631

expressing AtLCB1, AtLCB2a, AtssSPTa (Vector) and either AtORM1 or AtORM2. The 632

activity is measured through the accumulation of total LCB produced. Values shown 633

are the average of three independent assays ± SD. *P<0.05. **P<0.01. 634



22

635

Figure 3. Subcellular localization of AtORM1 and AtORM2 polypeptides and gene 636

expression of AtORM1 and AtORM2 in Arabidopsis. 637

(A)-(F) Subcellular localization of AtORM1- or AtORM2-YFP fusions co-expressed with 638

the ER marker-mCherry fusion construct. All constructs were transiently expressed in 639

N. benthamiana through Agrobacterium tumefaciens infiltration and viewed by confocal 640

microscopy. Green color in panels (A) and (D) shows AtORM1 and AtORM2 641

localization, respectively. The red color in panels (B) and (E) indicates ER marker 642

localization. The yellow color as seen in merged panels (C) and (F) indicates co-643

localization of AtORM1 and AtORM2 polypeptides, respectively, with the ER marker. 644

(G) Relative expression of AtORM1 and AtORM2. Tissues were collected from wild-645

type Col-0, and qPCR was used to determine AtORM1 and AtORM2 transcript levels. 646

Protein phosphatase 2A subunit A3 (PP2AA3) was used as a reference gene. Values 647

shown are ± SD for three independent measurements and indicate relative fold increase 648

of AtORM1 or AtORM2 compared to AtORM2 or AtORM1, respectively. 649

(H)-(Q) AtORM1 and AtORM2 promoter::GUS expression analysis. A ~1kb region 650

upstream of AtORM1 (H)-(L) or AtORM2 (M)-(Q) start codon was fused to the β-651

glucuronidase (GUS) gene and analyzed for expression in various organs and tissues 652

as previously described (Jefferson et al., 1987). 653

654

Figure 4. Phenotypes associated with AtORM1 and AtORM2 overexpression. 655

(A) High levels of AtORM1 and AtORM2 expression results in reduced plant size, early 656

leaf senescence, and early plant death. Representative pictures of plants grown under 657

the same conditions and of comparable age are shown in (A)-(F). Wild type (Col-0) is 658

shown in (A) and (D). AtORM1 overexpression line 1 is shown in panel (B) while 659

AtORM2 overexpression line 1 is shown in panel (E). AtORM1 overexpression line 2 is 660

shown in panel (C) while AtORM2 overexpression line 2 is shown in panel (F). The 661

phenotype correlates with ORM expression level, as lines with the strongest 662

overexpression display a phenotype, while relatively weaker overexpression lines do not 663

show a noticeable growth phenotype. 664



23

(G)-(H) Expression levels of AtORM1 (G) and AtORM2 (H) in overexpressing lines. 665

Tissue was collected from wild-type Col-0 and lines overexpressing AtORM1 and 666

AtORM2 grown under standard conditions. qPCR was used to determine relative 667

AtORM1 and AtORM2 transcript levels by comparison with Col-0. Protein phosphatase 668

2A subunit A3 (PP2AA3) was used as a reference gene. Values shown are ± SD for 669

three independent measurements and indicate relative fold increase of AtORM1 or 670

AtORM2 compared to wild type levels. 671

672

Figure 5. Modulation of AtORM1 and AtORM2 expression alters sensitivity to FB1 and 673

LCB and C16 ceramide accumulation. 674

(A) Altered AtORM1 and AtORM2 expression affects sensitivity of plants to FB1, a 675

competitive inhibitor of ceramide synthase. Seeds were sown on LS agar plates 676

supplemented with FB1 at 0.3 and 0.5 μM as indicated. The wild type (Col-0) is 677

extremely sensitive to FB1 at 0.5 μM and less affected at 0.3 μM. Upregulation of 678

AtORM1 and AtORM2 by transgenic overexpression (OE) causes an FB1 resistant 679

phenotype, whereas RNAi suppression of AtORM by RNAi causes an FB1 sensitive 680

phenotype (ORM RNAi). Images were taken 14 days after seeds were sowed and are 681

representative of three independent experiments. 682

(B) Altered AtORM1 and AtORM2 affects accumulation of cytotoxic free LCB and LCB-683

phosphate (LCB-P) levels in response to FB1 treatment. Wild-type plants show 684

increased total LCB levels when treated with FB1. Compared with the wild type, 685

AtORM1 and AtORM2 overexpression (OE) plants display FB1 resistance and reduced 686

total LCB level. Alternatively, AtORM RNAi suppression plants display FB1 sensitivity 687

and increased total LCB level. Electrospray ionization-tandem mass spectrometry 688

analyses were performed with three independent biological replicates ± SD. Plants 689

were grown on LS plates ± FB1 for two weeks before tissue collection. DW, dry weight. 690

*P<0.05. **P<0.01. 691

(C) Total C16 ceramide levels are affected by modulation of AtORM expression. 692

AtORM1 overexpression plants show decreased accumulation of C16 ceramide when 693

compared with wild type comparatively grown on LS ± FB1, while ORM RNAi plants 694



24

show increased accumulation of C16 ceramide. Analyses were performed as described 695

in (B) with three independent biological replicates ± SD. DW, dry weight. *P<0.05. 696

697

Figure 6. Ceramide synthase activity is altered by modulation of AtORM1 and AtORM2 698

expression. 699

(A) Altered AtORM1 and AtORM2 expression affects Class I ceramide synthase activity. 700

Class I ceramide synthase activity was assayed on microsomal protein prepared from 701

hydroponically grown root tissue using 16:0-CoA and d18:0 as substrates. Increases in 702

ORM expression resulted in decreases in Class I ceramide synthase activity, while RNAi 703

suppression of AtORM resulted in an increase in activity of this enzyme. Analyses were 704

performed with three independent biological replicates ± SD. *P<0.05, **P<0.01. 705

(B) Increases in AtORM1 and AtORM2 expression resulted in an increases in Class II ceramide 706

synthase activity, while RNAi suppression of AtORM resulted in a decrease in activity of this 707

enzyme class. Activity assays were conducted with microsomes from hydroponically grown root 708

tissue using 24:0-CoA and t18:0 substrates. Analyses were performed with three independent 709

biological replicates ± SD. **P<0.01. 710

711

Figure 7. Model of coordinate regulation of sphingolipid synthesis by AtORM1 and 712

AtORM2 and ceramide synthase activity. 713

The figure shown represents the core synthesis pathway of ceramides in Arabidopsis. 714

Serine palmitoyltransferase (SPT) catalyzes a condensation reaction between serine 715

and palmitoyl-CoA, leading to the production of 3-ketosphinganine which is reduced to 716

dihydroxy LCB (d18:0) through 3-ketosphinganine reductase activity. Dihydroxy LCBs 717

can be used by Class I ceramide synthase (LOH2) along with 16:0-CoA to produce C16 718

ceramides. Alternatively, dihydroxy LCBs can be hydroxylated by LCB C-4 hydroxylase 719

to form trihydroxy LCBs (t18:0) that are used by Class II ceramide synthases (LOH1 720

and LOH3) along with very long-chain fatty acyl-CoAs, primarily 24:0- and 26:0-CoA, to 721

produce ceramides containing very long-chain fatty acids (VLCFAs). In the model 722

shown, modulation of AtORM expression leads to alterations in ceramide synthase 723

activity. AtORM RNAi suppression, indicated by “GO”, results in increased SPT activity 724

and increased generation of LCBs with a concomitant increase in Class I activity and a 725



25

decrease in Class II activity. Conversely, overexpression of AtORM1 and AtORM2, 726

indicated by “STOP”, results in decreased SPT activity and reduced LCB generation 727

with a concomitant decrease in Class I activity and an increase in Class II activity to 728

ensure production of sufficient levels of ceramides with VLCFAs to support growth. A 729

role of LCB C-4 hydroxylase activity in mediating relative flux through Class I and II 730

ceramide synthases, particularly in response to enhanced LCB synthesis, is also 731

possible. 732

733

734

735

REFERENCES 736

Bach L, Gissot L, Marion J, Tellier F, Moreau P, Satiat-Jeunemaitre B, Palauqui 737

JC, Napier JA, Faure JD (2011) Very-long-chain fatty acids are required for cell 738

plate formation during cytokinesis in Arabidopsis thaliana. J Cell Sci 124: 3223-739

3234 740

Bach L, Michaelson LV, Haslam R, Bellec Y, Gissot L, Marion J, Da Costa M, 741

Boutin JP, Miquel M, Tellier F, Domergue F, Markham JE, Beaudoin F, 742

Napier JA, Faure JD (2008) The very-long-chain hydroxy fatty acyl-CoA 743

dehydratase PASTICCINO2 is essential and limiting for plant development. Proc 744

Natl Acad Sci USA 105: 14727-14731 745

Bayer EM, Mongrand S, Tilsner J (2014) Specialised membrane domains of 746

plasmodesmata, plant intercellular nanopores. Frontiers in Plant Science 5: 507 747

Bi FC, Liu Z, Wu JX, Liang H, Xi XL, Fang C, Sun TJ, Yin J, Dai GY, Rong C, 748

Greenberg JT, Su WW, Yao N (2014) Loss of ceramide kinase in Arabidopsis 749

impairs defenses and promotes ceramide accumulation and mitochondrial H2O2 750

bursts. Plant Cell 26: 3449-3467 751

Breslow DK (2013) Sphingolipid homeostasis in the endoplasmic reticulum and 752

beyond. Cold Spring Harb Perspect Biol 5: a013326 753

Breslow DK, Collins SR, Bodenmiller B, Aebersold R, Simons K, Shevchenko A, 754

Ejsing CS, Weissman JS (2010) Orm family proteins mediate sphingolipid 755

homeostasis. Nature 463: 1048-1053 756



26

Cacas JL, Bure C, Grosjean K, Gerbeau-Pissot P, Lherminier J, Rombouts Y, 757

Maes E, Bossard C, Gronnier J, Furt F, Fouillen L, Germain V, Bayer E, 758

Cluzet S, Robert F, Schmitter JM, Deleu M, Lins L, Simon-Plas F, Mongrand 759

S (2016) Re-visiting plant plasma membrane lipids in tobacco: a focus on 760

sphingolipids. Plant Physiol 170: 367-384 761

Cacas JL, Furt F, Le Guedard M, Schmitter JM, Bure C, Gerbeau-Pissot P, Moreau 762

P, Bessoule JJ, Simon-Plas F, Mongrand S (2012) Lipids of plant membrane 763

rafts. Prog Lipid Res 51: 272-299 764

Chen M, Han G, Dietrich CR, Dunn TM, Cahoon EB (2006) The essential nature of 765

sphingolipids in plants as revealed by the functional identification and 766

characterization of the Arabidopsis LCB1 subunit of serine palmitoyltransferase. 767

Plant Cell 18: 3576-3593 768

Chen M, Markham JE, Dietrich CR, Jaworski JG, Cahoon EB (2008) Sphingolipid 769

long-chain base hydroxylation is important for growth and regulation of 770

sphingolipid content and composition in Arabidopsis. Plant Cell 20: 1862-1878 771

Chueasiri C, Chunthong K, Pitnjam K, Chakhonkaen S, Sangarwut N, 772

Sangsawang K, Suksangpanomrung M, Michaelson LV, Napier JA, 773

Muangprom A (2014) Rice ORMDL controls sphingolipid homeostasis affecting 774

fertility resulting from abnormal pollen development. PloS one 9: e106386 775

Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated 776

transformation of Arabidopsis thaliana. Plant J 16: 735-743 777

Conn SJ, Hocking B, Dayod M, Xu B, Athman A, Henderson S, Aukett L, Conn V, 778

Shearer MK, Fuentes S, Tyerman SD, Gilliham M (2013) Protocol: optimising 779

hydroponic growth systems for nutritional and physiological analysis of 780

Arabidopsis thaliana and other plants. Plant Methods 9: 4 781

Dietrich CR, Han G, Chen M, Berg RH, Dunn TM, Cahoon EB (2008) Loss-of-782

function mutations and inducible RNAi suppression of Arabidopsis LCB2 genes 783

reveal the critical role of sphingolipids in gametophytic and sporophytic cell 784

viability. Plant J 54: 284-298 785



27

Fresques T, Niles B, Aronova S, Mogri H, Rakhshandehroo T, Powers T (2015) 786

Regulation of ceramide synthase by casein kinase 2-dependent phosphorylation 787

in Saccharomyces cerevisiae. J Biol Chem 290: 1395-1403 788

Gable K, Gupta SD, Han G, Niranjanakumari S, Harmon JM, Dunn TM (2010) A 789

disease-causing mutation in the active site of serine palmitoyltransferase causes 790

catalytic promiscuity. J Biol Chem 285: 22846-22852 791

Greenberg JT, Silverman FP, Liang H (2000) Uncoupling salicylic acid-dependent cell 792

death and defense-related responses from disease resistance in the arabidopsis 793

mutant acd5. Genetics 156: 341-350 794

Gururaj C, Federman RS, Chang A (2013) Orm proteins integrate multiple signals to 795

maintain sphingolipid homeostasis. J Biol Chem 288: 20453-20463 796

Han G, Gable K, Yan L, Natarajan M, Krishnamurthy J, Gupta SD, Borovitskaya A, 797

Harmon JM, Dunn TM (2004) The topology of the Lcb1p subunit of yeast serine 798

palmitoyltransferase. J Biol Chem 279: 53707-53716 799

Han G, Gupta SD, Gable K, Niranjanakumari S, Moitra P, Eichler F, Brown RH, Jr., 800

Harmon JM, Dunn TM (2009) Identification of small subunits of mammalian 801

serine palmitoyltransferase that confer distinct acyl-CoA substrate specificities. 802

Proc Natl Acad Sci USA 106: 8186-8191 803

Han S, Lone MA, Schneiter R, Chang A (2010) Orm1 and Orm2 are conserved 804

endoplasmic reticulum membrane proteins regulating lipid homeostasis and 805

protein quality control. Proc Natl Acad Sci USA 107: 5851-5856 806

Hanada K (2003) Serine palmitoyltransferase, a key enzyme of sphingolipid 807

metabolism. Biochim Biophys Acta 1632: 16-30 808

Hirokawa T, Boon-Chieng S, Mitaku S (1998) SOSUI: classification and secondary 809

structure prediction system for membrane proteins. Bioinformatics 14: 378-379 810

Hjelmqvist L, Tuson M, Marfany G, Herrero E, Balcells S, Gonzalez-Duarte R 811

(2002) ORMDL proteins are a conserved new family of endoplasmic reticulum 812

membrane proteins. Genome Biology 3: RESEARCH0027 813

Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as 814

a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901-815

3907 816



28

Kimberlin AN, Majumder S, Han G, Chen M, Cahoon RE, Stone JM, Dunn TM, 817

Cahoon EB (2013) Arabidopsis 56-amino acid serine palmitoyltransferase-818

interacting proteins stimulate sphingolipid synthesis, are essential, and affect 819

mycotoxin sensitivity. Plant Cell 25: 4627-4639 820

Kohlwein SD, Eder S, Oh CS, Martin CE, Gable K, Bacikova D, Dunn T (2001) 821

Tsc13p is required for fatty acid elongation and localizes to a novel structure at 822

the nuclear-vacuolar interface in Saccharomyces cerevisiae. Mol Cell Biol 21: 823

109-125 824

Liang H, Yao N, Song JT, Luo S, Lu H, Greenberg JT (2003) Ceramides modulate 825

programmed cell death in plants. Genes & Development 17: 2636-2641 826

Luttgeharm KD, Cahoon EB, Markham JE (2015) A mass spectrometry-based 827

method for the assay of ceramide synthase substrate specificity. Anal Biochem 828

478: 96-101 829

Luttgeharm KD, Cahoon EB, Markham JE (2016) Substrate specificity, kinetic 830

properties and inhibition by fumonisin B1 of ceramide synthase isoforms from 831

Arabidopsis. Biochem J 473: 593-603 832

Luttgeharm KD, Chen M, Mehra A, Cahoon RE, Markham JE, Cahoon EB (2015) 833

Overexpression of Arabidopsis ceramide synthases differentially affects growth, 834

sphingolipid metabolism, programmed cell death, and mycotoxin resistance. 835

Plant Physiol 169: 1108-1117 836

Lynch DV, Fairfield SR (1993) Sphingolipid long-chain base synthesis in plants: 837

characterization of serine palmitoyltransferase activity in squash fruit 838

microsomes. Plant Physiol 103: 1421-1429 839

Markham JE, Jaworski JG (2007) Rapid measurement of sphingolipids from 840

Arabidopsis thaliana by reversed-phase high-performance liquid chromatography 841

coupled to electrospray ionization tandem mass spectrometry. Rapid Commun 842

Mass Spectrom 21: 1304-1314 843

Markham JE, Lynch DV, Napier JA, Dunn TM, Cahoon EB (2013) Plant 844

sphingolipids: function follows form. Curr Opin Plant Biol 16: 350-357 845

Markham JE, Molino D, Gissot L, Bellec Y, Hematy K, Marion J, Belcram K, 846

Palauqui JC, Satiat-Jeunemaitre B, Faure JD (2011) Sphingolipids containing 847



29

very-long-chain fatty acids define a secretory pathway for specific polar plasma 848

membrane protein targeting in Arabidopsis. Plant Cell 23: 2362-2378 849

Melser S, Molino D, Batailler B, Peypelut M, Laloi M, Wattelet-Boyer V, Bellec Y, 850

Faure JD, Moreau P (2011) Links between lipid homeostasis, organelle 851

morphodynamics and protein trafficking in eukaryotic and plant secretory 852

pathways. Plant Cell Rep 30: 177-193 853

Msanne J, Chen M, Luttgeharm KD, Bradley AM, Mays ES, Paper JM, Boyle DL, 854

Cahoon RE, Schrick K, Cahoon EB (2015) Glucosylceramides are critical for 855

cell-type differentiation and organogenesis, but not for cell viability in 856

Arabidopsis. Plant J 84: 188-201 857

Muir A, Ramachandran S, Roelants FM, Timmons G, Thorner J (2014) TORC2-858

dependent protein kinase Ypk1 phosphorylates ceramide synthase to stimulate 859

synthesis of complex sphingolipids. Elife 3 860

Mullen TD, Hannun YA, Obeid LM (2012) Ceramide synthases at the centre of 861

sphingolipid metabolism and biology. Biochem J 441: 789-802 862

Nühse TS, Stensballe A, Jensen ON, Peck SC (2004) Phosphoproteomics of the 863

Arabidopsis plasma membrane and a new phosphorylation site database. Plant 864

Cell 16: 2394-2405 865

Perraki A, Cacas JL, Crowet JM, Lins L, Castroviejo M, German-Retana S, 866

Mongrand S, Raffaele S (2012) Plasma membrane localization of Solanum 867

tuberosum remorin from group 1, homolog 3 is mediated by conformational 868

changes in a novel C-terminal anchor and required for the restriction of potato 869

virus X movement. Plant Physiol 160: 624-637 870

Roelants FM, Breslow DK, Muir A, Weissman JS, Thorner J (2011) Protein kinase 871

Ypk1 phosphorylates regulatory proteins Orm1 and Orm2 to control sphingolipid 872

homeostasis in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 108: 873

19222-19227 874

Saucedo-Garcia M, Guevara-Garcia A, Gonzalez-Solis A, Cruz-Garcia F, Vazquez-875

Santana S, Markham JE, Lozano-Rosas MG, Dietrich CR, Ramos-Vega M, 876

Cahoon EB, Gavilanes-Ruiz M (2011) MPK6, sphinganine and the LCB2a gene 877

from serine palmitoyltransferase are required in the signaling pathway that 878



30

mediates cell death induced by long chain bases in Arabidopsis. New Phytol 191: 879

943-957 880

Shi L, Bielawski J, Mu J, Dong H, Teng C, Zhang J, Yang X, Tomishige N, Hanada 881

K, Hannun YA, Zuo J (2007) Involvement of sphingoid bases in mediating 882

reactive oxygen intermediate production and programmed cell death in 883

Arabidopsis. Cell Research 17: 1030-1040 884

Simanshu DK, Zhai X, Munch D, Hofius D, Markham JE, Bielawski J, Bielawska A, 885

Malinina L, Molotkovsky JG, Mundy JW, Patel DJ, Brown RE (2014) 886

Arabidopsis accelerated cell death 11, ACD11, is a ceramide-1-phosphate 887

transfer protein and intermediary regulator of phytoceramide levels. Cell Rep 6: 888

388-399 889

Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, 890

Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein 891

using bicinchoninic acid. Anal Biochem 150: 76-85 892

Sperling P, Franke S, Luthje S, Heinz E (2005) Are glucocerebrosides the 893

predominant sphingolipids in plant plasma membranes? Plant Physiol Biochem 894

43: 1031-1038 895

Stiban J, Tidhar R, Futerman AH (2010) Ceramide synthases: roles in cell physiology 896

and signaling. Adv Exp Med Biol 688: 60-71 897

Sugiyama N, Nakagami H, Mochida K, Daudi A, Tomita M, Shirasu K, Ishihama Y 898

(2008) Large-scale phosphorylation mapping reveals the extent of tyrosine 899

phosphorylation in Arabidopsis. Mol Syst Biol 4: 193 900

Sun Y, Miao Y, Yamane Y, Zhang C, Shokat KM, Takematsu H, Kozutsumi Y, 901

Drubin DG (2012) Orm protein phosphoregulation mediates transient 902

sphingolipid biosynthesis response to heat stress via the Pkh-Ypk and Cdc55-903

PP2A pathways. Mol Biol Cell 23: 2388-2398 904

Teng C, Dong H, Shi L, Deng Y, Mu J, Zhang J, Yang X, Zuo J (2008) Serine 905

palmitoyltransferase, a key enzyme for de novo synthesis of sphingolipids, is 906

essential for male gametophyte development in Arabidopsis. Plant Physiol 146: 907

1322-1332 908



31

Ternes P, Feussner K, Werner S, Lerche J, Iven T, Heilmann I, Riezman H, 909

Feussner I (2011) Disruption of the ceramide synthase LOH1 causes 910

spontaneous cell death in Arabidopsis thaliana. The New phytologist 192: 841-911

854 912

von Heijne G (1992) Membrane protein structure prediction. Hydrophobicity analysis 913

and the positive-inside rule. J Mol Biol 225: 487-494 914

915



A

D

+ Long-Chain Base Synthetic Complete

BY4741

orm1Δ

orm2Δ

+AtORM1

+AtORM2

+ScORM2

Inp

ut

IP-F

lag

Inp

ut

IP-F

lag

AtLCB1-Flag

Myc-AtLCB2a

HA-AtORM2

HA-AtssSPTa

ELO3

E

B

C

***

ScORM1 MTELDYQGTAEAASTSYSRNQTDLKPFPSAGSASSSIKTTEPVKDHRRRRSSSIISHVEPETFEDENDQQLLPNM 75 ScORM2 MIDRTKNESPAFEESPLTPNVSNLKPFPSQ-----SNKISTPVTDHRRRRSSSVISHVEQETFEDENDQQMLPNM 70 AtORM1 -----------------------------------------------------------MANLYVKAVPPPDMNR 16 AtORM2 --------------------------------------------------------------MYVRALPTTDVNR 13 ORMDL1 ---------------------------------------------------------------MNVGVAHSEVNP 12 ORMDL2 ---------------------------------------------------------------MNVGVAHSEVNP 12 ORMDL3 ---------------------------------------------------------------MNVGTAHSEVNP 12 ScORM1 NATWVDQRGAWIIHVVIIILLKLFYNLFPGVTTEWSWTLTNMTYVIGSYVMFHLIKGTPFDF-NGGAYDNLTMWE 149 ScORM2 NATWVDQRGAWLIHIVVIVLLRLFYSLFG-STPKWTWTLTNMTYIIGFYIMFHLVKGTPFDF-NGGAYDNLTMWE 143 AtORM1 NTEWFMYPGVWTTYMLILFFGWLVVLSVSGCSPGMAWTVVNLAHFVVTYHSFHWMKGTPFAD-DQGIYNGLTWWE 90 AtORM2 NTEWFTYPGVWTTYILILFFSWLLVLSVFHCSPGIAWTIVHLAHFTVTYHSFHWKKGTPFGD-DQGVYNRLTWWE 87 ORMDL1 NTRVMNSRGMWLTYALGVGLLHIVLLSIPFFSVPVAWTLTNIIHNLGMYVFLHAVKGTPFETPDQGKARLLTHWE 87 ORMDL2 NTRVMNSRGIWLAYIILVGLLHMVLLSIPFFSIPVVWTLTNVIHNLATYVFLHTVKGTPFETPDQGKARLLTHWE 87 ORMDL3 NTRVMNSRGIWLSYVLAIGLLHIVLLSIPFVSVPVVWTLTNLIHNMGMYIFLHTVKGTPFETPDQGKARLLTHWE 87 ScORM1 QIDDETLYTPSRKFLISVPITLFLVSTHYAHYDLKLFSWNCFLTTFGAVVPKLPVTHRLRISIPGITGRAQIS 223 ScORM2 QINDETLYTPTRKFLLIVPIVLFLISNQYYRNDMTLFLSNLAVTVLIGVVPKLGITHRLRISIPGITGRAQIS 216 AtORM1 QMDNGQQLTRNRKFLTLVPVVLYLIASHTTDYRHPWLFLN-TLAVMVLVVAKFPNMHKVRIFGINGDK----- 157 AtORM2 QIDNGKQLTRNRKFLTVVPVVLYLIASHTTDYQHPMLFLN-TLAVFVMVVAKFPHMHKVRIFGINGDQ----- 154 ORMDL1 QLDYGVQFTSSRKFFTISPIILYFLASFYTKYDPTHFILN-TASLLSVLIPKMPQLHGVRIFGINKY------ 153 ORMDL2 QMDYGLQFTSSRKFLSISPIVLYLLASFYTKYDAAHFLIN-TASLLSVLLPKLPQFHGVRVFGINKY------ 153 ORMDL3 QMDYGVQFTASRKFLTITPIVLYFLTSFYTKYDQIHFVLN-TVSLMSVLIPKLPQLHGVRIFGINKY------ 153

* * * * * * * *

AtLCB1-Flag

Myc-AtLCB2a

HA-AtORM1

HA-AtssSPTa

ELO3

Figure 1. Arabidopsis ORMs physically interact with the Arabidopsis core serine palmitoyltransferase

(SPT) components and complement a S. cerevisiae ORM2 knockout mutant. (A) Amino acid sequence alignment for ORM polypeptides from Saccharomyces cerevisiae (ScORM1,

ScORM2), Arabidopsis thaliana (AtORM1, AtORM2), and Homo sapiens (ORMDL1, ORMDL2,

ORMDL3). The alignments shows the N-terminal extension, found only in yeast, responsible for

reversible phosphorylation (at residues marked with asterisks) that modulates SPT activity (Breslow et

al., 2010). The dotted lines mark the positions of 4 potential transmembrane domains identified by

hydropathy analyses. (B) Topology mapping of ScORM2. Glycosylation cassettes (GC) were inserted after the indicated amino

acid and the GC-tagged proteins were expressed in yeast. Increased mobility following treatment of

microsomes with Endoglycosidase H (EndoH) revealed that the GCs at residue 100 and 169 are

glycosylated and therefore reside in the lumen of the ER. (C) Model of ORM protein topology. The figure shows the experimentally determined membrane topology

of ScORM2 as shown in (B). (D) Co-immunoprecipitation of FLAG-tagged AtLCB1 in yeast expressing AtLCB1-FLAG, AtLCB2a-Myc,

AtssSPTa-HA, and either AtORM1-HA or AtORM2-HA. Solubilized yeast microsomes were incubated

with anti-FLAG beads and protein was eluted with FLAG peptide. Solubilized microsomes (Input) and

the eluent (IP-FLAG) were analyzed by SDS-PAGE and the polypeptides were detected by

immunoblotting. Elo3p was used as a negative control. (E) AtORM1 and AtORM2 complement the sensitivity of the S. cerevisiae orm2D mutant to exogenous

long-chain bases. Shown are plates containing synthetic complete media lacking or supplemented with

the long-chain base phytosphingosine at a concentration of 15 µM. As shown, plates contain dilutions of

the parental cell line BY4741, S. cerevisiae orm1D mutant, S. cerevisiae orm2D mutant, and the S.

cerevisiae orm2D complemented with either wild-type copy of the S. cerevisiae ORM2 (+ScORM2),

AtORM1 (+AtORM1), or AtORM2 (+AtORM2). www.plantphysiol.orgon May 21, 2018 - Published by Downloaded from

Copyright © 2016 American Society of Plant Biologists. All rights reserved.


0

10

20

30

40

50

60

70

80

Vector AtORM1 AtORM2

pm

ol L

CB

/O.D

. C

ells

**

**

A

B C

0

500

1000

1500

2000

2500

Vector AtORM1 AtORM2

pm

ol L

CB

/O.D

. C

ells

D

**

*

Ve

cto

r

AtO

RM

1

AtO

RM

2

AtLCB1C144W

AtLCB2a

AtORM1 or

AtORM2

AtssSPTa

Figure 2. AtORM1 and AtORM2 are negative regulators of SPT activity.

(A) Schematic representation of the core SPT complex consisting of AtLCB1, AtLCB2a/b, AtssSPT, and

AtORM. The complex resides in the ER membrane and catalyzes a condensation reaction between serine and

palmitoyl-CoA to produce LCB. The C144W (HSAN1) mutation in AtLCB1 allows SPT to use alanine as well as

serine as a substrate. The deoxy-LCB produced with alanine is referred to as deoxy-sphinganine (DoxSA) and

lacks the hydroxyl group that is needed for LCB degradation and for conversion to the glycosphingolipids

glucosylceramides in the ER or glycosylinositolphosphoceramides in the Golgi.

(B) In vivo AtSPT activity was measured in a yeast mutant that lacks endogenous SPT. Cells expressing

AtLCB1C144W, AtLCB2a, AtssSPTa with or without AtORM1 or AtORM2 were used to demonstrate an inhibitory

effect on SPT activity. The activity is measured by accumulation of DoxSA, produced by the AtLCB1C144W-

containing mutant SPT enzyme. The DoxSA product is not naturally produced and is not degraded. Values

shown are the average of three independent assays ± SD. **P<0.01.

(C) Immunoblot of yeast microsomes expressing AtLCB1C144W-FLAG, AtLCB2a-Myc, AtORM1/2-HA, and

AtssSPTa-HA. Anti-FLAG, anti-Myc, and anti-HA antibodies were used for detection. The results show that the

C144W mutation in AtLCB1 does not affect the interaction of SPT components and interacting proteins.

(D) In vivo AtSPT activity measured in cells of yeast lacking endogenous SPT but expressing AtLCB1,

AtLCB2a, AtssSPTa (Vector) and either AtORM1 or AtORM2. The activity is measured through the

accumulation of total LCB produced. Values shown are the average of three independent assays ± SD.

*P<0.05. **P<0.01. www.plantphysiol.orgon May 21, 2018 - Published by Downloaded from



0

1

2

3

4

5

6

Flower Leaf Stem Silique Root Pollen

Fo

ld D

iffe

ren

ce

ORM1 ORM2

G

H I

J K L

M N

O P Q

A B C

D E F

Figure 3. Subcellular localization of AtORM1 and AtORM2 polypeptides and gene expression of

AtORM1 and AtORM2 in Arabidopsis.

(A)-(F) Subcellular localization of AtORM1 or AtORM2 YFP fusion constructs co-expressed with the ER marker-

mCherry fusion construct. All constructs were transiently expressed in N. benthamiana through Agrobacterium

tumefaciens infiltration and viewed by confocal microscopy. Green color in panels (A) and (D) shows AtORM1

and AtORM2 localization, respectively. The red color in panels (B) and (E) indicates ER marker localization. The

yellow color as seen in merged panels (C) and (F) indicates co-localization of AtORM1 and AtORM2,

respectively, with the ER marker.

(G) Relative expression of AtORM1 and AtORM2. Tissues were collected from wild-type Col-0, and

qPCR was used to determine AtORM1 and AtORM2 transcript levels. Protein phosphatase 2A subunit

A3 (PP2AA3) was used as a reference gene. Values shown are ± SD for three independent

measurements and indicate relative fold increase of AtORM1 or AtORM2 compared to AtORM2 or

AtORM1, respectively.

(H)-(Q) AtORM1 and AtORM2 promoter::GUS expression analysis. A ~1kb region upstream of

AtORM1 (H)-(L) or AtORM2 (M)-(Q) start codon was fused to the b-glucuronidase (GUS) gene and

analyzed for expression in various tissues as previously described (Jefferson et al., 1987).



A

0

20

40

60

80

100

120

Col-0 OEORM1-1

OEORM1-2

Fo

ld In

cre

ase

0

100

200

300

400

500

600

700

800

900

1000

Col-0 OEORM2-1

OEORM2-2

Fo

ld In

cre

ase

G H

B C

D E F

Figure 4. Phenotypes associated with AtORM1 and AtORM2 overexpression.

(A) High levels of AtORM1 and AtORM2 expression results in reduced plant size, early leaf senescence,

and early plant death. Representative pictures of plants grown under the same conditions and of

comparable are shown in (A)-(F). Wild type (Col-0) is shown in (A) and (D). AtORM1 overexpression line

1 is shown in panel (B) while AtORM2 overexpression line 1 is shown in panel (E). AtORM1

overexpression line 2 is shown in panel (C) while AtORM2 overexpression line 2 is shown in panel (F).

The phenotype correlates with ORM expression level, as lines with the strongest overexpression display

a phenotype, while relatively weaker overexpression lines do not show a noticeable growth phenotype.

(G)-(H) Expression levels of AtORM1 (G) and AtORM2 (H) in overexpressing lines. Tissue was collected

from wild-type Col-0 and lines overexpressing AtORM1 and AtORM2 grown under standard conditions.

qPCR was used to determine relative AtORM1 and AtORM2 transcript levels by comparison with Col-0.

Protein phosphatase 2A subunit A3 (PP2AA3) was used as a reference gene. Values shown are ± SD for

three independent measurements and indicate relative fold increase of AtORM1 or AtORM2 compared to

wild type levels. www.plantphysiol.orgon May 21, 2018 - Published by Downloaded from



0.5 mM FB1 0.3 mM FB1 0 mM FB1

Col-0

ORM1

OE

ORM2

OE

ORM

RNAi

nm

ol L

CB

or

LC

BP

/g D

W

A

[FB1]

mM

0

1000

2000

3000

4000

5000

6000

7000

8000

0 0.3 0.5 0 0.3 0.5 0 0.3 0.5 0 0.3 0.5

LCB LCBP

Col-0 OE ORM1 OE ORM2 ORM RNAi

B

0

200

400

600

800

1000

1200

1400

1600

0 0.3 0.5 0 0.3 0.5 0 0.3 0.5 0 0.3 0.5[FB1]

mM

C

* * *

*

* ** **

*

** **

**

nm

ol C

16

ce

ram

ide/g

DW

Col-0 OE ORM1 OE ORM2 ORM RNAi

Figure 5. Modulation of AtORM1 and AtORM2

expression alters sensitivity to FB1 and LCB and C16

ceramide accumulation.

(A) Altered AtORM1 and AtORM2 expression affects

sensitivity of plants to FB1, a competitive inhibitor of

ceramide synthase. Seeds were sown on LS agar

plates supplemented with FB1 at 0.3 and 0.5 mM as

indicated. The wild type (Col-0) is extremely sensitive

to FB1 at 0.5 mM and less affected at 0.3 mM.

Upregulation of AtORM1 and AtORM2 by transgenic

overexpression (OE) causes an FB1 resistant

phenotype, whereas RNAi suppression of AtORM by

RNAi causes an FB1 sensitive phenotype (ORM

RNAi). Images were taken 14 days after seeds were

sowed and are representative of three independent

experiments.

(B) Altered AtORM1 and AtORM2 affects accumulation

of cytotoxic free LCB and LCB-phosphate (LCB-P)

levels in response to FB1 treatment. Wild-type plants

show increased total LCB levels when treated with

FB1. Compared with the wild type, AtORM1 and

AtORM2 overexpression (OE) plants display FB1

resistance and reduced total LCB level. Alternatively,

AtORM RNAi suppression plants display FB1

sensitivity and increased total LCB level. Electrospray

ionization-tandem mass spectrometry analyses were

performed with three independent biological replicates

± SD. Plants were grown on LS plates ± FB1 for two

weeks before tissue collection. DW, dry weight.

*P<0.05. **P<0.01.

(C) Total C16 ceramide levels are affected by

modulation of AtORM expression. AtORM1

overexpression plants show decreased accumulation

of C16 ceramide when compared with wild type

comparatively grown on LS ± FB1, while ORM RNAi

plants show increased accumulation of C16 ceramide.

Analyses were performed as described in (B) with

three independent biological replicates ± SD. DW, dry

weight. *P<0.05.



0

50

100

150

200

250

300

Col-0 OEORM1

OEORM2

ORMRNAi

pm

ol d

18

:0_

c1

6:0

ce

ram

ide

/min

/mg

pro

t.

B A

0

50

100

150

200

250

300

350

400

Col-0 OEORM1

OEORM2

ORMRNAi

pm

ol t1

8:0

_c2

4:0

ce

ram

ide

/min

/mg

pro

t.

**

**

**

** **

*

Figure 6. Ceramide synthase activity is altered by modulation of AtORM1 and AtORM2

expression.

(A) Altered AtORM1 and AtORM2 expression affects Class I ceramide synthase activity. Class I

ceramide synthase activity was assayed on microsomal protein prepared from hydroponically

grown root tissue using 16:0-CoA and d18:0 as substrates. Increases in ORM expression resulted

in decreases in Class I ceramide synthase activity, while RNAi suppression of AtORM resulted in

an increase in activity of this enzyme. Analyses were performed with three independent biological

replicates ± SD. *P<0.05, **P<0.01.

(B) Increases in AtORM1 and AtORM2 expression resulted in an increases in Class II ceramide

synthase activity, while RNAi suppression of AtORM resulted in a decrease in activity of this

enzyme class. Activity assays were conducted with microsomes from hydroponically grown root

tissue using 24:0-CoA and t18:0 substrates. Analyses were performed with three independent

biological replicates ± SD. **P<0.01.

Class I Class II



Serine + Palmitoyl-CoA

3-Ketosphinganine

Serine

Palmitoyltransferase

(SPT)

Dihydroxy LCB Trihydroxy LCB

C16 FA/Dihydroxy LCB

Ceramide

3-Ketosphinganine

Reductase

16:0-CoA

STOP GO

LCB C-4

Hydroxylase

24:0- &

26:0-CoA

Class I Ceramide

Synthase

(LOH2)

Class II Ceramide

Synthases

(LOH1, LOH3)

VLCFA/Trihydroxy LCB

Ceramide

ORM

RNAi

ORM

OE

Figure 7. Model of coordinate regulation of sphingolipid synthesis by AtORM1 and AtORM2 and

ceramide synthase activity.

The figure shown represents the core synthesis pathway of ceramides in Arabidopsis. Serine

palmitoyltransferase (SPT) catalyzes a condensation reaction between serine and palmitoyl-CoA

leading to the production of 3-ketosphinganine which is reduced to dihydroxy LCB (d18:0) through

3-ketosphinganine reductase activity. Dihydroxy LCBs can be used by Class I ceramide synthase

(LOH2) along with 16:0-CoA to produce C16 ceramides. Alternatively, dihydroxy LCBs can be

hydroxylated by LCB C-4 hydroxylase to form trihydroxy LCBs (t18:0) that are used by Class II

ceramide synthases (LOH1 and LOH3) along with primarily 24:0- and 26:0-CoA to produce

ceramides containing very long-chain fatty acids. In the model shown, modulation of AtORM

expression leads to alterations in ceramide synthase activity. AtORM RNAi suppression, indicated

by “GO”, results in increased SPT activity and increased generation of LCBs with a concomitant

increase in Class I activity and a decrease in Class II activity. Conversely, overexpression of

AtORM1 and AtORM2, indicated by “STOP”, results in decreased SPT activity and reduced LCB

generation with a concomitant decrease in Class I activity and an increase in Class II activity to

ensure production of sufficient levels of ceramides with very long-chain fatty acids to support

growth. A role of LCB C-4 hydroxylase activity in mediating relative flux through Class I and II

ceramide synthases, particularly in response to enhanced LCB synthesis, is also possible.



Parsed CitationsBach L, Gissot L, Marion J, Tellier F, Moreau P, Satiat-Jeunemaitre B, Palauqui JC, Napier JA, Faure JD (2011) Very-long-chain fattyacids are required for cell plate formation during cytokinesis in Arabidopsis thaliana. J Cell Sci 124: 3223-3234

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Bach L, Michaelson LV, Haslam R, Bellec Y, Gissot L, Marion J, Da Costa M, Boutin JP, Miquel M, Tellier F, Domergue F, MarkhamJE, Beaudoin F, Napier JA, Faure JD (2008) The very-long-chain hydroxy fatty acyl-CoA dehydratase PASTICCINO2 is essential andlimiting for plant development. Proc Natl Acad Sci USA 105: 14727-14731


Bayer EM, Mongrand S, Tilsner J (2014) Specialised membrane domains of plasmodesmata, plant intercellular nanopores.Frontiers in Plant Science 5: 507


Bi FC, Liu Z, Wu JX, Liang H, Xi XL, Fang C, Sun TJ, Yin J, Dai GY, Rong C, Greenberg JT, Su WW, Yao N (2014) Loss of ceramidekinase in Arabidopsis impairs defenses and promotes ceramide accumulation and mitochondrial H2O2 bursts. Plant Cell 26: 3449-3467


Breslow DK (2013) Sphingolipid homeostasis in the endoplasmic reticulum and beyond. Cold Spring Harb Perspect Biol 5: a013326Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Breslow DK, Collins SR, Bodenmiller B, Aebersold R, Simons K, Shevchenko A, Ejsing CS, Weissman JS (2010) Orm familyproteins mediate sphingolipid homeostasis. Nature 463: 1048-1053


Cacas JL, Bure C, Grosjean K, Gerbeau-Pissot P, Lherminier J, Rombouts Y, Maes E, Bossard C, Gronnier J, Furt F, Fouillen L,Germain V, Bayer E, Cluzet S, Robert F, Schmitter JM, Deleu M, Lins L, Simon-Plas F, Mongrand S (2016) Re-visiting plant plasmamembrane lipids in tobacco: a focus on sphingolipids. Plant Physiol 170: 367-384


Cacas JL, Furt F, Le Guedard M, Schmitter JM, Bure C, Gerbeau-Pissot P, Moreau P, Bessoule JJ, Simon-Plas F, Mongrand S(2012) Lipids of plant membrane rafts. Prog Lipid Res 51: 272-299


Chen M, Han G, Dietrich CR, Dunn TM, Cahoon EB (2006) The essential nature of sphingolipids in plants as revealed by thefunctional identification and characterization of the Arabidopsis LCB1 subunit of serine palmitoyltransferase. Plant Cell 18: 3576-3593


Chen M, Markham JE, Dietrich CR, Jaworski JG, Cahoon EB (2008) Sphingolipid long-chain base hydroxylation is important forgrowth and regulation of sphingolipid content and composition in Arabidopsis. Plant Cell 20: 1862-1878


Chueasiri C, Chunthong K, Pitnjam K, Chakhonkaen S, Sangarwut N, Sangsawang K, Suksangpanomrung M, Michaelson LV,Napier JA, Muangprom A (2014) Rice ORMDL controls sphingolipid homeostasis affecting fertility resulting from abnormal pollendevelopment. PloS one 9: e106386


Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. PlantJ 16: 735-743


Conn SJ, Hocking B, Dayod M, Xu B, Athman A, Henderson S, Aukett L, Conn V, Shearer MK, Fuentes S, Tyerman SD, Gilliham M www.plantphysiol.orgon May 21, 2018 - Published by Downloaded from


http://www.ncbi.nlm.nih.gov/pubmed?term=Bach L%2C Gissot L%2C Marion J%2C Tellier F%2C Moreau P%2C Satiat%2DJeunemaitre B%2C Palauqui JC%2C Napier JA%2C Faure JD %282011%29 Very%2Dlong%2Dchain fatty acids are required for cell plate formation during cytokinesis in Arabidopsis thaliana%2E J Cell Sci 124%3A 3223%2D3234&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Bach L, Gissot L, Marion J, Tellier F, Moreau P, Satiat-Jeunemaitre B, Palauqui JC, Napier JA, Faure JD (2011) Very-long-chain fatty acids are required for cell plate formation during cytokinesis in Arabidopsis thaliana. J Cell Sci 124: 3223-3234

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Bach&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Very-long-chain fatty acids are required for cell plate formation during cytokinesis in Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Very-long-chain fatty acids are required for cell plate formation during cytokinesis in Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Bach&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Bach L%2C Michaelson LV%2C Haslam R%2C Bellec Y%2C Gissot L%2C Marion J%2C Da Costa M%2C Boutin JP%2C Miquel M%2C Tellier F%2C Domergue F%2C Markham JE%2C Beaudoin F%2C Napier JA%2C Faure JD %282008%29 The very%2Dlong%2Dchain hydroxy fatty acyl%2DCoA dehydratase PASTICCINO2 is essential and limiting for plant development%2E Proc Natl Acad Sci USA 105%3A 14727%2D14731&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Bach L, Michaelson LV, Haslam R, Bellec Y, Gissot L, Marion J, Da Costa M, Boutin JP, Miquel M, Tellier F, Domergue F, Markham JE, Beaudoin F, Napier JA, Faure JD (2008) The very-long-chain hydroxy fatty acyl-CoA dehydratase PASTICCINO2 is essential and limiting for plant development. Proc Natl Acad Sci USA 105: 14727-14731

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Bach&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=The very-long-chain hydroxy fatty acyl-CoA dehydratase PASTICCINO2 is essential and limiting for plant development&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=The very-long-chain hydroxy fatty acyl-CoA dehydratase PASTICCINO2 is essential and limiting for plant development&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Bach&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Bayer EM%2C Mongrand S%2C Tilsner J %282014%29 Specialised membrane domains of plasmodesmata%2C plant intercellular nanopores%2E Frontiers in Plant Science 5%3A 507&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Bayer EM, Mongrand S, Tilsner J (2014) Specialised membrane domains of plasmodesmata, plant intercellular nanopores. Frontiers in Plant Science 5: 507

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Bayer&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Specialised membrane domains of plasmodesmata, plant intercellular nanopores.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Specialised membrane domains of plasmodesmata, plant intercellular nanopores.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Bayer&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Bi FC%2C Liu Z%2C Wu JX%2C Liang H%2C Xi XL%2C Fang C%2C Sun TJ%2C Yin J%2C Dai GY%2C Rong C%2C Greenberg JT%2C Su WW%2C Yao N %282014%29 Loss of ceramide kinase in Arabidopsis impairs defenses and promotes ceramide accumulation and mitochondrial H2O2 bursts%2E Plant Cell 26%3A 3449%2D3467&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Bi FC, Liu Z, Wu JX, Liang H, Xi XL, Fang C, Sun TJ, Yin J, Dai GY, Rong C, Greenberg JT, Su WW, Yao N (2014) Loss of ceramide kinase in Arabidopsis impairs defenses and promotes ceramide accumulation and mitochondrial H2O2 bursts. Plant Cell 26: 3449-3467

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Bi&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Loss of ceramide kinase in Arabidopsis impairs defenses and promotes ceramide accumulation and mitochondrial H2O2 bursts&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Loss of ceramide kinase in Arabidopsis impairs defenses and promotes ceramide accumulation and mitochondrial H2O2 bursts&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Bi&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Breslow DK %282013%29 Sphingolipid homeostasis in the endoplasmic reticulum and beyond%2E Cold Spring Harb Perspect Biol 5%3A a013326&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Breslow DK (2013) Sphingolipid homeostasis in the endoplasmic reticulum and beyond. Cold Spring Harb Perspect Biol 5: a013326

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Breslow&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Sphingolipid homeostasis in the endoplasmic reticulum and beyond.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Sphingolipid homeostasis in the endoplasmic reticulum and beyond.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Breslow&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Breslow DK%2C Collins SR%2C Bodenmiller B%2C Aebersold R%2C Simons K%2C Shevchenko A%2C Ejsing CS%2C Weissman JS %282010%29 Orm family proteins mediate sphingolipid homeostasis%2E Nature 463%3A 1048%2D1053&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Breslow DK, Collins SR, Bodenmiller B, Aebersold R, Simons K, Shevchenko A, Ejsing CS, Weissman JS (2010) Orm family proteins mediate sphingolipid homeostasis. Nature 463: 1048-1053

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Breslow&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Orm family proteins mediate sphingolipid homeostasis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Orm family proteins mediate sphingolipid homeostasis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Breslow&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Cacas JL%2C Bure C%2C Grosjean K%2C Gerbeau%2DPissot P%2C Lherminier J%2C Rombouts Y%2C Maes E%2C Bossard C%2C Gronnier J%2C Furt F%2C Fouillen L%2C Germain V%2C Bayer E%2C Cluzet S%2C Robert F%2C Schmitter JM%2C Deleu M%2C Lins L%2C Simon%2DPlas F%2C Mongrand S %282016%29 Re%2Dvisiting plant plasma membrane lipids in tobacco%3A a focus on sphingolipids%2E Plant Physiol 170%3A 367%2D384&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Cacas JL, Bure C, Grosjean K, Gerbeau-Pissot P, Lherminier J, Rombouts Y, Maes E, Bossard C, Gronnier J, Furt F, Fouillen L, Germain V, Bayer E, Cluzet S, Robert F, Schmitter JM, Deleu M, Lins L, Simon-Plas F, Mongrand S (2016) Re-visiting plant plasma membrane lipids in tobacco: a focus on sphingolipids. Plant Physiol 170: 367-384

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Cacas&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Re-visiting plant plasma membrane lipids in tobacco: a focus on sphingolipids&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Re-visiting plant plasma membrane lipids in tobacco: a focus on sphingolipids&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Cacas&as_ylo=2016&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Cacas JL%2C Furt F%2C Le Guedard M%2C Schmitter JM%2C Bure C%2C Gerbeau%2DPissot P%2C Moreau P%2C Bessoule JJ%2C Simon%2DPlas F%2C Mongrand S %282012%29 Lipids of plant membrane rafts%2E Prog Lipid Res 51%3A 272%2D299&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Cacas JL, Furt F, Le Guedard M, Schmitter JM, Bure C, Gerbeau-Pissot P, Moreau P, Bessoule JJ, Simon-Plas F, Mongrand S (2012) Lipids of plant membrane rafts. Prog Lipid Res 51: 272-299

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Cacas&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Lipids of plant membrane rafts&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Lipids of plant membrane rafts&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Cacas&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Chen M%2C Han G%2C Dietrich CR%2C Dunn TM%2C Cahoon EB %282006%29 The essential nature of sphingolipids in plants as revealed by the functional identification and characterization of the Arabidopsis LCB1 subunit of serine palmitoyltransferase%2E Plant Cell 18%3A 3576%2D3593&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Chen M, Han G, Dietrich CR, Dunn TM, Cahoon EB (2006) The essential nature of sphingolipids in plants as revealed by the functional identification and characterization of the Arabidopsis LCB1 subunit of serine palmitoyltransferase. Plant Cell 18: 3576-3593

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Chen&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=The essential nature of sphingolipids in plants as revealed by the functional identification and characterization of the Arabidopsis LCB1 subunit of serine palmitoyltransferase&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=The essential nature of sphingolipids in plants as revealed by the functional identification and characterization of the Arabidopsis LCB1 subunit of serine palmitoyltransferase&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Chen&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Chen M%2C Markham JE%2C Dietrich CR%2C Jaworski JG%2C Cahoon EB %282008%29 Sphingolipid long%2Dchain base hydroxylation is important for growth and regulation of sphingolipid content and composition in Arabidopsis%2E Plant Cell 20%3A 1862%2D1878&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Chen M, Markham JE, Dietrich CR, Jaworski JG, Cahoon EB (2008) Sphingolipid long-chain base hydroxylation is important for growth and regulation of sphingolipid content and composition in Arabidopsis. Plant Cell 20: 1862-1878

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Chen&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Sphingolipid long-chain base hydroxylation is important for growth and regulation of sphingolipid content and composition in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Sphingolipid long-chain base hydroxylation is important for growth and regulation of sphingolipid content and composition in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Chen&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Chueasiri C%2C Chunthong K%2C Pitnjam K%2C Chakhonkaen S%2C Sangarwut N%2C Sangsawang K%2C Suksangpanomrung M%2C Michaelson LV%2C Napier JA%2C Muangprom A %282014%29 Rice ORMDL controls sphingolipid homeostasis affecting fertility resulting from abnormal pollen development%2E PloS one 9%3A e106386&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Chueasiri C, Chunthong K, Pitnjam K, Chakhonkaen S, Sangarwut N, Sangsawang K, Suksangpanomrung M, Michaelson LV, Napier JA, Muangprom A (2014) Rice ORMDL controls sphingolipid homeostasis affecting fertility resulting from abnormal pollen development. PloS one 9: e106386

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Chueasiri&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Rice ORMDL controls sphingolipid homeostasis affecting fertility resulting from abnormal pollen development.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Rice ORMDL controls sphingolipid homeostasis affecting fertility resulting from abnormal pollen development.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Chueasiri&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Clough SJ%2C Bent AF %281998%29 Floral dip%3A a simplified method for Agrobacterium%2Dmediated transformation of Arabidopsis thaliana%2E Plant J 16%3A 735%2D743&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735-743

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Clough&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Clough&as_ylo=1998&as_allsubj=all&hl=en&c2coff=1


(2013) Protocol: optimising hydroponic growth systems for nutritional and physiological analysis of Arabidopsis thaliana and otherplants. Plant Methods 9: 4


Dietrich CR, Han G, Chen M, Berg RH, Dunn TM, Cahoon EB (2008) Loss-of-function mutations and inducible RNAi suppression ofArabidopsis LCB2 genes reveal the critical role of sphingolipids in gametophytic and sporophytic cell viability. Plant J 54: 284-298


Fresques T, Niles B, Aronova S, Mogri H, Rakhshandehroo T, Powers T (2015) Regulation of ceramide synthase by casein kinase2-dependent phosphorylation in Saccharomyces cerevisiae. J Biol Chem 290: 1395-1403


Gable K, Gupta SD, Han G, Niranjanakumari S, Harmon JM, Dunn TM (2010) A disease-causing mutation in the active site of serinepalmitoyltransferase causes catalytic promiscuity. J Biol Chem 285: 22846-22852


Greenberg JT, Silverman FP, Liang H (2000) Uncoupling salicylic acid-dependent cell death and defense-related responses fromdisease resistance in the arabidopsis mutant acd5. Genetics 156: 341-350


Gururaj C, Federman RS, Chang A (2013) Orm proteins integrate multiple signals to maintain sphingolipid homeostasis. J BiolChem 288: 20453-20463


Han G, Gable K, Yan L, Natarajan M, Krishnamurthy J, Gupta SD, Borovitskaya A, Harmon JM, Dunn TM (2004) The topology of theLcb1p subunit of yeast serine palmitoyltransferase. J Biol Chem 279: 53707-53716


Han G, Gupta SD, Gable K, Niranjanakumari S, Moitra P, Eichler F, Brown RH, Jr., Harmon JM, Dunn TM (2009) Identification ofsmall subunits of mammalian serine palmitoyltransferase that confer distinct acyl-CoA substrate specificities. Proc Natl Acad SciUSA 106: 8186-8191


Han S, Lone MA, Schneiter R, Chang A (2010) Orm1 and Orm2 are conserved endoplasmic reticulum membrane proteins regulatinglipid homeostasis and protein quality control. Proc Natl Acad Sci USA 107: 5851-5856


Hanada K (2003) Serine palmitoyltransferase, a key enzyme of sphingolipid metabolism. Biochim Biophys Acta 1632: 16-30Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Hirokawa T, Boon-Chieng S, Mitaku S (1998) SOSUI: classification and secondary structure prediction system for membraneproteins. Bioinformatics 14: 378-379


Hjelmqvist L, Tuson M, Marfany G, Herrero E, Balcells S, Gonzalez-Duarte R (2002) ORMDL proteins are a conserved new familyof endoplasmic reticulum membrane proteins. Genome Biology 3: RESEARCH0027


Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker inhigher plants. EMBO J 6: 3901-3907


Kimberlin AN, Majumder S, Han G, Chen M, Cahoon RE, Stone JM, Dunn TM, Cahoon EB (2013) Arabidopsis 56-amino acid serinepalmitoyltransferase-interacting proteins stimulate sphingolipid synthesis, are essential, and affect mycotoxin sensitivity. Plant


http://www.ncbi.nlm.nih.gov/pubmed?term=Conn SJ%2C Hocking B%2C Dayod M%2C Xu B%2C Athman A%2C Henderson S%2C Aukett L%2C Conn V%2C Shearer MK%2C Fuentes S%2C Tyerman SD%2C Gilliham M %282013%29 Protocol%3A optimising hydroponic growth systems for nutritional and physiological analysis of Arabidopsis thaliana and other plants%2E Plant Methods 9%3A 4&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Conn SJ, Hocking B, Dayod M, Xu B, Athman A, Henderson S, Aukett L, Conn V, Shearer MK, Fuentes S, Tyerman SD, Gilliham M (2013) Protocol: optimising hydroponic growth systems for nutritional and physiological analysis of Arabidopsis thaliana and other plants. Plant Methods 9: 4

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Conn&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Protocol: optimising hydroponic growth systems for nutritional and physiological analysis of Arabidopsis thaliana and other plants.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Protocol: optimising hydroponic growth systems for nutritional and physiological analysis of Arabidopsis thaliana and other plants.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Conn&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Dietrich CR%2C Han G%2C Chen M%2C Berg RH%2C Dunn TM%2C Cahoon EB %282008%29 Loss%2Dof%2Dfunction mutations and inducible RNAi suppression of Arabidopsis LCB2 genes reveal the critical role of sphingolipids in gametophytic and sporophytic cell viability%2E Plant J 54%3A 284%2D298&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Dietrich CR, Han G, Chen M, Berg RH, Dunn TM, Cahoon EB (2008) Loss-of-function mutations and inducible RNAi suppression of Arabidopsis LCB2 genes reveal the critical role of sphingolipids in gametophytic and sporophytic cell viability. Plant J 54: 284-298

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Dietrich&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Loss-of-function mutations and inducible RNAi suppression of Arabidopsis LCB2 genes reveal the critical role of sphingolipids in gametophytic and sporophytic cell viability&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Loss-of-function mutations and inducible RNAi suppression of Arabidopsis LCB2 genes reveal the critical role of sphingolipids in gametophytic and sporophytic cell viability&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Dietrich&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Fresques T%2C Niles B%2C Aronova S%2C Mogri H%2C Rakhshandehroo T%2C Powers T %282015%29 Regulation of ceramide synthase by casein kinase 2%2Ddependent phosphorylation in Saccharomyces cerevisiae%2E J Biol Chem 290%3A 1395%2D1403&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Fresques T, Niles B, Aronova S, Mogri H, Rakhshandehroo T, Powers T (2015) Regulation of ceramide synthase by casein kinase 2-dependent phosphorylation in Saccharomyces cerevisiae. J Biol Chem 290: 1395-1403

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Fresques&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Regulation of ceramide synthase by casein kinase 2-dependent phosphorylation in Saccharomyces cerevisiae&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Regulation of ceramide synthase by casein kinase 2-dependent phosphorylation in Saccharomyces cerevisiae&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Fresques&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Gable K%2C Gupta SD%2C Han G%2C Niranjanakumari S%2C Harmon JM%2C Dunn TM %282010%29 A disease%2Dcausing mutation in the active site of serine palmitoyltransferase causes catalytic promiscuity%2E J Biol Chem 285%3A 22846%2D22852&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Gable K, Gupta SD, Han G, Niranjanakumari S, Harmon JM, Dunn TM (2010) A disease-causing mutation in the active site of serine palmitoyltransferase causes catalytic promiscuity. J Biol Chem 285: 22846-22852

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Gable&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=A disease-causing mutation in the active site of serine palmitoyltransferase causes catalytic promiscuity&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=A disease-causing mutation in the active site of serine palmitoyltransferase causes catalytic promiscuity&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Gable&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Greenberg JT%2C Silverman FP%2C Liang H %282000%29 Uncoupling salicylic acid%2Ddependent cell death and defense%2Drelated responses from disease resistance in the arabidopsis mutant acd5%2E Genetics 156%3A 341%2D350&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Greenberg JT, Silverman FP, Liang H (2000) Uncoupling salicylic acid-dependent cell death and defense-related responses from disease resistance in the arabidopsis mutant acd5. Genetics 156: 341-350

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Greenberg&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Uncoupling salicylic acid-dependent cell death and defense-related responses from disease resistance in the arabidopsis mutant acd5&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Uncoupling salicylic acid-dependent cell death and defense-related responses from disease resistance in the arabidopsis mutant acd5&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Greenberg&as_ylo=2000&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Gururaj C%2C Federman RS%2C Chang A %282013%29 Orm proteins integrate multiple signals to maintain sphingolipid homeostasis%2E J Biol Chem 288%3A 20453%2D20463&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Gururaj C, Federman RS, Chang A (2013) Orm proteins integrate multiple signals to maintain sphingolipid homeostasis. J Biol Chem 288: 20453-20463

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Gururaj&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Orm proteins integrate multiple signals to maintain sphingolipid homeostasis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Orm proteins integrate multiple signals to maintain sphingolipid homeostasis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Gururaj&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Han G%2C Gable K%2C Yan L%2C Natarajan M%2C Krishnamurthy J%2C Gupta SD%2C Borovitskaya A%2C Harmon JM%2C Dunn TM %282004%29 The topology of the Lcb1p subunit of yeast serine palmitoyltransferase%2E J Biol Chem 279%3A 53707%2D53716&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Han G, Gable K, Yan L, Natarajan M, Krishnamurthy J, Gupta SD, Borovitskaya A, Harmon JM, Dunn TM (2004) The topology of the Lcb1p subunit of yeast serine palmitoyltransferase. J Biol Chem 279: 53707-53716

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Han&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=The topology of the Lcb1p subunit of yeast serine palmitoyltransferase&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=The topology of the Lcb1p subunit of yeast serine palmitoyltransferase&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Han&as_ylo=2004&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Han G%2C Gupta SD%2C Gable K%2C Niranjanakumari S%2C Moitra P%2C Eichler F%2C Brown RH%2C Jr%2E%2C Harmon JM%2C Dunn TM %282009%29 Identification of small subunits of mammalian serine palmitoyltransferase that confer distinct acyl%2DCoA substrate specificities%2E Proc Natl Acad Sci USA 106%3A 8186%2D8191&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Han G, Gupta SD, Gable K, Niranjanakumari S, Moitra P, Eichler F, Brown RH, Jr., Harmon JM, Dunn TM (2009) Identification of small subunits of mammalian serine palmitoyltransferase that confer distinct acyl-CoA substrate specificities. Proc Natl Acad Sci USA 106: 8186-8191


http://scholar.google.com/scholar?as_q=Identification of small subunits of mammalian serine palmitoyltransferase that confer distinct acyl-CoA substrate specificities&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Identification of small subunits of mammalian serine palmitoyltransferase that confer distinct acyl-CoA substrate specificities&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Han&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Han S%2C Lone MA%2C Schneiter R%2C Chang A %282010%29 Orm1 and Orm2 are conserved endoplasmic reticulum membrane proteins regulating lipid homeostasis and protein quality control%2E Proc Natl Acad Sci USA 107%3A 5851%2D5856&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Han S, Lone MA, Schneiter R, Chang A (2010) Orm1 and Orm2 are conserved endoplasmic reticulum membrane proteins regulating lipid homeostasis and protein quality control. Proc Natl Acad Sci USA 107: 5851-5856


http://scholar.google.com/scholar?as_q=Orm1 and Orm2 are conserved endoplasmic reticulum membrane proteins regulating lipid homeostasis and protein quality control&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Orm1 and Orm2 are conserved endoplasmic reticulum membrane proteins regulating lipid homeostasis and protein quality control&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Han&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Hanada K %282003%29 Serine palmitoyltransferase%2C a key enzyme of sphingolipid metabolism%2E Biochim Biophys Acta 1632%3A 16%2D30&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Hanada K (2003) Serine palmitoyltransferase, a key enzyme of sphingolipid metabolism. Biochim Biophys Acta 1632: 16-30

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Hanada&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Serine palmitoyltransferase, a key enzyme of sphingolipid metabolism&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Serine palmitoyltransferase, a key enzyme of sphingolipid metabolism&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Hanada&as_ylo=2003&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Hirokawa T%2C Boon%2DChieng S%2C Mitaku S %281998%29 SOSUI%3A classification and secondary structure prediction system for membrane proteins%2E Bioinformatics 14%3A 378%2D379&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Hirokawa T, Boon-Chieng S, Mitaku S (1998) SOSUI: classification and secondary structure prediction system for membrane proteins. Bioinformatics 14: 378-379

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Hirokawa&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=SOSUI: classification and secondary structure prediction system for membrane proteins&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=SOSUI: classification and secondary structure prediction system for membrane proteins&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Hirokawa&as_ylo=1998&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Hjelmqvist L%2C Tuson M%2C Marfany G%2C Herrero E%2C Balcells S%2C Gonzalez%2DDuarte R %282002%29 ORMDL proteins are a conserved new family of endoplasmic reticulum membrane proteins%2E Genome Biology 3%3A RESEARCH0027&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Hjelmqvist L, Tuson M, Marfany G, Herrero E, Balcells S, Gonzalez-Duarte R (2002) ORMDL proteins are a conserved new family of endoplasmic reticulum membrane proteins. Genome Biology 3: RESEARCH0027

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Hjelmqvist&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=ORMDL proteins are a conserved new family of endoplasmic reticulum membrane proteins.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=ORMDL proteins are a conserved new family of endoplasmic reticulum membrane proteins.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Hjelmqvist&as_ylo=2002&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Jefferson RA%2C Kavanagh TA%2C Bevan MW %281987%29 GUS fusions%3A beta%2Dglucuronidase as a sensitive and versatile gene fusion marker in higher plants%2E EMBO J 6%3A 3901%2D3907&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901-3907

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Jefferson&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Jefferson&as_ylo=1987&as_allsubj=all&hl=en&c2coff=1


Cell 25: 4627-4639Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Kohlwein SD, Eder S, Oh CS, Martin CE, Gable K, Bacikova D, Dunn T (2001) Tsc13p is required for fatty acid elongation andlocalizes to a novel structure at the nuclear-vacuolar interface in Saccharomyces cerevisiae. Mol Cell Biol 21: 109-125


Liang H, Yao N, Song JT, Luo S, Lu H, Greenberg JT (2003) Ceramides modulate programmed cell death in plants. Genes &Development 17: 2636-2641


Luttgeharm KD, Cahoon EB, Markham JE (2015) A mass spectrometry-based method for the assay of ceramide synthase substratespecificity. Anal Biochem 478: 96-101


Luttgeharm KD, Cahoon EB, Markham JE (2016) Substrate specificity, kinetic properties and inhibition by fumonisin B1 ofceramide synthase isoforms from Arabidopsis. Biochem J 473: 593-603


Luttgeharm KD, Chen M, Mehra A, Cahoon RE, Markham JE, Cahoon EB (2015) Overexpression of Arabidopsis ceramidesynthases differentially affects growth, sphingolipid metabolism, programmed cell death, and mycotoxin resistance. Plant Physiol169: 1108-1117


Lynch DV, Fairfield SR (1993) Sphingolipid long-chain base synthesis in plants: characterization of serine palmitoyltransferaseactivity in squash fruit microsomes. Plant Physiol 103: 1421-1429


Markham JE, Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom21: 1304-1314


Markham JE, Lynch DV, Napier JA, Dunn TM, Cahoon EB (2013) Plant sphingolipids: function follows form. Curr Opin Plant Biol 16:350-357


Markham JE, Molino D, Gissot L, Bellec Y, Hematy K, Marion J, Belcram K, Palauqui JC, Satiat-Jeunemaitre B, Faure JD (2011)Sphingolipids containing very-long-chain fatty acids define a secretory pathway for specific polar plasma membrane proteintargeting in Arabidopsis. Plant Cell 23: 2362-2378


Melser S, Molino D, Batailler B, Peypelut M, Laloi M, Wattelet-Boyer V, Bellec Y, Faure JD, Moreau P (2011) Links between lipidhomeostasis, organelle morphodynamics and protein trafficking in eukaryotic and plant secretory pathways. Plant Cell Rep 30: 177-193


Msanne J, Chen M, Luttgeharm KD, Bradley AM, Mays ES, Paper JM, Boyle DL, Cahoon RE, Schrick K, Cahoon EB (2015)Glucosylceramides are critical for cell-type differentiation and organogenesis, but not for cell viability in Arabidopsis. Plant J 84:188-201


Muir A, Ramachandran S, Roelants FM, Timmons G, Thorner J (2014) TORC2-dependent protein kinase Ypk1 phosphorylatesceramide synthase to stimulate synthesis of complex sphingolipids. Elife 3

Pubmed: Author and TitleCrossRef: Author and Title www.plantphysiol.orgon May 21, 2018 - Published by Downloaded from


http://www.ncbi.nlm.nih.gov/pubmed?term=Kimberlin AN%2C Majumder S%2C Han G%2C Chen M%2C Cahoon RE%2C Stone JM%2C Dunn TM%2C Cahoon EB %282013%29 Arabidopsis 56%2Damino acid serine palmitoyltransferase%2Dinteracting proteins stimulate sphingolipid synthesis%2C are essential%2C and affect mycotoxin sensitivity%2E Plant Cell 25%3A 4627%2D4639&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Kimberlin AN, Majumder S, Han G, Chen M, Cahoon RE, Stone JM, Dunn TM, Cahoon EB (2013) Arabidopsis 56-amino acid serine palmitoyltransferase-interacting proteins stimulate sphingolipid synthesis, are essential, and affect mycotoxin sensitivity. Plant Cell 25: 4627-4639

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Kimberlin&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Arabidopsis 56-amino acid serine palmitoyltransferase-interacting proteins stimulate sphingolipid synthesis, are essential, and affect mycotoxin sensitivity&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Arabidopsis 56-amino acid serine palmitoyltransferase-interacting proteins stimulate sphingolipid synthesis, are essential, and affect mycotoxin sensitivity&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kimberlin&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Kohlwein SD%2C Eder S%2C Oh CS%2C Martin CE%2C Gable K%2C Bacikova D%2C Dunn T %282001%29 Tsc13p is required for fatty acid elongation and localizes to a novel structure at the nuclear%2Dvacuolar interface in Saccharomyces cerevisiae%2E Mol Cell Biol 21%3A 109%2D125&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Kohlwein SD, Eder S, Oh CS, Martin CE, Gable K, Bacikova D, Dunn T (2001) Tsc13p is required for fatty acid elongation and localizes to a novel structure at the nuclear-vacuolar interface in Saccharomyces cerevisiae. Mol Cell Biol 21: 109-125

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Kohlwein&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Tsc13p is required for fatty acid elongation and localizes to a novel structure at the nuclear-vacuolar interface in Saccharomyces cerevisiae&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Tsc13p is required for fatty acid elongation and localizes to a novel structure at the nuclear-vacuolar interface in Saccharomyces cerevisiae&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kohlwein&as_ylo=2001&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Liang H%2C Yao N%2C Song JT%2C Luo S%2C Lu H%2C Greenberg JT %282003%29 Ceramides modulate programmed cell death in plants%2E Genes %26 Development 17%3A 2636%2D2641&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Liang H, Yao N, Song JT, Luo S, Lu H, Greenberg JT (2003) Ceramides modulate programmed cell death in plants. Genes & Development 17: 2636-2641

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Liang&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Ceramides modulate programmed cell death in plants&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Ceramides modulate programmed cell death in plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Liang&as_ylo=2003&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Luttgeharm KD%2C Cahoon EB%2C Markham JE %282015%29 A mass spectrometry%2Dbased method for the assay of ceramide synthase substrate specificity%2E Anal Biochem 478%3A 96%2D101&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Luttgeharm KD, Cahoon EB, Markham JE (2015) A mass spectrometry-based method for the assay of ceramide synthase substrate specificity. Anal Biochem 478: 96-101

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Luttgeharm&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=A mass spectrometry-based method for the assay of ceramide synthase substrate specificity&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=A mass spectrometry-based method for the assay of ceramide synthase substrate specificity&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Luttgeharm&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Luttgeharm KD%2C Cahoon EB%2C Markham JE %282016%29 Substrate specificity%2C kinetic properties and inhibition by fumonisin B1 of ceramide synthase isoforms from Arabidopsis%2E Biochem J 473%3A 593%2D603&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Luttgeharm KD, Cahoon EB, Markham JE (2016) Substrate specificity, kinetic properties and inhibition by fumonisin B1 of ceramide synthase isoforms from Arabidopsis. Biochem J 473: 593-603


http://scholar.google.com/scholar?as_q=Substrate specificity, kinetic properties and inhibition by fumonisin B1 of ceramide synthase isoforms from Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Substrate specificity, kinetic properties and inhibition by fumonisin B1 of ceramide synthase isoforms from Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Luttgeharm&as_ylo=2016&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Luttgeharm KD%2C Chen M%2C Mehra A%2C Cahoon RE%2C Markham JE%2C Cahoon EB %282015%29 Overexpression of Arabidopsis ceramide synthases differentially affects growth%2C sphingolipid metabolism%2C programmed cell death%2C and mycotoxin resistance%2E Plant Physiol 169%3A 1108%2D1117&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Luttgeharm KD, Chen M, Mehra A, Cahoon RE, Markham JE, Cahoon EB (2015) Overexpression of Arabidopsis ceramide synthases differentially affects growth, sphingolipid metabolism, programmed cell death, and mycotoxin resistance. Plant Physiol 169: 1108-1117


http://scholar.google.com/scholar?as_q=Overexpression of Arabidopsis ceramide synthases differentially affects growth, sphingolipid metabolism, programmed cell death, and mycotoxin resistance&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Overexpression of Arabidopsis ceramide synthases differentially affects growth, sphingolipid metabolism, programmed cell death, and mycotoxin resistance&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Luttgeharm&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Lynch DV%2C Fairfield SR %281993%29 Sphingolipid long%2Dchain base synthesis in plants%3A characterization of serine palmitoyltransferase activity in squash fruit microsomes%2E Plant Physiol 103%3A 1421%2D1429&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Lynch DV, Fairfield SR (1993) Sphingolipid long-chain base synthesis in plants: characterization of serine palmitoyltransferase activity in squash fruit microsomes. Plant Physiol 103: 1421-1429

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Lynch&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Sphingolipid long-chain base synthesis in plants: characterization of serine palmitoyltransferase activity in squash fruit microsomes&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Sphingolipid long-chain base synthesis in plants: characterization of serine palmitoyltransferase activity in squash fruit microsomes&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Lynch&as_ylo=1993&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Markham JE%2C Jaworski JG %282007%29 Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed%2Dphase high%2Dperformance liquid chromatography coupled to electrospray ionization tandem mass spectrometry%2E Rapid Commun Mass Spectrom 21%3A 1304%2D1314&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Markham JE, Jaworski JG (2007) Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom 21: 1304-1314

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Markham&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Markham&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Markham JE%2C Lynch DV%2C Napier JA%2C Dunn TM%2C Cahoon EB %282013%29 Plant sphingolipids%3A function follows form%2E Curr Opin Plant Biol 16%3A 350%2D357&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Markham JE, Lynch DV, Napier JA, Dunn TM, Cahoon EB (2013) Plant sphingolipids: function follows form. Curr Opin Plant Biol 16: 350-357


http://scholar.google.com/scholar?as_q=Plant sphingolipids: function follows form&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Plant sphingolipids: function follows form&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Markham&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Markham JE%2C Molino D%2C Gissot L%2C Bellec Y%2C Hematy K%2C Marion J%2C Belcram K%2C Palauqui JC%2C Satiat%2DJeunemaitre B%2C Faure JD %282011%29 Sphingolipids containing very%2Dlong%2Dchain fatty acids define a secretory pathway for specific polar plasma membrane protein targeting in Arabidopsis%2E Plant Cell 23%3A 2362%2D2378&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Markham JE, Molino D, Gissot L, Bellec Y, Hematy K, Marion J, Belcram K, Palauqui JC, Satiat-Jeunemaitre B, Faure JD (2011) Sphingolipids containing very-long-chain fatty acids define a secretory pathway for specific polar plasma membrane protein targeting in Arabidopsis. Plant Cell 23: 2362-2378


http://scholar.google.com/scholar?as_q=Sphingolipids containing very-long-chain fatty acids define a secretory pathway for specific polar plasma membrane protein targeting in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Sphingolipids containing very-long-chain fatty acids define a secretory pathway for specific polar plasma membrane protein targeting in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Markham&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Melser S%2C Molino D%2C Batailler B%2C Peypelut M%2C Laloi M%2C Wattelet%2DBoyer V%2C Bellec Y%2C Faure JD%2C Moreau P %282011%29 Links between lipid homeostasis%2C organelle morphodynamics and protein trafficking in eukaryotic and plant secretory pathways%2E Plant Cell Rep 30%3A 177%2D193&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Melser S, Molino D, Batailler B, Peypelut M, Laloi M, Wattelet-Boyer V, Bellec Y, Faure JD, Moreau P (2011) Links between lipid homeostasis, organelle morphodynamics and protein trafficking in eukaryotic and plant secretory pathways. Plant Cell Rep 30: 177-193

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Melser&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Links between lipid homeostasis, organelle morphodynamics and protein trafficking in eukaryotic and plant secretory pathways&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Links between lipid homeostasis, organelle morphodynamics and protein trafficking in eukaryotic and plant secretory pathways&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Melser&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Msanne J%2C Chen M%2C Luttgeharm KD%2C Bradley AM%2C Mays ES%2C Paper JM%2C Boyle DL%2C Cahoon RE%2C Schrick K%2C Cahoon EB %282015%29 Glucosylceramides are critical for cell%2Dtype differentiation and organogenesis%2C but not for cell viability in Arabidopsis%2E Plant J 84%3A 188%2D201&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Msanne J, Chen M, Luttgeharm KD, Bradley AM, Mays ES, Paper JM, Boyle DL, Cahoon RE, Schrick K, Cahoon EB (2015) Glucosylceramides are critical for cell-type differentiation and organogenesis, but not for cell viability in Arabidopsis. Plant J 84: 188-201

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Msanne&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Glucosylceramides are critical for cell-type differentiation and organogenesis, but not for cell viability in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Glucosylceramides are critical for cell-type differentiation and organogenesis, but not for cell viability in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Msanne&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Muir A%2C Ramachandran S%2C Roelants FM%2C Timmons G%2C Thorner J %282014%29 TORC2%2Ddependent protein kinase Ypk1 phosphorylates ceramide synthase to stimulate synthesis of complex sphingolipids%2E Elife 3&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Muir A, Ramachandran S, Roelants FM, Timmons G, Thorner J (2014) TORC2-dependent protein kinase Ypk1 phosphorylates ceramide synthase to stimulate synthesis of complex sphingolipids. Elife 3


Google Scholar: Author Only Title Only Author and Title

Mullen TD, Hannun YA, Obeid LM (2012) Ceramide synthases at the centre of sphingolipid metabolism and biology. Biochem J 441:789-802


Nühse TS, Stensballe A, Jensen ON, Peck SC (2004) Phosphoproteomics of the Arabidopsis plasma membrane and a newphosphorylation site database. Plant Cell 16: 2394-2405


Perraki A, Cacas JL, Crowet JM, Lins L, Castroviejo M, German-Retana S, Mongrand S, Raffaele S (2012) Plasma membranelocalization of Solanum tuberosum remorin from group 1, homolog 3 is mediated by conformational changes in a novel C-terminalanchor and required for the restriction of potato virus X movement. Plant Physiol 160: 624-637


Roelants FM, Breslow DK, Muir A, Weissman JS, Thorner J (2011) Protein kinase Ypk1 phosphorylates regulatory proteins Orm1and Orm2 to control sphingolipid homeostasis in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 108: 19222-19227


Saucedo-Garcia M, Guevara-Garcia A, Gonzalez-Solis A, Cruz-Garcia F, Vazquez-Santana S, Markham JE, Lozano-Rosas MG,Dietrich CR, Ramos-Vega M, Cahoon EB, Gavilanes-Ruiz M (2011) MPK6, sphinganine and the LCB2a gene from serinepalmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in Arabidopsis.New Phytol 191: 943-957


Shi L, Bielawski J, Mu J, Dong H, Teng C, Zhang J, Yang X, Tomishige N, Hanada K, Hannun YA, Zuo J (2007) Involvement ofsphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis. Cell Research17: 1030-1040


Simanshu DK, Zhai X, Munch D, Hofius D, Markham JE, Bielawski J, Bielawska A, Malinina L, Molotkovsky JG, Mundy JW, Patel DJ,Brown RE (2014) Arabidopsis accelerated cell death 11, ACD11, is a ceramide-1-phosphate transfer protein and intermediaryregulator of phytoceramide levels. Cell Rep 6: 388-399


Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985)Measurement of protein using bicinchoninic acid. Anal Biochem 150: 76-85


Sperling P, Franke S, Luthje S, Heinz E (2005) Are glucocerebrosides the predominant sphingolipids in plant plasma membranes?Plant Physiol Biochem 43: 1031-1038


Stiban J, Tidhar R, Futerman AH (2010) Ceramide synthases: roles in cell physiology and signaling. Adv Exp Med Biol 688: 60-71Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Sugiyama N, Nakagami H, Mochida K, Daudi A, Tomita M, Shirasu K, Ishihama Y (2008) Large-scale phosphorylation mappingreveals the extent of tyrosine phosphorylation in Arabidopsis. Mol Syst Biol 4: 193


Sun Y, Miao Y, Yamane Y, Zhang C, Shokat KM, Takematsu H, Kozutsumi Y, Drubin DG (2012) Orm protein phosphoregulationmediates transient sphingolipid biosynthesis response to heat stress via the Pkh-Ypk and Cdc55-PP2A pathways. Mol Biol Cell 23:2388-2398


Teng C, Dong H, Shi L, Deng Y, Mu J, Zhang J, Yang X, Zuo J (2008) Serine palmitoyltransferase, a key enzyme for de novo www.plantphysiol.orgon May 21, 2018 - Published by Downloaded from


http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Muir&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=TORC2-dependent protein kinase Ypk1 phosphorylates ceramide synthase to stimulate synthesis of complex sphingolipids.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=TORC2-dependent protein kinase Ypk1 phosphorylates ceramide synthase to stimulate synthesis of complex sphingolipids.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Muir&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Mullen TD%2C Hannun YA%2C Obeid LM %282012%29 Ceramide synthases at the centre of sphingolipid metabolism and biology%2E Biochem J 441%3A 789%2D802&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Mullen TD, Hannun YA, Obeid LM (2012) Ceramide synthases at the centre of sphingolipid metabolism and biology. Biochem J 441: 789-802

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Mullen&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Ceramide synthases at the centre of sphingolipid metabolism and biology&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Ceramide synthases at the centre of sphingolipid metabolism and biology&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Mullen&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=N�hse TS%2C Stensballe A%2C Jensen ON%2C Peck SC %282004%29 Phosphoproteomics of the Arabidopsis plasma membrane and a new phosphorylation site database%2E Plant Cell 16%3A 2394%2D2405&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=N�hse TS, Stensballe A, Jensen ON, Peck SC (2004) Phosphoproteomics of the Arabidopsis plasma membrane and a new phosphorylation site database. Plant Cell 16: 2394-2405

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=N�hse&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Phosphoproteomics of the Arabidopsis plasma membrane and a new phosphorylation site database&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Phosphoproteomics of the Arabidopsis plasma membrane and a new phosphorylation site database&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=N�hse&as_ylo=2004&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Perraki A%2C Cacas JL%2C Crowet JM%2C Lins L%2C Castroviejo M%2C German%2DRetana S%2C Mongrand S%2C Raffaele S %282012%29 Plasma membrane localization of Solanum tuberosum remorin from group 1%2C homolog 3 is mediated by conformational changes in a novel C%2Dterminal anchor and required for the restriction of potato virus X movement%2E Plant Physiol 160%3A 624%2D637&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Perraki A, Cacas JL, Crowet JM, Lins L, Castroviejo M, German-Retana S, Mongrand S, Raffaele S (2012) Plasma membrane localization of Solanum tuberosum remorin from group 1, homolog 3 is mediated by conformational changes in a novel C-terminal anchor and required for the restriction of potato virus X movement. Plant Physiol 160: 624-637

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Perraki&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Perraki&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Roelants FM%2C Breslow DK%2C Muir A%2C Weissman JS%2C Thorner J %282011%29 Protein kinase Ypk1 phosphorylates regulatory proteins Orm1 and Orm2 to control sphingolipid homeostasis in Saccharomyces cerevisiae%2E Proc Natl Acad Sci U S A 108%3A 19222%2D19227&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Roelants FM, Breslow DK, Muir A, Weissman JS, Thorner J (2011) Protein kinase Ypk1 phosphorylates regulatory proteins Orm1 and Orm2 to control sphingolipid homeostasis in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 108: 19222-19227

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Roelants&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Protein kinase Ypk1 phosphorylates regulatory proteins Orm1 and Orm2 to control sphingolipid homeostasis in Saccharomyces cerevisiae&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Protein kinase Ypk1 phosphorylates regulatory proteins Orm1 and Orm2 to control sphingolipid homeostasis in Saccharomyces cerevisiae&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Roelants&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Saucedo%2DGarcia M%2C Guevara%2DGarcia A%2C Gonzalez%2DSolis A%2C Cruz%2DGarcia F%2C Vazquez%2DSantana S%2C Markham JE%2C Lozano%2DRosas MG%2C Dietrich CR%2C Ramos%2DVega M%2C Cahoon EB%2C Gavilanes%2DRuiz M %282011%29 MPK6%2C sphinganine and the LCB2a gene from serine palmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in Arabidopsis%2E New Phytol 191%3A 943%2D957&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Saucedo-Garcia M, Guevara-Garcia A, Gonzalez-Solis A, Cruz-Garcia F, Vazquez-Santana S, Markham JE, Lozano-Rosas MG, Dietrich CR, Ramos-Vega M, Cahoon EB, Gavilanes-Ruiz M (2011) MPK6, sphinganine and the LCB2a gene from serine palmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in Arabidopsis. New Phytol 191: 943-957

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Saucedo-Garcia&hl=en&c2coff=1


http://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Saucedo-Garcia&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Shi L%2C Bielawski J%2C Mu J%2C Dong H%2C Teng C%2C Zhang J%2C Yang X%2C Tomishige N%2C Hanada K%2C Hannun YA%2C Zuo J %282007%29 Involvement of sphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis%2E Cell Research 17%3A 1030%2D1040&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Shi L, Bielawski J, Mu J, Dong H, Teng C, Zhang J, Yang X, Tomishige N, Hanada K, Hannun YA, Zuo J (2007) Involvement of sphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis. Cell Research 17: 1030-1040

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Shi&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Involvement of sphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Involvement of sphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Shi&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Simanshu DK%2C Zhai X%2C Munch D%2C Hofius D%2C Markham JE%2C Bielawski J%2C Bielawska A%2C Malinina L%2C Molotkovsky JG%2C Mundy JW%2C Patel DJ%2C Brown RE %282014%29 Arabidopsis accelerated cell death 11%2C ACD11%2C is a ceramide%2D1%2Dphosphate transfer protein and intermediary regulator of phytoceramide levels%2E Cell Rep 6%3A 388%2D399&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Simanshu DK, Zhai X, Munch D, Hofius D, Markham JE, Bielawski J, Bielawska A, Malinina L, Molotkovsky JG, Mundy JW, Patel DJ, Brown RE (2014) Arabidopsis accelerated cell death 11, ACD11, is a ceramide-1-phosphate transfer protein and intermediary regulator of phytoceramide levels. Cell Rep 6: 388-399

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Simanshu&hl=en&c2coff=1


http://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Simanshu&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Smith PK%2C Krohn RI%2C Hermanson GT%2C Mallia AK%2C Gartner FH%2C Provenzano MD%2C Fujimoto EK%2C Goeke NM%2C Olson BJ%2C Klenk DC %281985%29 Measurement of protein using bicinchoninic acid%2E Anal Biochem 150%3A 76%2D85&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150: 76-85

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Smith&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Measurement of protein using bicinchoninic acid&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Measurement of protein using bicinchoninic acid&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Smith&as_ylo=1985&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Sperling P%2C Franke S%2C Luthje S%2C Heinz E %282005%29 Are glucocerebrosides the predominant sphingolipids in plant plasma membranes%3F Plant Physiol Biochem 43%3A 1031%2D1038&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Sperling P, Franke S, Luthje S, Heinz E (2005) Are glucocerebrosides the predominant sphingolipids in plant plasma membranes? Plant Physiol Biochem 43: 1031-1038

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Sperling&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Are glucocerebrosides the predominant sphingolipids in plant plasma membranes&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Are glucocerebrosides the predominant sphingolipids in plant plasma membranes&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Sperling&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Stiban J%2C Tidhar R%2C Futerman AH %282010%29 Ceramide synthases%3A roles in cell physiology and signaling%2E Adv Exp Med Biol 688%3A 60%2D71&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Stiban J, Tidhar R, Futerman AH (2010) Ceramide synthases: roles in cell physiology and signaling. Adv Exp Med Biol 688: 60-71

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Stiban&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Ceramide synthases: roles in cell physiology and signaling&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Ceramide synthases: roles in cell physiology and signaling&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Stiban&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Sugiyama N%2C Nakagami H%2C Mochida K%2C Daudi A%2C Tomita M%2C Shirasu K%2C Ishihama Y %282008%29 Large%2Dscale phosphorylation mapping reveals the extent of tyrosine phosphorylation in Arabidopsis%2E Mol Syst Biol 4%3A 193&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Sugiyama N, Nakagami H, Mochida K, Daudi A, Tomita M, Shirasu K, Ishihama Y (2008) Large-scale phosphorylation mapping reveals the extent of tyrosine phosphorylation in Arabidopsis. Mol Syst Biol 4: 193

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Sugiyama&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Large-scale phosphorylation mapping reveals the extent of tyrosine phosphorylation in Arabidopsis.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Large-scale phosphorylation mapping reveals the extent of tyrosine phosphorylation in Arabidopsis.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Sugiyama&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Sun Y%2C Miao Y%2C Yamane Y%2C Zhang C%2C Shokat KM%2C Takematsu H%2C Kozutsumi Y%2C Drubin DG %282012%29 Orm protein phosphoregulation mediates transient sphingolipid biosynthesis response to heat stress via the Pkh%2DYpk and Cdc55%2DPP2A pathways%2E Mol Biol Cell 23%3A 2388%2D2398&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Sun Y, Miao Y, Yamane Y, Zhang C, Shokat KM, Takematsu H, Kozutsumi Y, Drubin DG (2012) Orm protein phosphoregulation mediates transient sphingolipid biosynthesis response to heat stress via the Pkh-Ypk and Cdc55-PP2A pathways. Mol Biol Cell 23: 2388-2398

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Sun&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Orm protein phosphoregulation mediates transient sphingolipid biosynthesis response to heat stress via the Pkh-Ypk and Cdc55-PP2A pathways&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Orm protein phosphoregulation mediates transient sphingolipid biosynthesis response to heat stress via the Pkh-Ypk and Cdc55-PP2A pathways&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Sun&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1


synthesis of sphingolipids, is essential for male gametophyte development in Arabidopsis. Plant Physiol 146: 1322-1332Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Ternes P, Feussner K, Werner S, Lerche J, Iven T, Heilmann I, Riezman H, Feussner I (2011) Disruption of the ceramide synthaseLOH1 causes spontaneous cell death in Arabidopsis thaliana. The New phytologist 192: 841-854


von Heijne G (1992) Membrane protein structure prediction. Hydrophobicity analysis and the positive-inside rule. J Mol Biol 225:487-494



http://www.ncbi.nlm.nih.gov/pubmed?term=Teng C%2C Dong H%2C Shi L%2C Deng Y%2C Mu J%2C Zhang J%2C Yang X%2C Zuo J %282008%29 Serine palmitoyltransferase%2C a key enzyme for de novo synthesis of sphingolipids%2C is essential for male gametophyte development in Arabidopsis%2E Plant Physiol 146%3A 1322%2D1332&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Teng C, Dong H, Shi L, Deng Y, Mu J, Zhang J, Yang X, Zuo J (2008) Serine palmitoyltransferase, a key enzyme for de novo synthesis of sphingolipids, is essential for male gametophyte development in Arabidopsis. Plant Physiol 146: 1322-1332

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Teng&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Serine palmitoyltransferase, a key enzyme for de novo synthesis of sphingolipids, is essential for male gametophyte development in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Serine palmitoyltransferase, a key enzyme for de novo synthesis of sphingolipids, is essential for male gametophyte development in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Teng&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Ternes P%2C Feussner K%2C Werner S%2C Lerche J%2C Iven T%2C Heilmann I%2C Riezman H%2C Feussner I %282011%29 Disruption of the ceramide synthase LOH1 causes spontaneous cell death in Arabidopsis thaliana%2E The New phytologist 192%3A 841%2D854&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Ternes P, Feussner K, Werner S, Lerche J, Iven T, Heilmann I, Riezman H, Feussner I (2011) Disruption of the ceramide synthase LOH1 causes spontaneous cell death in Arabidopsis thaliana. The New phytologist 192: 841-854

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Ternes&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Disruption of the ceramide synthase LOH1 causes spontaneous cell death in Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Disruption of the ceramide synthase LOH1 causes spontaneous cell death in Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Ternes&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=von Heijne G %281992%29 Membrane protein structure prediction%2E Hydrophobicity analysis and the positive%2Dinside rule%2E J Mol Biol 225%3A 487%2D494&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=von Heijne G (1992) Membrane protein structure prediction. Hydrophobicity analysis and the positive-inside rule. J Mol Biol 225: 487-494

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=von&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Membrane protein structure prediction&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Membrane protein structure prediction&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=von&as_ylo=1992&as_allsubj=all&hl=en&c2coff=1


Date post:	22-Mar-2018
Category:	Documents
Upload:	phamkiet
View:	219 times
Download:	2 times

1 Short Title: ORM Mediation of Sphingolipid Homeostasis 2 · PDF file59 SPT activity when...

Documents