Corresponding author - plantphysiol.org · 162 and labeled with the environment-sensitive probe di-4-ANEPPDHQ (Grosjean et al., 163 2015). Fluorescence spectroscopy experiments showed

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RESEARCH ARTICLE 3

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Running head: Plasma membrane lipids in tobacco 6

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Corresponding author: 10

Sébastien Mongrand 11

Laboratoire de Biogenèse Membranaire (LBM), UMR 5200, CNRS-University of 12

Bordeaux, 71 avenue Edouard Bourlaux, 33883 Villenave d’Ornon Cedex, France 13

Tel: 033 663266000 14

Fax 033 5 56 51 83 61 15

e-mail: [email protected] 16

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Research Area: 19

Membranes, Transport and Biogenetics: Associate Editor Anna Amtmann (Glasgow) 20

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Secondary Research Area: 22

Biochemistry and Metabolism: Associate Editor Julian Hibberd (Cambridge) and Cathie 23

Martin (Norwich) 24

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Plant Physiology Preview. Published on October 30, 2015, as DOI:10.1104/pp.15.00564

Copyright 2015 by the American Society of Plant Biologists

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RE-VISITING PLANT PLASMA MEMBRANE LIPIDS IN TOBACCO: A FOCUS ON 27

SPHINGOLIPIDS 28

29

Jean-Luc Cacas1,3, Corinne Buré2, Kevin Grosjean3, Patricia Gerbeau-Pissot3, Jeannine 30

Lherminier4, Yoann Rombouts5,6, Emmanuel Maes5,6, Claire Bossard1,7, Julien Gronnier1, 31

Fabienne Furt1, Laetitia Fouillen1, Véronique Germain1, Emmanuelle Bayer1, Stéphanie 32

Cluzet8, Franck Robert4, Jean-Marie Schmitter2, Magali Deleu7, Laurence Lins7, 33

Françoise Simon-Plas4, Sébastien Mongrand1 34

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1, Laboratoire de Biogenèse Membranaire (LBM), CNRS-University of Bordeaux, UMR 37

5200. 71 avenue Edouard Bourlaux, F-33883 Villenave d’Ornon Cedex, France 38

2, Chimie Biologie des Membranes et Nanoobjets CBMN - UMR 5248 Centre de 39

Génomique Fonctionnelle BP 68 Université de Bordeaux. 146, rue Léo Saignat F-33076 40

Bordeaux Cedex, France 41

3, Université de Bourgogne UMR1347 Agroécologie, ERL 6300 CNRS, 17 Rue Sully, BP 42

86510, F-21065 Dijon Cedex, France 43

4, INRA UMR1347 Agroécologie, ERL 6300 CNRS, 17 Rue Sully, BP 86510, F-21065 44

Dijon Cedex, France 45

5, Université de Lille 1, Unité de Glycobiologie Structurale et Fonctionnelle, Villeneuve 46

d'Ascq, France 47

6, CNRS, UMR 8576, Villeneuve d'Ascq, France 48

7, Laboratoire de Biophysique Moléculaire aux Interfaces (LBMI), Université de Liège, 2, 49

Passage des Déportés, B-5030 Gembloux, Belgium 50

8, ISVV, Groupe d’Etude des Substances Végétales à Activité Biologique (GESVAB), 51

University of Bordeaux, EA 3675, F-33140 Villenave-d’Ornon, 3 avenue Espeleta, F-52

33400 Talence, France 53

54

One-sentence summaries: 55

This paper shows that GIPCs are major lipids of the PM forming raft in the outer leaflet, 56

yet extending into the inner one through VLCFA, thereby creating interdigitation across 57

the membrane. 58

59



3

FOOTNOTES 60

Financial source: 61

We acknowledge the French Agence Nationale de la Recherche (ANR), programme 62

blanc “PANACEA” NT09_517917, contracts to SM and FSP. Lipidomic analyses were 63

performed on Bordeaux Metabolome Facility-MetaboHUB (ANR-11-INBS-0010), 64

contracts to SM and LF (http://www.biomemb.cnrs.fr/page_8.html). JLC was supported 65

by ANR projet blanc “PANACEA” NT09_517917. SM, LF, MD and LL are supported 66

by the ARC FIELD project “Finding Interesting Elicitor LipiDs”, and financial support of 67

FSR, University of Liege. MD and LL thank the Belgian Funds for Scientific Research 68

(FNRS) for their position as Senior Research Associate. Financial support from the 69

TGIR-RMN-THC Fr3050 CNRS for conducting the research is gratefully acknowledged. 70

71

Present address: 72

FF, Worcester Polytechnic Institute, Department of Biology and Biotechnology, 100 73

Institute Road, Worcester, MA 01609, USA 74

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4

ABSTRACT 76

Lipid composition of plasma membrane (PM) and the corresponding Detergent-Insoluble 77

Membrane (DIM) fraction were analyzed with a specific focus on highly polar 78

sphingolipids so-called Glycosyl-Inositol-Phosphoryl-Ceramides (GIPCs). Using tobacco 79

Bright Yellow-2 cell suspension and tobacco leaves (Nicotiana tabacum), evidences were 80

provided that GIPCs represent up to 40 mol% of the PM lipids. Comparative analysis of 81

DIMs with the PM showed an enrichment of 2-hydroxylated Very Long Chain Fatty Acid 82

(VLCFA)-containing GIPCs and polyglycosylated GIPCs in the DIMs. Purified 83

antibodies raised against these GIPCs were further used for immunogold-electron 84

microscopy strategy, revealing the distribution of polyglycosylated GIPCs in domains of 85

35±7nm in the plane of the PM. Biophysical studies also showed strong interactions 86

between GIPCs and sterols, and suggested a role for VLCFA in the interdigitation 87

between the two PM composing-monolayers. The ins and outs of lipid asymmetry, raft 88

formation and interdigitation in plant membrane biology are finally discussed. 89

90



5

INTRODUCTION 91

Eukaryotic Plasma Membranes (PMs) are composed of three main classes of lipids: 92

glycerolipids, sphingolipids and sterols, which may account for up to 100,000 different 93

molecular species (Yetukuri et al., 2008); (Shevchenko and Simons, 2010). Overall, all 94

glycerolipids share the same molecular moieties in plants, animals and fungi. By contrast, 95

sterols and sphingolipids are different and specific to each kingdom. For instance, the 96

plant PM contain an important number of sterols among which β-sitosterol, stigmasterol 97

and campesterol predominate (Furt et al., 2011). In addition to free sterols, phytosterols 98

can be conjugated to form Steryl Glycosides (SG) and Acyl Steryl Glycosides (ASG) that 99

represent up to ca. 15% of the tobacco PM (Furt et al., 2010). As for sphingolipids, 100

sphingomyelin (SM) the major phospho-sphingolipid in animals, which harbors a 101

phosphocholine as polar head, is not detected in plants. Glycosyl Inositol 102

PhosphorylCeramides (GIPCs) are the major class of sphingolipids in plants, but they are 103

absent in animals (Sperling and Heinz, 2003; Pata et al., 2010). Sphingolipidomic 104

approaches identified up to 200 plant sphingolipids (reviewed in (Pata et al., 2010) and 105

(Cacas et al., 2013). 106

Although GIPCs belong to one of the earliest classes of plant sphingolipids that have 107

been identified in the late 50’s (Carter et al., 1958), only a few GIPCs were structurally 108

characterized to date because of their high polarity, and a limited solubility in typical 109

lipid extraction solvents. For these reasons, they were systematically omitted from 110

published plant PM lipid composition. GIPCs are formed by addition of an inositol 111

phosphate to the ceramide moiety, the inositol head group of which can then undergo 112

several glycosylation steps. The dominant glycan structure, composed of a Hexose-113

Glucuronic acid (GlcA) linked to the inositol, is called series A. Polar heads containing 3 114

to 7 sugars, so-called series B to F, have been identified and appeared to be species 115

specific (Bure et al., 2011);(Cacas et al., 2013) (Mortimer et al., 2013). The ceramide 116

moiety of GIPCs consists of a long chain base (LCB), mainly t18:0 (called 117

phytosphingosine) or t18:1 compounds (for review, see (Pata et al., 2010) to which is 118

amidified a very long chain fatty acid (VLCFA); the latter of which being mostly 2-119

hydroxylated (hVLCFA) with odd or even number of carbon atoms. In plants, little is 120

known about the subcellular localization of GIPCs. It is, however, assumed that they 121



6

would be highly represented in PM (Worrall et al., 2003; Lynch & Dunn, 2004; Sperling 122

et al., 2005) even if this remains to be experimentally proven. The main argument 123

supporting such an assumption is the strong enrichment of tri-hydroxylated LCB (t18:n) 124

in DIM fractions (Borner et al., 2005); (Lefebvre et al., 2007), LCB known to be 125

predominant in GIPC’s core structure as aforementioned. 126

In addition to this chemical complexity, lipids are not evenly distributed within the PM. 127

Sphingolipids and sterols can preferentially interact with each other and segregate to form 128

microdomains dubbed membrane raft (Simons and Toomre, 2000). The “membrane raft” 129

hypothesis suggests that lipids play a regulatory role in mediating protein clustering 130

within the bilayer by undergoing phase separation into liquid-disordered (Ld) and liquid-131

ordered (Lo) phases. The Lo phase, termed membrane raft, was described as enriched in 132

sterol and saturated sphingolipids, and is characterized by tight lipid packing. Proteins, 133

which have differential affinities for each phase, may become enriched in, or excluded 134

from, the Lo phase domains to optimize the rate of protein-protein interactions and 135

maximize signaling processes. In animals, rafts have been implicated in a huge range of 136

cellular processes, such as hormone signaling, membrane trafficking in polarized 137

epithelial cells, T cell activation, cell migration, life cycle of influenza and HIV viruses 138

(Simons and Gerl, 2010) (Simons and Ikonen, 1997). In plants, evidence are raising that 139

rafts are also involved in signal transduction processes and membrane trafficking 140

(reviewed in (Simon-Plas et al., 2011); (Cacas et al., 2012b); (Mongrand et al., 2010). 141

Moreover, lipids are not evenly distributed between the two leaflets of the PM either. 142

Within PM of eukaryotic cells, sphingolipids are primarily located in the outer monolayer 143

whereas unsaturated phospholipids are predominantly exposed on the cytosolic leaflet. 144

This asymmetrical distribution has been well established in human red blood cells, in 145

which the outer leaflet contains SM, phosphatidylcholine (PC) and a variety of 146

glycolipids like gangliosides. By contrast, the cytoplasmic leaflet is composed mostly of 147

phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI) 148

and its phosphorylated derivatives (Devaux and Morris, 2004). With regards to 149

sphingolipids and glycerolipids, the asymmetry of the former is established during their 150

biosynthesis and that of the latter requires ATPases such as the amino-phospholipid 151

translocase that transports lipids from the outer to the inner leaflet, as well as Multiple 152



7

Drug Resistance proteins that transport PC in the opposite direction (Devaux and Morris, 153

2004). This ubiquitous scheme encountered in animal cells could apply in plant cells as 154

proposed by (Tjellstrom et al., 2010). Indeed, the authors showed that there is a 155

pronounced transversal lipid asymmetry in root at the PM. Phospholipids and 156

galactolipids dominate the cytosolic leaflet whereas the apoplastic leaflet is enriched in 157

sphingolipids and sterols. 158

From such a high diversity of the plant PM thus arises the question of the respective 159

contribution of lipids to membrane sub-organization. Our groups recently tackled this 160

aspect by characterizing the order level of liposomes prepared from various plant lipids 161

and labeled with the environment-sensitive probe di-4-ANEPPDHQ (Grosjean et al., 162

2015). Fluorescence spectroscopy experiments showed that, among phytosterols, 163

campesterol exhibits the strongest ability to order model membranes. In agreement with 164

these data, spatial analysis of membrane organization through multispectral confocal 165

microscopy pointed the strong ability of campesterol to promote Lo domain formation 166

and organize their spatial distribution at the membrane surface. Conjugated sterols also 167

exhibit a striking ability to order membranes. In addition, GIPCs enhance the sterol-168

induced ordering effect by emphasizing the formation and increasing the size of sterol-169

dependent ordered domains. 170

The aim of this study was to re-investigate the lipid composition and organization of the 171

PM with a particular focus on GIPCs using tobacco leaves and BY-2 cell cultures as 172

models. Analyzing all membrane lipid classes at once, including sphingolipids, is 173

challenging because they all display dramatically different chemical polarity going from 174

very apolar (like free sterols) to highly polar molecules (like polyglycosylated GIPCs). 175

Most lipid extraction techniques published thus far use chloroform/methanol mixture and 176

phase partition to remove contaminants, resulting in the loss GIPCs, which remain either 177

in the aqueous phase, unextracted in the insoluble pellet or at the interphase (Markham et 178

al., 2006). In order to gain access to both glycerolipids and sphingolipids species at a 179

glance, we developed a protocol whereby the esterifed or amidified FAs were hydrolyzed 180

from the glycerobackbone (glycerolipids) or the long chain base (sphingolipids) of 181

membrane lipids, respectivley. FA were then analyzed by GC-MS with appropriate 182

internal standards for quantification. We further proposed that the use of Methyl tert-183



8

butyl ether (MTBE) ensures the extraction of all classes of plant polar lipids. Our results 184

indicate that GIPCs represent up to 40 mol% of total tobacco PM lipids. Interestingly, 185

polyglycolyslated GIPCs are 5-fold enriched in DIMs of BY-2 cell when compared to 186

PM. Further investigation brought us to develop a preparative purification procedure that 187

allowed to obtaining enough material for raising antibodies against GIPCs. Using 188

immunogold-labeling on PM vesicles, it was found that polyglycosylated GIPCs cluster 189

in membrane nanodomains, strengthening the idea that lateral nano-segregation of 190

sphingolipids takes place at the PM in plants. Multispectral confocal microscopy was 191

performed on vesicles prepared using GIPCs, phospholipids and sterols, and labeled with 192

the environment-sensitive probe di-4-ANEPPDHQ. Our results show that, despite a 193

different fatty acid and polar head composition, GIPCs extracted from tobacco leaves and 194

BY-2 cells have a similar intrinsic propensity of enhancing vesicle global order together 195

with sterols. Assuming that GIPCs are mostly present in the outer leaflet of the PM, 196

interactions between sterols and sphingolipids was finally studied by Langmuir 197

monolayer and the area of a single molecule of GIPC, or in interaction with phytosterols, 198

was calculated. Using the calculation docking method, the energy of interaction between 199

GIPCs and phytosterols was determined. A model was proposed in which GIPCs and 200

phytosterols interact together to form Lo domains, and in which the VLCFAs of GIPCs 201

promote interdigitation of the two membrane leaflets. The implication of domain 202

formation and asymmetrical distribution of lipids at the PM in plants is also discussed. 203

Finally, we propose a model that re-considers the intricate organization of the plant PM 204

bilayer. 205

206



9

RESULTS 207

GIPCs are enriched in plasma membrane microdomains 208

Because FA-containing lipids, i.e. glycerolipids, acylated sterylglucosides and GIPCs, 209

exhibit marked different FA compositions, total FA distribution of microsomal, PM and 210

DIM fractions isolated from BY2 cells and tobacco leaves was determined to test for the 211

assumption that GIPCs mainly reside in PM. Samples were transesterified in hot 212

methanol/sulfuric acid solution to fully release both FA-esterified glycerolipids and 213

sterylglucoside, and FA-amidified sphingolipids. Total FA content was then quantified by 214

GC-MS (see Supplemental Fig. S1, for a typical GC-MS spectrum). The Fig. 1A shows 215

that, in both tobacco leaves and BY-2 cells, the combined percentage of VLCFA and 216

hVLCFA increases from ca. 3% in total tissue to ca. 55% in DIM at the expense of 217

LCFAs, i.e. 16 and 18 carbon atom-long FA (Fig. 1A; refer to Supplemental Fig. S2 for 218

the detailed FA content). Comparison of FA contents of DIM fractions floating in the 219

sucrose gradient, and Detergent-Soluble Membrane (DSM) fractions in the bottom of the 220

gradient revealed a higher level of LCFAs in DSM, which correlates with a lower 221

percentage of (h)VLCFA in this fraction (Supplemental Fig. S3). 222

In order to understand the origin of (h)VLCFA, we analyzed the structure of the different 223

family of plant PM lipids. We previously showed that VLCFA and hVLCFA are absent 224

from tobacco glycerolipids, except for a few percent of 20:0/22:0 in PS (Mongrand et al., 225

2004; Lefebvre et al., 2007). Further extraction and structural analyses revealed that 226

tobacco ASGs consist of saturated FA with mainly 16 and 18 carbon atoms and common 227

sterols of the PM (Supplemental Fig. S4). GluCER that accounts for only 5-10 mol% of 228

PM (Furt et al., 2010) is acylated by h16:0 as major hydroxylated FA (Supplemental Fig. 229

S5). Therefore, we hypothesized that VLCFA and hVLCFA, present in high amount in 230

PM and DIM, likely originate from GIPCs. 231

To test this hypothesis, we purified total GIPCs from tobacco leaf and BY-2 cells as 232

described in (Bure et al., 2011), and determined their FA and LCB content by GC-MS. 233

hVLCFA and VLCFA content is highly comparable in DIMs and pure GIPCs (Fig. 1B), 234

with an even higher proportion of hVLCFA in DIMs purified from BY2 cells, suggesting 235

that hVLCFA-containing GIPCs are most likely to be present in membrane 236

microdomains. Besides, levels of the two LCB t18:0 and t18:1, which are mostly present 237



10

in GIPCs (Bure et al., 2011), strongly increase in DIMs when compared to PM, reaching 238

80% of total LCBs in DIMs (Supplemental Fig. S6, and Borner et al., 2005). This is also 239

in good agreement with a strong enrichment of GIPCs in DIMs. Note that h16:0 is 240

enriched in DIMs (Fig. 1B). Logically, this FA is found in tobacco gluCER (Fig. S5), a 241

sphingolipid enriched in tobacco DIMs (Mongrand et al., 2004); (Lefebvre et al., 2007). 242

To further characterize the enrichment of GIPCs in DIMs, we first compared the total 243

GIPCs extracted from leaf and BY-2 cells by MALDI-TOF mass spectrometry (Bure, et 244

al. 2011). Leaf GIPCs mostly contain GIPCs of series A (with 2 sugars) and BY-2 245

contain in addition to series A, a vast array of polyglycosylated GIPCs of the B to E 246

series (Fig. 2), as previously described (Bure, et al. 2011). The detailed molecular species 247

of the polar head and LCB/FA combination is provided in Supplemental Fig. S7 with the 248

nomenclature described in (Bure et al., 2011). We next used HP-TLC to separate the 249

different series (Kaul and Lester, 1975) (Fig. 2B), scratched the corresponding silica 250

bands and quantified the FAMES by GC-MS after transmethylation/silylation (Fig. 3 and 251

4). Note that the series A partitioned into two bands called PhytoSphingoLipid: PSL I (N-252

Acetyl glucosamine(GlcNAc)-GlcA-IPC) and PSL II (Glucosamine(GlcN)-GlcA-IPC) as 253

previously established (Kaul and Lester, 1975). 254

GIPC of series A were found in both PM and DIM fractions of tobacco leaves 255

(Supplemental Fig. S8), but the fact that BY-2 cells contain different series of GIPCs 256

prompted us to determine whether polyglycosylated GIPCs were enriched in DIMs of 257

BY-2. We performed HP-TLC coupled to GC-MS, as described above. GIPCs of series B 258

were enriched three times in DIMs when compared to PM, reaching 17% of total GIPCs 259

in BY-2 DIM (Fig. 3). 260

Lipid composition of tobacco plant PM based on the latter set of data was combined to 261

previous findings (Furt et al., 2010), and the global lipid composition of PM and DIM 262

fractions was recalculated taking into account GIPC concentrations (Fig. 3). As expected, 263

glycerolipids were depleted in DIM fractions when compared to PM, whereas the exact 264

opposite trends was observed for sphingolipids, whether these fractions were prepared 265

from tobacco leaves (Fig. 4A) or BY-2 cells (Fig. 4B). Remarkably, GIPCs that have 266

long been omitted for technical reasons in PM composition, represent up to 45 and 30 267

mol% of total PM lipids isolated from leaves and BY-2 cell suspensions, respectively. 268



11

Furthermore, DIM fractions purified from both BY-2 cells and photosynthetic tissues 269

display a huge proportion of GIPCs that reaches 60 mol%, suggesting that the 270

contribution to sphingolipid enrichment in PM microdomains is mainly due to GIPCs. It 271

is also worth noting that the sum of sterols and sphingolipids averages 90 and 88 mol% in 272

DIM of tobacco leaves (Fig. 3A right) and that of BY-2 cells (Fig. 3B right), respectively. 273

We reasoned that if GIPCs are exclusively located in the outer leaflet of PM (see 274

discussion section), the presence of more than 50 % of GIPCs in DIMs suggests a higher 275

solubilization of the inner leaflet by the TritonX-100. 276

Hence, from these data arise the question of the lipid-to-protein ratio, commonly thought 277

to be closed to 1 for plant PM. This ratio was experimentally reinvestigated using BY-2 278

PM samples. One hundred micrograms of PM vesicles were extracted by the “gold-279

standard” Folch protocol, i.e. chloroform/methanol 2/1 (v/v) extraction. As previously 280

described by (Markham et al., 2006), almost half of the (h)VLCFA-containing GIPCs 281

were lost in the lower aqueous phase (Fig. S9). This phase was therefore evaporated to 282

remove solvents, resuspended in pure water and GIPCs were re-extracted with butanol-1 283

(which extract 98% of plant GIPC from water as previously shown in Bure et al., 2011). 284

Importantly, no FA was recovered in water after this double extraction (Fig. S9). In 285

addition, when comparing direct transesterification to Folch protocol followed by 286

butanol-1 extraction, the estimated lipid recovery yield using FA levels as proxy was 287

close to 100%, indicating full extraction of lipids irrespectively of their polarity. Based 288

on these results, the lipid-to-protein ratio was calculated to be 1.3 ± 0,07 for BY-2 PM. 289

290

We further decided to test other solvents for lipid extraction to get a simple, quantitative 291

and unbiased recovery of lipid species from plants. Methyl tert-butyl ether (MTBE) 292

extraction was tested, because it has been shown to allow faster and cleaner lipid 293

recovery (Matyash et al., 2008). Its low density forms the upper layer organic phase 294

during phase separation, which simplifies its recovery (Fig. S10A). We thus compared 295

Folch protocol (Extraction #1 in Experimental section), MTBE extraction (Extraction #2) 296

and Markham protocol (extraction #3) developed to fully extract plant sphingolipids 297

(Markham et al., 2006). Rigorous testing demonstrated that the extraction in hot 298

isopropanol followed by one of the three extractions was suitable to extract total polar 299



12

lipids of plant samples (see Experimental section). Nevertheless, Markham’s extraction 300

(#3) diplays the disadvantage to contain large amount of water, hardly evaporated, and 301

protein contamination in the organic phase because of the absence of liquid-liquid phase 302

separation. In the Folch extraction (#1), inconvenience resides in the fact that the higher 303

density of chloroform forms the lower phase in the two-phase partitioning system, and a 304

glass pipette or a needle must cross the aqueous phase to collect the lipid-containing one. 305

By contrast, lipid extraction by upper phase MTBE/methanol/water “extraction #2” 306

greatly simplifies sample handling. We therefore proposed the MTBE method as a 307

method of choice to extract total plant polar lipids. 308

309

Purification of GIPC series from BY-2 cells and production of antibodies against 310

polyglycosylated GIPCs 311

Pure GIPCs are not commercially available, neither are the corresponding molecular tools 312

dedicated to their study, like fluorescently-labeled lipids or specific antibodies. We thus 313

purified several milligrams of GIPCs in order to immunize rabbits and raise antibodies to 314

be used in immunolabeling experiments. Because of their anionic phosphate groups, 315

GIPCs can be purified by anion-exchange chromatography on diethylaminoethyl (DEAE) 316

cellulose. This approach has the double advantage of allowing sample cleanup and 317

concentration. 318

The preparative purification procedure was carried out according to Kaul and Lester 319

(1975) with slight modifications described in the section Material and Methods. Home-320

packed DEAE cellulose chromatographic column was used for that purpose and GIPCs 321

were eluted with increasing concentrations of ammonium acetate dissolved in 322

chloroform/methanol/water (30/60/8, v/v/v). Under our experimental conditions, GIPCs 323

of series A (namely PSL1 and PSL2) were successfully separated from polyglycosylated 324

GIPC of series B-F; the former eluting in fractions 41-45, whereas the latter eluted in 325

fractions 46-49 (Supplemental Fig. S11A). Glycerolipid contaminations were discarded 326

from these fractions by methylamine treatment, which hydrolyses ester bonds (Markham 327

et al., 2006). Fractions were then dialyzed to remove ammonium acetate and their 328

concentration and purity estimated by GC-MS and MALDI-TOF (Supplemental Fig. 329

S11B). They were subsequently used for preparing liposomes supplemented with 330



13

bacterial lipid A known to boost rabbit immunity (Richards et al., 1998). The two 331

immune sera obtained following rabbit injection with GIPC of series A did not react with 332

the DEAE-purified fractions used for the immunization protocol (data not shown). By 333

contrast, one immune serum (rabbit #46) raised against polyglycosylated GIPCs of series 334

B-F clearly reacted with the chromatographic fractions used for the immunization 335

protocol, whereas no reaction was observed with the corresponding pre-immune serum 336

(Figure 5A). ELISA performed on PM showed strong signal increase between negative 337

controls (preimmune serum) and final serum or purified IgG antibodies (Supplemental 338

Fig. S12). No specific signal was detected with antibodies against GIPCs on hydrophobic 339

membranes that have been spotted with all eight phosphoinositides and seven other 340

biological important lipids (“PIP strip”, Supplemental Fig. S13). Finally, to further test 341

the specificity of the antibodies on the different GIPCs series purified from BY-2 cells, 342

Eastern blots were directly performed on HP-TLC plates containing PM lipids extracted 343

with the MTBE protocol described above. Rabbit polyclonal antibodies were able to 344

recognize polyglycosylated GIPCs of series B-F, but not those of series A (Fig. 5B). 345

346

Polyglycosylated GIPCs cluster within nanodomains in tobacco PM 347

Since polyglycosylated GIPCs are enriched in BY-2 DIM fractions (Fig. 4B), the 348

possibility of visualizing GIPC-enriched clusters was challenged by transmission electron 349

microscopy using anti-GIPC antibodies-based immunogold labelling experiments. PM 350

vesicles were purified from BY-2 cells, directly deposited onto microscope grids 351

allowing for the exposure of large membrane sheets. The grids were then pretreated to 352

prevent nonspecific binding, incubated first with primary antibodies against 353

polyglycosylated GIPCs and then with secondary IgG conjugated to colloidal gold 354

particles. Preparations were negatively stained with ammonium molybdate to reveal the 355

vesicle morphology and observed by transmission electron microscopy. 356

Statistical analysis was performed on 49 independent gold-labeled PM vesicles to analyze 357

the putative clustering of gold particles. The mean and standard deviation were calculated 358

for different parameters: diameter of PM vesicles, area of PM vesicle, number of gold per 359

PM vesicle, number of gold particles per cluster, size of gold particle cluster and distance 360

between two neighboring clusters. The mean labeling density was quantified to be 7 gold 361



14

particles per vesicle. Groups of particles were composed of an average of 4 gold particles 362

(from 3 to 7 particles were clustered). We calculated that 88% (n=3) of the gold particles 363

showed a clustered distribution throughout the vesicle surface with an average of 4 gold 364

particles, and with an average cluster diameter of 35 nm ± 7nm (Fig. 6A,B). Only 12% of 365

the gold particles exhibited a random distribution on the PM surface. Ripley’s K-function 366

analysis indicated that the gold pattern was aggregated, since K(r) values of experimental 367

data laid clearly above the Poisson simulation curve corresponding to completely random 368

pattern (Fig 6C). Negative controls including omission of the primary antibody or use of 369

pre-immune serum exhibited a very weak labeling. Positive controls carried out using 370

antibodies raised against the proton pump ATPase (PMA) showed a heavy labeling of the 371

PM (Raffaele et al., 2009) (Supplemental Fig. S14). Therefore, GIPCs (series B-F) 372

exhibit an aggregated pattern within the PM of BY-2 cells with a mean size of 35nm. 373

374

Order level of model membranes prepared with GIPCs isolated from leaves or cell 375

cultures 376

The ability of GIPCs to change membrane order and organize Lo domain was 377

investigated using the environment-sensitive probe di-4-ANEPPDHQ, as described in 378

(Grosjean et al., 2015). Briefly, membrane organization alters the fluorescence emission 379

spectrum of the probe that emits in both red (635 to 655 nm) and green spectral regions 380

(545 to 565 nm). Increase in green fluorescence emission correlates with a higher average 381

order level of the membrane. Conversely, an emission shift towards red wavelengths 382

indicates a decrease in the relative amount of ordered domains in the lipid bilayer. The 383

Red-to-Green fluorescence ratio of the Membrane (RGM) thus reflects the relative 384

proportions of Ld/Lo phases within membranes (Nichols et al. 1986). 385

Previously, we showed that plant sphingolipids, especially GIPCs, enhanced the sterol-386

induced ordering effect by increasing the size of sterol-dependent ordered domains 387

(Grosjean et al., 2015). In order to test for the impact of GIPC’s composition on global 388

membrane organization, Large Unilamellar Vesicles (LUV) were prepared using distinct 389

combination of lipids, incubated in the presence of di-4-ANEPPDHQ and the RGM 390

calculated upon spectral acquisition. The phospholipids dioleoylphosphatidylcholine 391

(DOPC) and dipalmitoylphosphatidylcholine (DPPC), the phytosterol (campesterol or a 392



15

sterol mix isolated from BY2 cells (TM)), and GIPCs were used for producing LUV. We 393

compared GIPCs of series A isolated from tobacco leaves, and GIPCs of series A-F, 394

which include polyglycosylated molecules, purified from BY-2 cell suspensions. 395

Importantly, the ceramide moiety of tobacco leaves mostly contains hVLCFA, whereas 396

that of BY2 suspension harbors an equimolar mix of VLCFA and hVLCFA. 397

Results of Fig. 7 confirmed that GIPCs have no particular ability to modify the order 398

level of model membranes containing only phospholipids and suggest that the size of 399

their sugar head and the hydroxylation of VLCFA do not change this capacity. When 400

33% of campesterol or a sterol mixture mimicking the one found in tobacco BY-2 PM 401

(TM, Tobacco Mix) were added to phospholipids and GIPCs, a significant and similar 402

decrease of the RGM was observed (Fig. 7). Such decrease of RGM confirm the major 403

involvement of sterol in increasing membrane order level, and suggest that 17% of 404

polyglycosylated (Fig. 3) or the presence of 50% of VLCFA (Fig. 1B) in GIPCs does not 405

drastically modify the ability of sphingolipids to order membrane with sterols. 406

407

Molecular simulation modeling and biophysical analysis reveals GIPC/Sterol 408

interaction and interdigitation of GIPC’s VLCFA between the two leaflets of the 409

PM 410

Based on the literature (see discussion) and immunolabelling experiments (Fig. 6), we 411

reasonably hypothesized that GIPCs may preferentially reside in the apoplastic leaflet. 412

Therefore, we conducted biophysical experiments and energetic calculation to 413

characterize outer leaflet organization of plant PM, i.e. structure, organization and 414

behavior. 415

The Langmuir trough technique applied on a monolayer model at air–water interface has 416

been extensively used for characterizing interfacial organization of lipids and lipid–lipid 417

interactions at the micrometric level (Deleu, et al. 2014). The GIPC compression 418

isotherm (Fig. 8A) showed a low and relatively constant surface pressure at large 419

molecular areas, corresponding to a gaseous state. Compression of a pure GIPC 420

monolayer induced a progressive increase in surface pressure, indicating the appearance 421

of a liquid-expanded (LE) state, which is characterized by a certain degree of cooperative 422

interaction between the molecules at the interface (Fig. 8A). This was confirmed by the 423



16

value of the two-dimensional compressibility modulus (Cs−1 = 31.9 mN/m in the 180-70 424

Å²/molecule region), which is lower than the highest value (100mN/m) for a LE film 425

(Rideal, 1963). At the onset of the LE state, corresponding to the more expanded 426

configuration, the molecule occupies a mean interfacial area of 209.9±3.6 Å²/molecule. 427

This increase in surface pressure was followed by a small plateau of quite constant 428

surface pressure and by a sharp increase in surface pressure at low areas per molecule. 429

This indicated that GIPC monolayers can adopt a more condensed state (Cs−1>80mN/m) 430

under high compression. In this state, the lipids occupy a mean molecular area of 431

66.0±11.3 Å²/molecule and adopt probably a vertical orientation at the interface. 432

We next assessed the interaction between GIPCs and sitosterol; information can be 433

obtained by a thermodynamic analysis of the compression isotherms of the mixed GIPC-434

sitosterol monolayers. Within a mixed monolayer, if the two components are immiscible 435

(or ideally miscible), the area occupied by the mixed film will be the sum of the areas of 436

the separate components (obeyed the additivity rule) (Maget-Dana, 1999). Any deviation 437

from the additivity rule can be attributed to specific interaction between the two 438

components (Maget-Dana, 1999; Fang et al., 2003). Whatever the surface pressure 439

considered (10, 20 or 30 mN/m), the mean molecular area of mixed monolayer GIPC/Sito 440

(85/15) was significantly lower than the theoretical value calculated from the additivity 441

rule (Fig. 8B). The negative deviation of the area, together with the negative excess of 442

free energy of mixing (ΔGex) (Fig. 8C) suggests a strong attractive interaction between 443

the two components (Maget-Dana, 1999; Gaines, 1966; Eeman et al., 2005). Moreover, 444

the negative value of ΔGM (Fig. 8C) indicated that the mixed GIPC/sitosterol monolayer 445

is thermodynamically stable. 446

Molecular modelling of the GIPC monolayer and membrane insertion 447

We finally used a simple theoretical docking method, called Hypermatrix (HM). This 448

method is particularly useful to compare the specific interaction of a molecule of interest 449

with lipids and with itself, and hence helps to understand its organization according to the 450

different interacting forces. The analysis of the assembly of GIPC molecules with 451

t18:0/h24:0 in a monolayer showed that the calculated interaction is mainly driven by 452

hydrophobic energy (Fig. 9C). The mean interfacial area occupied by one molecule in the 453

monolayer is 69±10 Å² (Fig. 9C). This is in very good agreement with the area measured 454



17

experimentally using the Langmuir technique in high compression conditions, suggesting 455

that the calculated structure of GIPC corresponds to this configuration. When the 456

interaction between GIPCs and sitosterol was analyzed, the energy of the interaction was 457

comparable to that of GIPC monomolecular layer, with a slight increase in Van der 458

Waals interactions (Fig. 9C). This suggests a good steric fitting between the two 459

molecules, as shown on figure 9A. This molecular fitting can also be correlated to the 460

fact that the mean area calculated in mixed GIPC/sitosterol monolayers was lower than 461

the area of individual molecules (Fig. 9C), again in very good agreement with the 462

experimental assays on monolayer (Fig. 8B). 463

To analyze the behavior of GIPCs with t18:0/h24:0 into a lipid membrane, we calculated 464

its insertion into a simplified implicit bilayer (IMPALA method) (Ducarme et al., 1998) 465

and compared it to gluCER with d18:2/h16:0. Fig. 9B clearly evidenced a significant 466

difference between gluCER and GIPC: 1/ the size of the polar heads and the positioning 467

of acyl chains are strikingly different; 2/ the saturated VLCFA of GIPCs runs out of the 468

middle of the bilayer and interdigitates by at least 6-7 carbon atoms within the second 469

leaflet. 470

471



18

DISCUSSION 472

GIPCs are by far the major lipids of the plant plasma membrane 473

In this work, we reinvestigated the lipid composition of PM and ordered domain isolated 474

as Detergent-Insoluble Membranes (DIMs), with a particular focus on GIPCs. The latter 475

class of sphingolipids has long been neglected because it is not extracted by conventional 476

lipid extraction procedures (Fig. S9 and Sperling et al., 2005; Markham et al., 2006). 477

Here, we showed that GIPCs represent up to 30-40 mol% of the total PM lipids of 478

tobacco plants and therefore represent the bulk of PM outer leaflet lipids, with 60-80% of 479

total outer leaflet lipids (Fig. 3 and 10). Taking into account this striking result, we 480

recalculated the lipid-to-protein ratio of plant PM and found a ratio of 1.3. Hence, bearing 481

in mind that PM contains a high protein density, it is tempting to propose that plant PM 482

should not be considered as system where proteins are floating in a “sea” of lipids, but as 483

a lipid–protein composite in which a very high density of transmembrane and anchored 484

proteins may modify order on nearby lipids (Jacobson et al., 2007). A recent publication 485

in plants showed for example that PM are subcompartmentalized into a plethora of 486

coexisting and diverse microdomains labeled by the different isoforms of the inner leaflet 487

plant raft protein REMORINs (Jarsch et al., 2014). The respective role of lipids and 488

proteins in this segregation of membrane compounds remains to be elucidated. 489

490

GIPC’s polar headgroups are much bulkier than phospholipid ones 491

The volume occupied by the glycosyl-phosphoinositol headgroup of GIPCs increases 492

with the complexity of the oligosaccharide chain. Our experimental data obtained by the 493

Langmuir monolayer technique point that the molecular area occupied by tobacco GIPCs 494

of series A varies from 66.0±11.3 to 209.9±3.6 Å²/molecule from a condensed to an 495

expanded state (Fig. 8A). This was further corroborated by our computational 496

calculations indicating a value of 69±10 Å²/molecule (Fig. 9C) in good agreement with 497

the values of the interfacial area either calculated or measured by Langmuir monolayer 498

technique, as reviewed in (Deleu et al., 2014). By contrast, phospholipids occupy 95-110 499

Å²/molecule in an expanded state and 45-55 Å²/molecule in a condensed state (Deleu et 500

al., 2001; Eeman et al., 2005) and sterols display an interfacial area of 38-48 Å²/molecule 501

(Eeman et al., 2005; Scheffer et al., 2005). Predictions based on the geometrical 502



19

properties of glycosphingolipid molecules indicate that the separation of a 503

glycosphingolipid-rich phase in a phospholipid bilayer would imply a minimization of 504

the interfacial free energy required to accommodate the amphipathic glycosphingolipid in 505

the bilayer. Therefore, the geometrical properties inherent to the bulky headgroup of 506

glycosphingolipids strongly favor phase separation and spontaneous membrane 507

curvature, for review (Sonnino and Prinetti, 2010). In animals, the extent of ganglioside 508

phase separation in glycerophospholipid bilayers depends on the surface area occupied by 509

the oligosaccharide group that is usually directly correlated with the number of sugar 510

residues (Masserini et al., 1989). Nevertheless, one must note that gangliosides are 511

present in very low amount in animal membrane (less than few percent), whereas plant 512

GIPCs represent the major sphingolipids of the PM. In that context, the biophysical 513

properties of the plant PM must be fully reinvestigated. 514

515

Role for GIPCs and sterols in coupling the inner and the outer leaflets of the PM 516

The asymmetry of lipids in the tobacco PM 517

A common feature of eukaryotic PMs is the non-random distribution of lipids in the two 518

leaflets of the membrane, called lipid asymmetry. Lipid asymmetry within the two PM 519

monolayers is responsible for different biophysical properties and influences numerous 520

cellular functions. The lipid asymmetry lies in the facts that glycerolipids are primarily 521

synthesized on the cytosolic side of cellular membranes whereas production of complex 522

sphingolipids is completed in the ER/Golgi, rendering the latter exposed to the outer 523

surface. In addition, sterols have higher affinity for sphingolipids than glycerolipids. This 524

out-of-equilibrium situation is maintained by the activity of lipid translocases, which 525

compensate for the slow spontaneous transverse diffusion of lipids (Devaux and Morris, 526

2004). 527

To build a model of plant PM, we used the results of the present paper, and those 528

obtained by Tjellström and collaborators (2010) who showed that there is a transversal 529

lipid asymmetry in root plant PM. They calculated that the distribution 530

cytosolic/apoplastic leaflet was 65:35 for phospholipids, 30:70 for total sterols, and 30:70 531

for GluCer. DGDG is exclusively located in the inner leaflet (Tjellstrom et al., 2010). 532

Here, we considered that the glycerophospholipid-rich inner leaflet is unsaturated and 533



20

that plant phosphatidylserine (PS) and polyphosphoninositides (e.g. PIP2) were 534

exclusively present in the inner leaflet, as described in animal models (Di Paolo and De 535

Camilli, 2006). As Filipin III labeling used to assess sterol distribution could not 536

discriminate between free sterols and sterol derivatives (ASG and SG) (Tjellstrom et al., 537

2010), we drew free sterols, SG and ASG equivalently distributed between the two 538

leaflets, with a molar ratio for sterols of 30:70, in:out. 539

We tried to experimentally access the distribution of GIPCs in the PM. We treated Right 540

Side Out (RSO) and In Side Out (ISO) vesicles with Sphingolipid ceramide N-deacylase 541

(SCD) enzyme able to hydrolyze GIPCs (Blaas and Humpf, 2013), as described with 542

phospholipase A2 for phospholipid asymmetrical distribution (Tjellstrom et al., 2010). In 543

our hands, no GIPC hydrolysis occurred (data not shown). Nevertheless, we reasonably 544

hypothesized that GIPCs are exclusively located in the apoplastic face for different 545

independent reasons: 1/ the two first steps of the GIPC synthesis (IPC synthase and 546

glucuronic transferase) occur in the Golgi apparatus (Wang et al., 2008); (Rennie et al., 547

2014); 2/ The mannosylation of series A of GIPC to build series B is dependent of the 548

GDP-mannose transporter GOLGI-LOCALIZED NUCLEOTIDE SUGAR 549

TRANSPORTER (GONST1), suggesting a luminal glycosylation of GIPCs so that the 550

polar heads is exposed in the outer leaflet of the PM after vesicular fusion (Mortimer et 551

al., 2013); 3/ GIPCs are structurally homologs to gangliosides, exclusively present in the 552

outer leaflet of the PM in animal cells (Sonnino and Prinetti, 2010); 4/ it is very unlikely 553

that GIPCs may spontaneously flip-flop in the PM because of the size and polarity of 554

their heads and 5/ immunogold labeling of GIPCs (series B-F) on PM vesicles seems to 555

be unilaterally distributed mainly outside of the PM vesicles (Fig. 6). 556

Saturated very long chain fatty acid of GIPCs interdigitate the two leaflets 557

A key question in understanding the functional role of the PM is whether lipids of the 558

outer leaflet are coupled to those of the inner leaflet. Plant GIPCs exhibit a high content 559

of VLCFA that can be hydroxylated on carbon 2. The presence of VLCFA in DIMs was 560

recently observed in bean and maize DIMs (Carmona-Salazar et al., 2015). Here, the 561

modeling approach suggests a strong and stable interdigitation of (h)VLCFA of GIPCs 562

from the outer leaflet into the inner leaflet by 6-7 carbon atoms (Fig. 9B). VLCFA are 563

also abundant in sphingolipids in animal cells. It has been proposed that lipid bilayer 564



21

organization of the stratum corneum could be stabilized by a partial interdigitation 565

between the two leaflets (Ruettinger et al., 2008). Interdigitation of long chain fatty acid 566

residues between complex lipids might thus represents a common feature in plants and 567

animals that allows a higher thermal stability of the outer leaflet, as described in artificial 568

asymmetrical liposomes prepared with animal lipids (Cheng et al., 2009). 569

570

GIPCs are able to organize in liquid-ordered domains with sterols 571

Tobacco cells produce several hundred GIPCs of different structures, (see Fig. 2, and 572

Cacas et al., 2013), with the same ceramide moiety and a variable glycan part. 573

Publication of the 70’s suggest that up to 20 sugars can be added to the GIPC core 574

structure, but little is known about such molecule (Kaul and Lester, 1975). In this paper, 575

we demonstrated that GIPCs with more than two sugars are enriched in DIMs (Fig 2C) 576

and these complex sphingolipids cluster in domains of 35 nm (Fig. 6). In addition, we 577

showed that 2-hydroxylated-containing GIPCs were enriched in DIMs (Fig. 1B). This 578

result is coherent with biophysical studies where raft phase separation is favored by the 579

fact that sphingolipids, as ceramide-based amphipathic lipids, can create a network of 580

hydrogen bonds due to the presence of the amide nitrogen, the carbonyl oxygen and the 581

hydroxyl group positioned in proximity of the water/ lipid interface of the bilayer 582

(Pascher, 1976). In addition, GIPC’s LCB being dominated by tri-hydroxylated LCBs 583

(Figs S6), the presence of two additional hydroxyl groups at the interface may be of 584

importance for sphingolipid/phytosterol interactions. The contribution of hydrogen bonds 585

between lipids stabilizing a more rigid segregated phase in the bilayer is energetically 586

remarkable (Quinn and Wolf, 2009). We recently showed that GIPCs enhance the sterol-587

induced ordering effect by stimulating the formation of and increasing the size of sterol-588

dependent ordered membrane domains (Grosjean et al., 2015), suggesting a strong 589

interaction between phytosterols and GIPCs leading to a well defined Lo phase 590

separation. Docking calculation between phytosterols and GIPCs showed that the 591

interaction is mostly of hydrophobic and Van der Waals types (Fig. 9C). Hence, the closer 592

the molecules are, the stronger the interaction is. This aspect is also pointed by Langmuir 593

monolayers’ experiments, where we measured an attractive interaction between GIPCs 594

and phytosterols at a molar ratio of 85:15 (Fig. 8). Finally, interdigitated hydrocarbon 595



22

chains may play a role in the stabilization of lipid domains, as reviewed in (Sonnino and 596

Prinetti, 2010). 597

A model summarizing these data is presented in the figure 10. To build this model, the 598

molar composition of lipids from BY-2 PM (Fig. 4) and information presented above 599

were considered. This model emphasizes the strong enrichment of GIPCs in the 600

apoplastic phase of the PM. In accordance with this assumption, only little space would 601

be left in the outer leaflet for phospholipids, which consequently are concentrated on the 602

cytoplasmic leaflet. Polyglycosylated GIPCs cluster with sterols in domains of ca. 35nm 603

in the outer leaflet, and polyphosphoinositide-enriched domain are present in the inner 604

leaflet according to our previous work (Furt et al., 2010). 605

606

Are rafts in the two leaflets coupled? 607

Plant GIPCs are clearly involved in raft formation, and rafts exist in both external and 608

internal leaflets of the plant PM (Raffaele et al., 2009); (Furt et al., 2010); (Mongrand et 609

al., 2010). “Biological rafts” are likely of nanometer scale and certainly differ in size and 610

stability in the two monolayers. It is not known whether they overlap so that they are 611

coupled functionally and structurally (Eisenberg et al., 2006); (Subczynski and Kusumi, 612

2003). By exploring this possibility, one could shed light on how cues are transmitted 613

through the bilayer. Does the clustering of proteins or lipids in the outer leaflet trigger the 614

re-arrangement of downstream proteins or lipids in the inner leaflet (Kinases, 615

phosphatases, small G proteins, PIP2…), leading to signal transduction and 616

amplification? Can rafts in the outer leaflet enriched in GIPCs and sterols mirrored by 617

PIP2-enriched cytoplasmic leaflet rafts, as represented in figure 10? What could be the 618

role of FA interdigitation and lipid asymmetry in plants, and how is this process 619

regulated? Proteins -omitted in our model- will certainly influence raft composition, size, 620

shape and overall physical properties, independently of thermodynamic considerations of 621

the pure lipid phases (Devaux and Morris, 2004). This last aspect remains to be fully 622

elucidated in plant PM. 623

624

CONCLUSIONS 625



23

In plants, GIPCs have been shown to be involved in early stages of symbiosis (Hernandez 626

et al., 1995), in Golgi and ER integrity (Chen et al., 2008), growth and hypersensitive 627

response through salicylic acid production (Mortimer et al., 2013). A recent study on cell 628

wall rhamnogalacturonan II (RG-II) showed that GIPCs are able to bind RG-II, possibly 629

via a Boron bridge, and that they can favor the Boron-dependent dimerisation of RG-II 630

(Voxeur and Fry, 2014). Cell wall is an important feature in regulating protein lateral 631

mobility. In plant cells, turgor pressure tightly pressed PM against cell wall. Martinière 632

and collaborators, 2012 showed that this intimate connection affects protein lateral 633

mobility including the one in the inner leaflet. This suggests that the plant cell wall, and 634

by extension the continuum between the PM and the cell wall, influences protein lateral 635

mobility (Martiniere et al., 2012). This regulation of protein lateral mobility by the cell 636

wall certainly plays a role in plant cellular processes. GIPCs may also be important 637

determinants in cell signaling, cell-to-cell communication, plant defense and the sorting 638

of proteins, as it is also described for complex sphingolipids in animal development 639

(Worrall et al., 2003). The link between outer leaflet lipids and the cell wall also deserves 640

to be fully investigated. Finally, apoplastic leaflet that contains high ordered-forming 641

lipids (GIPC/phytosterols) likely represents physical barrier involved in maintenance of 642

thermal tolerance (Cheng et al., 2009), cell integrity and responses to pathogens. 643

Preparation of asymmetric vesicles that mimic the plant PM will be of great interest to 644

study this coupling, the effect of lipid raft formation and the distribution of 645

transmembrane protein helices (Cheng et al., 2009). In animals, alteration of lipid 646

asymmetry plays a prominent role during cell fusion, activation of the coagulation 647

cascade and, recognition and removal of apoptotic cell. Our work should pave the way to 648

address such questions in plants. 649

650



24

MATERIAL and METHODS 651

Materials—High-performance thin-layer chromatography (HP-TLC) plates were 652

Silicagel 60 F254 (Merck, Rahway, NJ). 653

654

Plant Materials—Leaves were obtained from 8-week-old tobacco plants (Nicotiana 655

tabacum cv. Xanthi) grown in a growth chamber at 25°C under 16/8-h day/night 656

conditions. Wild type BY-2 cells (Nicotiana. tabacum cv. Bright Yellow 2) and Vitis 657

vinifera L. cv ‘Cabernet Sauvignon’ (CS6) were grown as previously described in (Morel 658

et al., 2006) and (Cacas et al., 2013). 659

660

Preparation and Purity of Tobacco PM—All steps were performed at 4°C. PMs were 661

obtained after cell fractionation according to (Mongrand et al., 2004) by partitioning in an 662

aqueous polymer two-phase system with polyethylene glycol /dextran. 663

664

Fatty acids Analysis— Each sample was transmethylated at 110°C overnight in 665

methanol containing 5% (v/v) sulfuric acid and spiked with 10 µg of heptadecanoic acid 666

(c17:0) and 10 µg of 2-hydroxy-tetradecanoic acid (h14:0) as internal standards. After 667

cooling, 3 mL of NaCl (2.5%, w/v) were added, and the released fatty acyl chains were 668

extracted in hexane. Extracts were washed with 3 mL of saline solution (200 mM NaCl, 669

200 mM Tris, pH 8.0), dried under a gentle stream of nitrogen, and dissolved in 150 µL 670

of BSTFA (N,O-bis(trimethylsilyl) trifluoroacetamide) and TMCS 671

(trimethylchlorosilane). Free hydroxyl groups were derivatized at 110°C for 30 min, 672

surplus BSTFA-TMCS was evaporated under nitrogen, and samples were dissolved in 673

hexane for analysis using GC-MS under the same conditions as described in (Bure et al., 674

2011). Quantification of fatty acids and hydroxyl acids was based on peak areas, which 675

were derived from Total Ion Current (Rehman et al.), and using the respective internal 676

standards. 677

678

Sphingoid Base (LCB) Analysis—Samples were heated at 110C for 24 h with 4 mL of 679

dioxane (Sigma) plus 3.5 mL of 10% (w/v) aqueous Ba(OH)2 (Sigma). The sphingoid 680

bases were oxidized to their corresponding aldehydes by stirring the sample with 100 µL 681



25

of 0.2 M sodium periodate (Sigma) at room temperature for 1 h in the dark. The 682

aldehydes were recovered by hexane extraction and used directly for GC analysis as 683

described in (Cacas et al., 2012a). 684

685

Extraction of total polar lipids; Set up of lipid extraction protocol for total polar 686

lipids in plants—Membrane fractions (100-200µg) or grape cell culture (ca. 20 mg of 687

lyophilized materiel) were extracted according to three independent methods: Extraction 688

#1: 3.5 mL of chloroform/methanol/HCl (200/100/1, v/v/v) supplemented with 0.01% 689

butylatedhydroxytoluene (BHT) (w/v) were incubated with the sample. Then, 2mL of 690

NaCl 0.9% (w/v) were added, vortexed for 5 min and centrifuged. The lower organic 691

phase was collected, and the higher phase was re-extracted once with 4 mL of pure 692

chloroform. Extraction #2: 3.5 mL of Methyl tert-butyl ether (MTBE)/methanol/water 693

(100/30/25, v/v/v) supplemented with 0,01% BHT (w/v) were incubated with the sample. 694

Then, 2ml of NaCl 0.9% was added, vortexed for 5 min and centrifuged. The upper 695

organic phase was collected, and the lower phase was re-extracted once with 4 mL of 696

pure MTBE. In both extractions, the organic phases were combined and dried. The 697

aqueous phases were dried to remove any trace of organic solvent, resuspended in 1mL 698

of pure water, and GIPCs were back-extracted twice by 1 mL butanol-1. Extraction #3 699

adapted from (Markham et al., 2006): 3.5 mL of (lower phase of propan-2-700

ol/hexane/water, 55:20:25 (v/v/ v)) were incubated with the sample. The sample was 701

incubated at 60 °C for 15 min with occasional shaking. The extract was spun at 500 g 702

while still warm, and the supernatant was transferred to a fresh tube. The pellet was 703

extracted once more, each time with 3.5 mL of extraction solvent, and the supernatants 704

were combined and dried. The pellet was dried to remove any trace of organic solvent, 705

resuspended in 1mL of pure water, and GIPC were back-extracted twice by 1mL butanol-706

1. Extracted lipids were dissolved in chloroform /methanol/water (30/60/8, v/v/v) for 707

storage. Alternatively, before lipid extraction, biological samples were transferred to 708

isopropanol (3mL) with 0.01% BHT at 75 °C and incubated for 15 min to inhibit lipase 709

activity. 710

The rationale for the three lipid extraction protocols is presented in Fig. S10A in 711

supplemental data. We used lyophilized grape cell culture (Vitis vinifera), previously 712



26

shown to contain similar (h)VLFCA and GIPCs as tobacco cells (Cacas et al., 2013), see 713

Supplemental Fig. S10B. We first compared Folch protocol (Extraction #1) and MTBE 714

extraction (Extraction #2) followed by butanol-1 extraction of the aqueous phase, with 715

Markahm protocol (Extraction #3) shown to fully extract plant sphingolipids (Fig. 4B) 716

(Markham et al., 2006). Results of Fig. S10C middle showed that all the classes of lipids 717

were extracted with no significant differences between the three different protocols. We 718

performed a HP-TLC plate to test the integrity of polar lipids, and we observed, as often 719

described during plant lipid extraction, a major activation of Phospholipase D leading to 720

the conversion of phospholipids into Phosphatidic Acid, PA (Supplemental Fig. S10D). 721

To circumvent this problem, we boiled the lyophilized grape cells in hot isopropanol, and 722

further extracted with the three protocols. To our surprise, all the polar lipids including 723

VLCFA-containing GIPCs were extracted without the need of the second extraction step 724

by butanol-1 of the aqueous phase (Fig. S10C right). As expected, no degradation of 725

phospholipids was anymore observed on TLC plates (see Supplemental Fig. S10D). 726

727

Extraction and purification of GIPCs by DEAE chromatography —GIPCs were 728

purified according to (Bure et al., 2011) and (Grosjean et al., 2015) to obtain milligram 729

amount. Alternatively, GIPCs were purified DEAE chromatography. DEAE Sephadex 730

DE-52 (Whatman pre-swollem, µgranular) was suspended in chloroform/ methanol/water 731

(30:60:8, v/v/v) supplemented with 1M ammonium acetate. A glass column (40 cm high, 732

2.2 cm of diameter) was plugged by defatted cotton, filled with DEAE Sephadex and 733

washed with 600 mL of chloroform/ methanol/water (30:60:8, v/v/v). GIPC whitish pellet 734

extracted from BY-2 cell culture was dissolved in 10 ml of chloroform/methanol/water 735

(30:60:8, v/v/v) and loaded on the column. The column was washed with 800 ml of 736

chloroform/ methanol/water (30:60:8, v/v/v) for removal of neutral compounds: fractions 737

1-8. The fractions were sequentially eluted with 500 mL of chloroform/ methanol/water 738

(30:60:8, v/v/v) supplemented with 5 mM ammonium acetate: fractions 9-14, 10 mM 739

ammonium acetate: fractions 15-20; 25 mM ammonium acetate: fractions 21-28; 50 mM 740

ammonium acetate: fractions 29-35; 100 mM ammonium acetate: fractions 36-43; 250 741

mM ammonium acetate: fractions 44-49, see Fig. S11 in supplemental information. The 742

purification process was monitored by on HP-TLC impregnated with freshly prepared 0.2 743



27

M ammonium acetate dissolved in methanol, and chromatographed in chloroform/ 744

methanol/ NH4OH (4N in water) (9:7:2, v/v). Lipids were visualized by spraying plates 745

with primuline. GIPC-containing fractions were dissolved in water, and dialyzed against 746

water at 4 °C for two days to remove ammonium acetate (Spectra/Por Dialysis 747

Membrane, MWCO 3500). The water was changed every 6h. The desalted fractions were 748

dried and dissolved in a volume of 3 mL of chloroform/methanol/water (30:60:8, v/v/v), 749

and stored at 4°C. 750

751

Generation of rabbit polyclonal antibodies to GIPC—Preparation of liposomes: 752

Liposomes were prepared essentially as described previously (Richards et al., 1998). 753

Liposomes for the primary immunization were composed of purified BY-2 cell GIPCs 754

(series A or mix of series B-E), phosphatidylcholine, phosphatidylglycerol, cholesterol in 755

mole ratios of 0.9 / 0.1 / 0.75. Lipid A was included in the liposomes at 20 nmol of lipid 756

A per µmol of phospholipid. Lipids were dried from chloroform/Methanol/water 30/60/8 757

solution. The liposomes were swollen in 1 mL of TBS by vigorous shaking in a vortex 758

mixer, and sonicated at room temperature for 30 min. Immunization of rabbits: Rabbits 759

were immunized four times at 0, 21, 42 days (COVALAB - France Biotechnologies, 760

France). Preimmune serum compare with 53 days post injection serum were analyzed. 761

762

Immunogold labeling of purified plant plasma membranes— Labeling was performed 763

on purified BY-2 cell PM vesicles according to (Noirot et al., 2014). Immunological 764

reaction on grids was performed for 1h with rabbit polyclonal antibody against 765

polyglycosylated GIPCs (46) diluted 1/40 which was revealed with a goat anti-rabbit IgG 766

conjugate (Aurion, Wageningen, The Netherlands) labeled to 6-nm colloidal gold 767

particles. Three independent experiments using three independent biochemical PM 768

purifications from BY-2 cells were recorded. For each experiment, 3 replicates of 769

immunolabeling and 2 replicates of each control sample (omission of the primary 770

antibody and use of the preimmune) were observed with a Hitachi H7500 transmission 771

electron microscope equipped with an AMT camera driven by AMT software. In order to 772

characterize the distribution of the detected antigen on PM vesicle surface, the density of 773

labeling was evaluated by counting the number of colloidal gold particles per labeled 774



28

vesicle. Groups of gold labeling were visualized and size(s) of the cluster(s) were 775

measured on each labeled vesicle with the AMT software. Proportions of gold particles in 776

groups and of isolated gold particles were evaluated. Counting and measurement were 777

performed on 49 pictures of PM vesicles from the three independent experiments. The 778

spatial distribution was determined as described in (Noirot et al., 2014) using the Ripley 779

function (Ripley, 1976). 780

781

Order Level Measurement of Artificial Membranes—Preparation of LUVs and 782

Fluorescence spectroscopy, membrane order level Measurement were as described in 783

(Grosjean et al., 2015) 784

785

Home made Lipid-blot— Immun-Blot PVDF Membrane (Bio-rad) were activated with 786

methanol for 30 sec. GIPC DEAE fractions (3µl) were deposited and let dried. 787

Membranes were further re-activated in methanol for 2 sec, blocked in TBS 788

supplemented with 5% de-fatted BSA for 1 h. Antibodies to GIPC (dilution 1/100) were 789

incubated for 1h at room temperature, membranes were then rinsed three times with TBS 790

supplemented with 0.1% (v/v) Tween 20, and revealed with anti-rabbit secondary 791

antibodies coupled to horse-radish peroxidase (1/100). 792

793

ELISA— All steps were performed at 37°C. ELISA plate were filled with or without 794

500ng of BY-2 cell PM vesicles in 0,1mL PBS for 2h, then blocked for 2h with 1% BSA 795

in PBS. Anti-GIPC antibodies (dilution 1/50 for sera or 1/100 for purified IgG) were 796

incubated for 1h and revealed with anti-rabbit secondary antibodies coupled to alkaline 797

phosphatase (1/5000). Reaction with p-Nitrophenyl Phosphate (0,01% in 10% 798

diethanolamine buffer) was read at 405 nm after 1h. 799

800

Langmuir trough—Total BY-2 GIPC (molecular weight, ca. 1260 g/mol) was used in 801

this study. A solution at 0.39mM in chloroform/methanol/water (30/60/8) was prepared. 802

Sitosterol was purchased from Avanti Polar Lipids (Alabaster, USA). It was dissolved at 803

0.39 mM in chloroform/methanol (2/1). The Π–A isotherms were recorded by means of 804

an automated Langmuir trough (KSV Minitrough, width: 75 mm, area: 24.225 mm², KSV 805



29

Instruments, Helsinki, Finland) equipped with a platinum plate attached to a Wilhelmy 806

type balance. The GIPC sample was heat up at 60°C for 15 minutes for a better 807

solubilization. Pure solutions and (0.15/0.85) molar mixtures of Sitosterol/GIPC were 808

spread (fixed volume of 30µL) as tiny droplets to produce a uniform monolayer on a 809

Tris/NaCl 10/150 mM (Millipore Co., Milford, MA) subphase adjusted at pH 7 with HCl. 810

After evaporation of the solvent (15 min), monolayers were compressed at a rate of 5 811

mm/min and at a temperature of 22 ± 1° °C. Before each experiment, the cleanliness of 812

the system was confirmed by checking the surface pressure over the surface compression 813

of the pure subphase. The reproducibility of the Π–A isotherms was checked by repeated 814

recordings and the relative standard deviation in surface pressure and area was found to 815

be ≤3%. 816

817

Molecular modeling approaches—The conformation of GIPC sitosterol and 818

glucosylceramide (d18:2(delta4, delta8)/h16:0) was calculated using the structure tree 819

procedure, as described elsewhere (Lins et al., 1996). The Hypermatrix (HM) docking 820

procedure was used to study the monolayer formed by GIPC and its interaction with 821

sitosterol, as already described (Lins et al., 1999) (Fa et al., 2007) (Bensikaddour et al., 822

2008) and reviewed recently, (Deleu et al., 2014). Briefly, one GIPC molecule is 823

positioned and fixed for the whole calculation at the center of the system, oriented at the 824

hydrophobic (Haimi et al.)/hydrophilic (phi) interface (Brasseur, 1990). The interacting 825

GIPC (for GIPC monolayer) or sitosterol (for mixed monolayer) is also oriented at the 826

pho/phi interface and, by rotations and translations, more than 107 positions of the 827

interacting molecule around the central molecule are calculated. The energy values 828

together with the coordinates of all assemblies are stored in a matrix and classified 829

according to decreasing values. The most stable matching is used to decide the position 830

of the first interacting molecule. The position of the second one is then defined as the 831

next most energetically favorable orientation stored in the matrix taking steric and 832

energetic constraints due to the presence of the first molecule into account. The process 833

ends when the central molecule is completely surrounded with the other molecule. In this 834

method, the lipid/water interface was taken into account by linearly varying the dielectric 835

constant ε between 3 (above the interface) and 30 (below the interface) and an empirical 836



30

equation for the hydrophobic energy is added in the force field, as described in (Lins and 837

Brasseur, 1995). The mean area occupied by one molecule in the complex was estimated 838

by projection on the x-y plane using a grid of 1 Å square. 839

To calculate the insertion of GIPCs or Soy bean glucosylceramide into an implicit 840

simplified bilayer, we used the IMPALA method described previously (Ducarme et al., 841

1998). Briefly, this method simulates the insertion of any molecule into a bilayer by 842

adding energy restraint functions to the usual energy description of molecules. The lipid 843

bilayer is defined by C(z), which represents an empirical function describing membrane 844

properties. This function is constant in the membrane plane (x- and y- axes) but varies 845

along the bilayer thickness (z-axis). Two restraints simulate the membrane, one the 846

bilayer hydrophobicity (Epho), and the other, the lipid perturbation (Elip). All the 847

equations were described elsewhere (Ducarme et al., 1998). The method was notably 848

successfully applied to small helical peptides of known configurations (Lins et al., 2001). 849

It provides insights of the behavior of peptide dynamics that cannot be obtained with 850

statistical approaches. All calculations were performed on a Linux station bi-xeon quad 851

core, using the home-designed Z-ultime software. 852

853



31

ACKNOWLEDGEMENTS 854

We thank Yohann Boutté and Patrick Moreau (LBM, Bordeaux) for critical reading of 855

the manuscript. We thank Michel Laguerre (IECB, Bordeaux) for the modelisation of 856

series A tobacco GIPC, Elodie Noirot (PF DimaCell, UMR Agroécologie, Dijon) and 857

Kiên Kiêu (INRA, UR341 Mathématiques et Informatique Appliquées, Jouy-en-Josas) 858

for spatial statistical analysis. We also acknowledge Paul Gouguet (LBM, Bordeaux) 859

Bernadette Codeville (Unité de Glycobiologie, Villeneuve d'Ascq) for the help in GIPC 860

purification, Veronique Aubert for her help in microscopy studies (PF DimaCell, UMR 861

Agroécologie), and Michel Ponchet (Institut Sophia Agrobiotech) for providing tobacco 862

leaves, and Yann Guérardel for the purification material (Unité de Glycobiologie, 863

Villeneuve d'Ascq). 864



32

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Vitiello, F., and Zanetta, J.P. (1978). Thin-layer chromatography of phospholipids. 1091

Journal of chromatography 166, 637-640. 1092



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Voxeur, A., and Fry, S.C. (2014). Glycosylinositol phosphorylceramides from Rosa cell 1093

cultures are boron-bridged in the plasma membrane and form complexes with 1094

rhamnogalacturonan II. The Plant journal : for cell and molecular biology 79, 1095

139-149. 1096

Wang, W., Yang, X., Tangchaiburana, S., Ndeh, R., Markham, J.E., Tsegaye, Y., 1097

Dunn, T.M., Wang, G.L., Bellizzi, M., Parsons, J.F., Morrissey, D., Bravo, 1098

J.E., Lynch, D.V., and Xiao, S. (2008). An inositolphosphorylceramide synthase 1099

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Worrall, D., Ng, C.K., and Hetherington, A.M. (2003). Sphingolipids, new players in 1102

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1106

1107



38

FIGURE LEGENDS 1108

Figure 1. Long chain (LCFA), very long chain (VLCFA) and 2-hydroxylated very 1109

long chain fatty acid (hVLCFA) content of tissue, microsomal, plasma membrane 1110

(PM) and Detergent-Insoluble Membranes (DIM) fractions from tobacco leaf or 1111

BY-2 cell culture 1112

A, Fatty Acids were released from biological samples by acid methanolysis; the resulting 1113

FAMEs were subsequently derivatized with BSTFA before GC/MS analysis. The data are 1114

expressed as the mean of three independent experiments. LCFA: Long Chain Fatty Acid 1115

with 16, 18 or 20 carbon atoms, VLCFA: Very Long Chain Fatty Acid with 22 to 26 1116

carbon atoms, hVLCFA, VLCFA with 22 to 26 carbon atoms hydroxylated in position 2; 1117

B, Histograms show the comparison between VLCFA and hVLCFA content of DIM and 1118

of GIPCs purified from tobacco leaves or BY-2 cell culture. The data are expressed as the 1119

mean of four independent experiments ±SD. 1120

1121

Figure 2. Analysis of GIPCs extracted from tobacco leaf and BY-2 cells by MALDI-1122

MS and HP-TLC. Polyglycosylated GIPCs are enriched in DIMs 1123

A, MALDI-MS analysis of GIPC extracts from BY-2 cells and tobacco leaf. Spectra were 1124

acquired in the negative ion mode using 2,6-dihydroxyacetophenone (DHA) as a matrix. 1125

GIPCs are grouped in series according to their number of saccharide units, from two 1126

(series A) to six (series E), see supplemental figure S7 for detailed analysis of the peaks; 1127

B, High Performance-Thin Layer Chromatography (HP-TLC) were used to separated the 1128

different series of GIPCs. Note that the series A show two bands called by Kaul and 1129

Lester 1975, PhytoSphingoLipid: PSL I and PSL II, corresponding to GlcNAc-GlcUA-1130

IPC and GlcNH2-GlcUA-IPC, respectively(Kaul and Lester, 1975). GIPCs extracted from 1131

Arabidopsis thaliana (At) and leek (Ap., Allium porrum) were used as HP-TLC standards 1132

for series A and B respectively, according to (Cacas et al., 2013). 1133

1134



39

Figure 3. Quantification of polyglycosylated GIPCs found in PM and DIMs of BY-2 1135

cells. 1136

Quantification by HP-TLC coupled to GC-MS of polyglycosylated GIPCs found in PM 1137

and DIMs of BY-2 cells. The data are expressed as the mean of three independent 1138

experiments as the percentage of total GIPCs found in PM and DIMs respectively ±SD. 1139

1140

Figure 4. Lipid content of tobacco leaf (A.) and BY-2 cell (B.) PM and DIMs 1141

Left: From the results presented in Fig. 1 and 2 and those obtained on phospholipids and 1142

sterols on the same plant materials (Furt et al., 2010), we were able to determine the lipid 1143

content of PM and DIM expressed as mol%. Right, The three main classes of lipids, 1144

namely phospholipids, sterols and sphingolipids were summed and represented as mol% 1145

of total lipids. The data are expressed as means of three independent experiments ± SD. 1146

Abbreviations are as followed: phosphatidylethanolamine (PE), phosphatidylcholine 1147

(PC), phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylinositol 4-phosphate 1148

(PI4P), phosphatidylinositol 4,5-bisphosphate (PI4,5P2), digalactosyldiacylglycerol 1149

(DGDG), Steryl-glucosides (SG), Acyl Steryl-glucosides (ASG), free sterols (Sterols), 1150

glucosylceramides (GluCER), GlycosylInositolPhosphorylCeramides (GIPCs). 1151

Right: sum of percentage of glycerolipids (PE, PC, PA, PI, PIP4P, PI4,5P2, DGDG) 1152

sterols (free sterols, SG, ASG) and sphingolipids (gluCER, GIPCs). 1153

1154

Figure 5. Test for the specificity of antibodies to polyglycosylated GIPCs 1155

A, Home–made PVDF dot-blots were done with DEAE fractions of GIPC purification, 1156

see supplemental Fig. S11. PVDF membranes were blotted with pre-immun serum 1157

(1/100) or with 53 days serum (1/100) immunized against polyglycosylated GIPCs, see 1158

experimental section; B, Lipids from BY-2 cell PM were separated on HP-TLC. The 1159

plates were blotted with pre-immun serum (1/100) or with 53 days serum (1/100) 1160

immunized against polyglycosylated GIPCs, see experimental section. 1161

1162

Figure 6. Polyglycosylated GIPCs locates in nanoscale membrane domains on BY-2 1163

cell PM vesicles. A, Transmission electron micrographs of negatively stained tobacco 1164

PM vesicles immunogold labeled on grids with purified antibodies to polyglycosylated 1165



40

GIPCs detected by 6 nm colloidal gold conjugated goat anti-rabbit. Circles indicate 1166

obvious membrane domains; Bars = 20 nm; B Total number of 49 independent gold-1167

labeled PM vesicles were analyzed for the statistics; C; Ripley’s K-function analysis of 1168

GIPCs distribution on the surface of PM vesicles: y axis K(r) is the average number of 1169

particles lying at a distance less than r (x axis), normalized by the mean particle density. 1170

This Ripley’s analysis of the labeled PM vesicles (black line) indicates clustering of the 1171

gold particles when compared to a theoretical simulation for a completely random 1172

(Poisson) point pattern (red dotted line). 1173

1174

Figure 7. Effect on membrane order level of tobacco leaf or BY-2 cell purified 1175

GIPCs, in combination with phospholipids and free sterols. 1176

The Red/Green Mean ratio of the membrane (RGM) of 1µm diameter LUVs of different 1177

compositions labeled with di-4ANEPPDHQ (3 µM) was measured by spectrofluorimetry 1178

in the presence of GIPCs isolated from tobacco BY-2 cells or tobacco leaves. Data shown 1179

are mean values ± SD, n=5 or more independent repetitions. The different letters indicate 1180

significantly different values (p-value < 0.05). 1181

1182

Figure 8. Surface pressure-area (Π-A) isotherms, at the air-aqueous phase interface, 1183

of pure GIPC () and sitosterol () monolayers and of mixed GIPC/sitosterol 1184

monolayer () prepared at GIPC a 0.85 molar ratio. 1185

A, The isotherms were recorded at 22 ± 1°C with an aqueous subphase composed by a 1186

Tris 10mM buffer at pH 7.0. Duplicate experiments using independent preparations 1187

yielded similar results; B, Comparison of the experimental (white bars) and theoretical 1188

(black bars) mean molecular areas at a surface pressure of 10 mN/m, 20 mN/m and 30 1189

mN/m for GIPC/sitosterol at a 0.85 GIPC molar ratio. The theoretical value is obtained 1190

according to the additivity rule : A12 = A1X1 + A2X2 where A12 is the mean molecular 1191

area for ideal mixing of the two components at a given Π, A1, and A2 are the molecular 1192

areas of the respective components in their pure monolayers at the same Π and X1 and 1193

X2 are the molar ratios of components 1 and 2 in the mixed monolayers; C, Excess free 1194

energy of mixing (ΔGex) (white bars), and free energy of mixing (ΔGM) (black bars) of 1195

the mixed monolayer GIPC/sitosterol at a 0.85 molar ratio of GIPC for various surface 1196



41

pressures. ΔGex and ΔGM were calculated according to the following equations (Maget-1197

Dana, 1999) (Eeman et al., 2005) : ΔGex = A12 dΠ0

Π

− X1 A1dΠ0

Π

− X2 A2 dΠ0

Π

where A is 1198

the mean molecular area, X is the molar fraction, subscripts 1 and 2 refer, respectively, to 1199

pure components 1 and 2 and 12 to their mixtures, and ΔGM =ΔGex +ΔGid where ΔGid is 1200

the free energy for ideal mixing and can be calculated from the following equation, 1201

ΔGid =RT X1 lnX1 + X2 lnX2( ) where R is the universal gas constant and T the absolute 1202

temperature. 1203

1204

Figure 9. Modeling approaches 1205

A, Theoretical interactions between 8 GIPC and 8 sitosterol molecules calculated by HM 1206

docking method. Sitosterol molecules are in green and GIPCs are coloured with carbon 1207

atoms in grey, oxygen in red, phosphorus in purple, nitrogen in blue and hydrogen in 1208

white; B, Most stable insertion of gluCER d18:2/h16:0 (left) and GIPCs t18:0/h24:0 1209

(Frazier and Alber, 2012) into an implicit bilayer calculated by IMPALA. The yellow 1210

plane represents the center of the bilayer; the mauve plane stands for the lipid polar 1211

head/acyl chain interface and the pink plane, for the water/lipid polar head interface; C, 1212

Interaction energies calculated for GIPCs and GIPCs/sitosterol (from Fig. 9A) 1213

monolayers. Epolar corresponds to polar and electrostatic interactions and Epho and 1214

Evdw, to hydrophobic and Van der Waals interactions, respectively. The mean calculated 1215

interfacial molecular areas for GIPCs alone or in interaction with sitosterol are also 1216

indicated. 1217

1218

Figure 10. Model for the organization of lipids in tobacco plasma membrane. 1219

To build this model, we took the molar composition of the BY-2 PM obtained in Fig. 4 1220

and used the data obtained by Tjellstrom and collaborators 2010, who were able to 1221

calculate the distribution of cytosolic/apoplastic lipids. We hypothesize that GIPCs are 1222

located exclusively in the apoplastic face, see text. 1223

1224



42

Supplemental Data 1225

The following materials are available in the online version of this article. 1226

1227

Supplemental Figure S1. Typical GC-MS spectrogram of total FAMES and sterols 1228

extracted from BY-2 cell PM. Two internal standards (IS) were added to quantify 1229

GIPC: 2-hydroxylated 14 carbon atom fatty acid (h14), and heptadecanoic acid (17:0). 1230

1231

Supplemental Figure S2. Fatty acid content of tissue, microsomal, plasma 1232

membrane (PM) and Detergent-Insoluble Membranes (DIM) fractions from tobacco 1233

leaves or BY-2 cell culture. A. Fatty Acids were released from biological samples by 1234

acid methanolysis; the resulting FAMEs were subsequently derivatized with BSTFA 1235

before GC/MS analysis. LCFA: Long Chain Fatty Acid with 16, 18 or 20 carbon atoms, 1236

VLCFA: Very Long Chain Fatty Acid with 22 to 26 carbon atoms, hVLCFA, 2-1237

hydroxylated Very Long Chain Fatty Acid with 22 to 26 carbon atoms. The data are 1238

expressed as the mean of three independent experiments ± SEs (Garssen et al.). 1239

B. Sum of LCFA: Long Chain Fatty Acid with 16, 18 or 20 carbon atoms, VLCFA: Very 1240

Long Chain Fatty Acid with 22 to 26 carbon atoms, hVLCFA, 2-hydroxylated Very Long 1241

Chain Fatty Acid with 22 to 26 carbon atoms expressed as nmol of FAMES per mg of 1242

proteins. The data are expressed as the mean of three independent experiments ± SEs 1243

(Garssen et al.). 1244

1245

Supplemental Figure S3. Fatty acid content of Detergent-Insoluble Membranes 1246

(DIM) vs. Detergent-Soluble Membranes (DSM) from tobacco leaf purified PM. 1247

Fatty Acids were released by acid methanolysis; the resulting FAMEs were subsequently 1248

derivatized with BSTFA before GC/MS analysis. LCFA: Long Chain Fatty Acid with 16, 1249

18 or 20 carbon atoms, VLCFA: Very Long Chain Fatty Acid with 22 to 26 carbon 1250

atoms, hVLCFA, 2-hydroxylated Very Long Chain Fatty Acid with 22 to 26 carbon 1251

atoms. The data are expressed as the mean of three independent experiments ± SEs 1252

(Garssen et al.). 1253

1254



43

Supplemental Figure S4. Fatty acid and sterol content of purified Acyl Steryl 1255

Glucosides (ASG) extracted from tobacco leaves or BY-2 cell culture. ASG was 1256

purified by HP-TLC (Lefebvre et al., 2007), scratched from the silica and submitted to 1257

either acid methanolysis for FAMES analysis, or to saponification for sterol analysis. 1258

After TMS derivatizasion, FAMES and sterols were quantified by GC-MS. The data are 1259

expressed as the mean of three independent experiments ± SEs (Garssen et al.). 1260

1261

Supplemental Figure S5. Fatty acid content of purified glucosyl ceramide (gluCER) 1262

extracted from tobacco leaves or BY-2 cell culture and purified by TLC. GluCER 1263

was purified by HP-TLC (Lefebvre et al., 2007), scratched from the silica and submitted 1264

to acid methanolysis for FAMES analysis, After BSTFA derivatizasion, FAMES were 1265

quantified by GC-MS. The data are expressed as the mean of three independent 1266

experiments ± SEs (Garssen et al.). 1267

1268

Supplemental Figure S6. LCB content of GIPCs, plasma membrane (PM) and 1269

Detergent-Insoluble Membranes (DIM) from tobacco leaves or BY-2 cell culture. A, 1270

LCBs were isolated by hydrolysis from GIPCs purified from leaf and BY-2 cells, 1271

converted to their fatty aldehydes by peroxydation and separated by GC, as described 1272

(Cacas et al., 2012a); B-C, LCBs content were determined in PM and DIM purified from 1273

tobacco leaves and BY-2 cells. Abbreviations are as follow: Peak nomenclature in the 1274

key is systematically based upon the 2-amino-acyl backbone of the LCB. t18:0: 2-1275

aminooctadecane-1,2,4-triol (trivial name phytosphingosine); t18:1(8Z): (Z)-2-1276

aminooctadec-8-ene-1,2,4-triol, (trivial name (8Z)-phytosphingenine); d18:1(4E): (E)-2-1277

aminooctadec-4-ene-1,2-diol (trivial name sphingosine); d18:2(4E/8Z,E): (E,Z)-2-1278

aminooctadeca-4,8-dienine-1,2-diol (trivial name (4E,8Z)-sphingadienine); d18:0: 2-1279

aminooctadecane-1,2-diol (trivial name sphinganine). 1280

1281

Supplemental Figure S7. MALDI-MS analysis of GIPC extracts from BY-2 cells. 1282

Spectra were acquired in the negative ion mode using 2,6-dihydroxyacetophenone (DHA) 1283

as a matrix. GIPCs are grouped in series according to their number of saccharide units, 1284

from two sugars (series A) to six (series E). 1285



44

1286

Supplemental Figure S8. MALDI-MS analysis of GIPC extracts purified from PM 1287

and DIMs extracted from tobacco leaves. Spectra were acquired in the negative ion 1288

mode using 2,6-dihydroxyacetophenone (DHA) as a matrix. 1289

1290

Supplemental Figure S9. Determination of lipid-to-protein ratio in plant PM. A, 100 1291

µg of BY-2 cell purified PM were extracted by protocol #1 (see experimental procedures) 1292

using chloroform/methanol/HCl, the aqueous phase were re-extracted by buranol-1. 1293

Histograms shows the total FA content recovered in each solvent fractions expressed as 1294

the mean ± SD of three independent experiments, compare with the direct 1295

transesterification of 100 µg of BY-2 cell purified PM. 1296

1297

Supplemental Figure S10. Fatty acid content of total lipids from grape cell culture. 1298

A, Fatty Acids were released from biological samples by acid methanolysis; the resulting 1299

FAMEs were subsequently derivatized with BSTFA before GC/MS analysis. LCFA: 1300

Long Chain Fatty Acid with 16, 18 or 20 carbon atoms, VLCFA: Very Long Chain Fatty 1301

Acid with 22 to 26 carbon atoms, hVLCFA, 2-hydroxylated Very Long Chain Fatty Acid 1302

with 22 to 26 carbon atoms. The data are expressed as the mean of three independent 1303

experiments ± SEs (Garssen et al.); B, Lipids from grape cell culture were extracted with 1304

or without preliminary hot isopropanol treatment. Polar lipis are further separated by HP-1305

TLC by the solvent migration described in (Vitiello and Zanetta, 1978). Abbreviations 1306

are as described in Fig. 3; C, Rationale for the three lipid extraction protocols; D, Fatty 1307

acid analyses of lyophilized grape cell culture by the three lipid extraction protocols with 1308

or without hot isopropanol pre-treatment, compare with the direct transesterification of 1309

grape cell culture (TOTAL, left). The data are expressed as means of three independent 1310

experiments ± SD. 1311

1312

Supplemental Figure S11. Purification of GIPCs from BY-2 cells by DEAE 1313

chromatography. A, Total BY-2 cell GIPC were separated by DEAE. The different 1314

fractions were eluted with increasing amount of ammonium acetate: fractions 9-49, see 1315

material and methods. The purification process was monitored by on HP-TLC. Lipids 1316



45

were visualized by spraying plates with primuline. GIPCBY2 are the starting material used 1317

as control standards for HP-TLC; B, Purified and dialyzed fractions (GIPC series A and 1318

B-F) were check for purifty by HP-TLC and MALDI-MS. Note the absence of residual 1319

glycerolipids and sterols, and the strong enrichment of series A in one hand (middle) and 1320

series B-F in the other hand. 1321

1322

Supplemental Figure S12. Test by ELISA of the specificity of antibodies against 1323

polyglycosylated GIPCs. ELISA were performed using BSA as negative control. Data 1324

represent mean value of four technical replicates. Vertical bars indicate standard error of 1325

the mean. 1326

1327

Supplemental Figure S13. Cross reactivity of antibodies against polyglycosylated 1328

GIPCs. A, The cross reactivity of antibodies against polyglycosylated GIPCs was 1329

performed on “PIP strips” according to manufacturers’s instructions (Echelon Bioscience, 1330

USA; http://www.echelon-inc.com). Membranes were first incubated with rabbit 1331

antibodies against polyglycosylated GIPCs (dilution 1/100 for 1h at RT) and further with 1332

horseradish peroxydase-conjugated secondary anti mouse antibody (dilution 1/15,000 for 1333

1h at RT); B, A negative control with preimmume serum, or without primary antibodies 1334

is shown at the bottom panel; C, Positive control is performed with antibodies against 1335

PI4,5P2 (Antibodies against native PI4,5P2 from bovine spinal cord (www.Assay 1336

designs.com); dilution 1/1,000 for 1h at RT). Note that these antibodies are 10-fold more 1337

diluted than antibodies against polyglycosylated GIPCs. 1338

1339

Supplemental Figure S14. Immumogold labeling controls of PM vesicles. 1340

A, omission of the primary antibody; B, use of the pre-immune serum of rabbit used for 1341

immunization of polyglycosylated GIPCs with the preimmune serum of rabbit used for 1342

immunization of polyglycosylated GIPCs; C, with antibodies against the proton pump 1343

ATPase PMA as used in (Raffaele et al., 2009) 1344

1345



Figure 1

0%

20%

40%

60%

80%

100%

Total Microsome PM DIM 0%

20%

40%

60%

80%

100%

Total Microsome PM DIM

LCFA hVLCFA VLCFA

Tobacco leaves BY-2 cells A

B Tobacco leaves

BY-2 cells

0 5

10 15 20 25 30 35 40 45 50

Mol

. %

DIM

GIPC

0

5

10

15

20

25

30

35

40

Mol

. %

DIM

GIPC

Figure 1. Long chain (LCFA), very long chain (VLCFA) and 2-hydroxylated very long chain fatty acid (hVLCFA) content of tissue, microsomal, plasma membrane (PM) and Detergent-Insoluble Membranes (DIM) fractions from tobacco leaf or BY-2 cell culture A, Fatty Acids were released from biological samples by acid methanolysis; the resulting FAMEs were subsequently derivatized with BSTFA before GC/MS analysis. The data are expressed as the mean of three independent experiments. LCFA: Long Chain Fatty Acid with 16, 18 or 20 carbon atoms, VLCFA: Very Long Chain Fatty Acid with 22 to 26 carbon atoms, hVLCFA, VLCFA with 22 to 26 carbon atoms hydroxylated in position 2; B, Histograms show the comparison between VLCFA and hVLCFA content of DIM and of GIPCs purified from tobacco leaves or BY-2 cell culture. The data are expressed as the mean of four independent experiments ±SD.



140723_BY2(3)_140513 0:B22 MS Raw

0

2000

4000

6000Inte

ns. [

a.u.

]

140723_Nt(4)_140513 0:A22 MS Raw

0

1000

2000

3000

4000

5000Inte

ns. [

a.u.

]

1200 1300 1400 1500 1600 1700 1800 1900m/z

Figure 2

A

D C

B

A Tobacco leaf

BY-2 cell

A MALDI-MS of total GIPCs

B HP-TLC of total GIPCs

E

}A ----B

GIP

C series

- PSL1

- PSL2

At. Nt leaf

A---

Ap. BY-2

--B ---D

GIP

C series

Glycerolipids + sterols

B---

----start

------E

}A

Figure 2. Analysis of GIPCs extracted from tobacco leaf and BY-2 cells by MALDI-MS and HP-TLC. Polyglycosylated GIPCs are enriched in DIMs A, MALDI-MS analysis of GIPC extracts from BY-2 cells and tobacco leaf. Spectra were acquired in the negative ion mode using 2,6-dihydroxyacetophenone (DHA) as a matrix. GIPCs are grouped in series according to their number of saccharide units, from two (series A) to six (series E), see supplemental figure S7 for detailed analysis of the peaks; B, High Performance-Thin Layer Chromatography (HP-TLC) were used to separated the different series of GIPCs. Note that the series A show two bands called by Kaul and Lester 1975, PhytoSphingoLipid: PSL I and PSL II, corresponding to GlcNAc-GlcUA-IPC and GlcNH2-GlcUA-IPC, respectively(Kaul and Lester, 1975). GIPCs extracted from Arabidopsis thaliana (At) and leek (Ap., Allium porrum) were used as HP-TLC standards for series A and B respectively, according to (Cacas et al., 2013).



0

20

40

60

80

100

series A series B series D series E-F

GIPC series of BY-2 PM and DIM %

of t

otal

GIP

C

Figure 3

Figure 3. Quantification of polyglycosylated GIPCs found in PM and DIMs of BY-2 cells. Quantification by HP-TLC coupled to GC-MS of polyglycosylated GIPCs found in PM and DIMs of BY-2 cells. The data are expressed as the mean of three independent experiments as the percentage of total GIPCs found in PM and DIMs respectively ±SD.



0

10

20

30

40

50

60

70

80

Mol

.%

PM

DIM

Glycerolipids Sterols Sphingolipids

A

Figure 4

Tobacco leaves

0

10

20

30

40

50

60

70

80

Mol

. %

PM

DIM

BY-2 cells

Glycerolipids Sphingolipids Sterols

0

10

20

30

40

50

60

70

Mol

.%

PM

DIM

B

0

10

20

30

40

50

60

70

Mol

. %

PM

DIM

Figure 4. Lipid content of tobacco leaf (A.) and BY-2 cell (B.) PM and DIMs Left: From the results presented in Fig. 1 and 2 and those obtained on phospholipids and sterols on the same plant materials (Furt et al., 2010), we were able to determine the lipid content of PM and DIM expressed as mol%. Right, The three main classes of lipids, namely phospholipids, sterols and sphingolipids were summed and represented as mol% of total lipids. The data are expressed as means of three independent experiments ± SD. Abbreviations are as followed: phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PI4P), phosphatidylinositol 4,5-bisphosphate (PI4,5P2), digalactosyldiacylglycerol (DGDG), Steryl-glucosides (SG), Acyl Steryl-glucosides (ASG), free sterols (Sterols), glucosylceramides (GluCER), GlycosylInositolPhosphorylCeramides (GIPCs). Right: sum of percentage of glycerolipids (PE, PC, PA, PI, PIP4P, PI4,5P2, DGDG) sterols (free sterols, SG, ASG) and sphingolipids (gluCER, GIPCs).



Figure 5 A B

Primulin pre-immune immune

GIPC

PL

gluCER

Neutral lipids

anti GIPC (1/100)

-----SG

anti GIPC (1/100)

pre-immun immun

45  49 45 49 DEAE fractions

A{

B--

Figure 5. Test for the specificity of antibodies to polyglycosylated GIPCs A, Home–made PVDF dot-blots were done with DEAE fractions of GIPC purification, see supplemental Fig. S10. PVDF membranes were blotted with pre-immun serum (1/100) or with 53 days serum (1/100) immunized against polyglycosylated GIPCs, see experimental section; B, Lipids from BY-2 cell PM were separated on HP-TLC. The plates were blotted with pre-immun serum (1/100) or with 53 days serum (1/100) immunized against polyglycosylated GIPCs, see experimental section.



Figure 6

!Cluster!size!of!vesicles!with!3!gold!particles!and!higher! 34.5!±!7.8nm!

Average!diameter!of!PM!vesicles! 246.3!nm!

Average!PM!vesicle!area! 0,047!µm2!Average!number!of!gold!per!PM!vesicle! 6.9!Average!number!of!gold!particles!per!cluster!! 4.1!Distance!between!two!neighboring!clusters! >!20!nm!!

B

A

C

Immunogold labeling on PM vesicles with antibodies to polyglycosylated GIPCs

Statistical analysis

Ripley’s Function analysis of GIPCs distribution

20 nm 20 nm 20 nm

Figure 6. Polyglycosylated GIPCs locates in nanoscale membrane domains in BY-2 cell PM vesicles. A, Transmission electron micrographs of negatively stained tobacco PM vesicles immunogold labeled on grids with purified antibodies to polyglycosylated GIPCs detected by 6 nm colloidal gold conjugated goat anti-rabbit. Circles indicate obvious membrane domains; Bars = 20 nm; B, Total number of 49 independent gold-labeled PM vesicles were analyzed for the statistics; C, Ripley’s K-function analysis of GIPCs distribution on the surface of PM vesicles: y axis (K(r) is the average number of particles lying at a distance less than r (x axis) from a typical particle, normalized by the mean particle density. This Ripley’s analysis of the labeled PM vesicles (black line) indicates clustering of the gold particles when compared to a theoretical simulation for a completely random (Poisson) point pattern (red dotted line).



Figure 7

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

a

b c

a

b c c

b a

RG

M

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Figure 7. Effect on membrane order level of tobacco leaf or BY-2 cell purified GIPCs, in combination with phospholipids and free sterols. The red/green ratio (Nichols et al.) of 1µm diameter LUVs of different compositions labeled with di-4ANEPPDHQ (3 µM) was measured by spectrofluorimetry in the presence of GIPCs isolated from tobacco BY-2 cells or tobacco leaves. Data shown are mean values ± SD, n=5 or more independent repetitions. The different letters indicate significantly different values (p-value < 0.05). RGM: Red/Green Mean ratio of the membrane.



Figure 8

0

10

20

30

40

50

60

70

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Figure 8. Surface pressure-area (Π-A) isotherms, at the air-aqueous phase interface, of pure GIPC (!) and sitosterol (!) monolayers and of mixed GIPC/sitosterol monolayer (!) prepared at GIPC a 0.85 molar ratio. A, The isotherms were recorded at 22 ± 1°C with an aqueous subphase composed by a Tris 10mM buffer at pH 7.0. Duplicate experiments using independent preparations yielded similar results; B, Comparison of the experimental (white bars) and theoretical (black bars) mean molecular areas at a surface pressure of 10 mN/m, 20 mN/m and 30 mN/m for GIPC/sitosterol at a 0.85 GIPC molar ratio. The theoretical value is obtained according to the additivity rule : A12 = A1X1 + A2X2 where A12 is the mean molecular area for ideal mixing of the two components at a given Π, A1, and A2 are the molecular areas of the respective components in their pure monolayers at the same Π and X1 and X2 are the molar ratios of components 1 and 2 in the mixed monolayers; C, Excess free energy of mixing (DGex) (white bars), and free energy of mixing (DGM) (black bars) of the mixed monolayer GIPC/sitosterol at a 0.85 molar ratio of GIPC for various surface pressures. DGex and DGM were calculated according to the following equations (Maget-Dana, 1999) (Eeman et al., 2005) : where A is the mean molecular area, X is the molar fraction, subscripts 1 and 2 refer, respectively, to pure components 1 and 2 and 12 to their mixtures, and where DGid is the free energy for ideal mixing and can be calculated from the following equation, where R is the universal gas constant and T the absolute temperature.



A

B

Figure 9

gluCER GIPC series A

GIPC series A

sitosterol

Figure 9. Modeling approaches A, Theoretical interactions between 8 GIPC and 8 sitosterol molecules calculated by HM docking method. Sitosterol molecules are in green and GIPCs are coloured with carbon atoms in grey, oxygen in red, phosphorus in purple, nitrogen in blue and hydrogen in white; B, Most stable insertion of gluCER d18:2/h16:0 (left) and GIPCs t18:0/h24:0 (Frazier and Alber, 2012) into an implicit bilayer calculated by IMPALA. The yellow plane represents the center of the bilayer; the mauve plane stands for the lipid polar head/acyl chain interface and the pink plane, for the water/lipid polar head interface; C, Interaction energies calculated for GIPCs and GIPCs/sitosterol (from Fig. 9A) monolayers. Epolar corresponds to polar and electrostatic interactions and Epho and Evdw, to hydrophobic and Van der Waals interactions, respectively. The mean calculated interfacial molecular areas for GIPCs alone or in interaction with sitosterol are also indicated.

!

!

Molecule! Etot! Evdw! Epolar! Epho! Mean!area!(Å2)!GIPC! :23.5±2! :7.1±1! :2.3±0.4! :14.1±1.2! 68.8±10!GIPC/sitosterol! :20.9±1.3! :10.2±1! :0.4±0.1! :10.3±1.1! 57.6±8!

C



Figure 10

Apoplastic side

Cytoplasmic side Inner leaflet raft

Outer leaflet raft

Figure 10. Model for the organization of lipids in tobacco plasma membrane. To build this model, we took the molar composition of the BY-2 PM obtained in Fig. 4 and used the data obtained by Tjellstrom and collaborators 2010, who were able to calculate the distribution of cytosolic vs. apoplastic lipids. We hypothesize that GIPCs are located exclusively in the apoplastic face, see text.



Parsed CitationsBensikaddour, H., Fa, N., Burton, I., Deleu, M., Lins, L., Schanck, A., Brasseur, R., Dufrene, Y.F., Goormaghtigh, E., and Mingeot-Leclercq, M.P. (2008). Characterization of the interactions between fluoroquinolone antibiotics and lipids: a multitechniqueapproach. Biophysical journal 94, 3035-3046.

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Blaas, N., and Humpf, H.U. (2013). Structural profiling and quantitation of glycosyl inositol phosphoceramides in plants with Fouriertransform mass spectrometry. Journal of agricultural and food chemistry 61, 4257-4269.


Borner, G.H., Sherrier, D.J., Weimar, T., Michaelson, L.V., Hawkins, N.D., Macaskill, A., Napier, J.A., Beale, M.H., Lilley, K.S., andDupree, P. (2005). Analysis of detergent-resistant membranes in Arabidopsis. Evidence for plasma membrane lipid rafts. Plantphysiology 137, 104-116.


Brasseur, R. (1990). TAMMO: theoretical analysis of membrane molecular organisation,. In Mol. Descr. Biol. Membr. Componentsby Comput. Conform. Anal., (CRC Press, Boca Raton), pp. pp. 203-219.


Bure, C., Cacas, J.L., Wang, F., Gaudin, K., Domergue, F., Mongrand, S., and Schmitter, J.M. (2011). Fast screening of highlyglycosylated plant sphingolipids by tandem mass spectrometry. Rapid communications in mass spectrometry : RCM 25, 3131-3145.


Cacas, J.L., Melser, S., Domergue, F., Joubes, J., Bourdenx, B., Schmitter, J.M., and Mongrand, S. (2012a). Rapid nanoscalequantitative analysis of plant sphingolipid long-chain bases by GC-MS. Analytical and bioanalytical chemistry 403, 2745-2755.


Cacas, J.L., Bure, C., Furt, F., Maalouf, J.P., Badoc, A., Cluzet, S., Schmitter, J.M., Antajan, E., and Mongrand, S. (2013). Biochemicalsurvey of the polar head of plant glycosylinositolphosphoceramides unravels broad diversity. Phytochemistry 96, 191-200.


Cacas, J.L., Furt, F., Le Guedard, M., Schmitter, J.M., Bure, C., Gerbeau-Pissot, P., Moreau, P., Bessoule, J.J., Simon-Plas, F., andMongrand, S. (2012b). Lipids of plant membrane rafts. Progress in lipid research 51, 272-299.


Carmona-Salazar, L., El Hafidi, M., Gutierrez-Najera, N., Noyola-Martinez, L., Gonzalez-Solis, A., and Gavilanes-Ruiz, M. (2015).Fatty acid profiles from the plasma membrane and detergent resistant membranes of two plant species. Phytochemistry 109, 25-35.


Carter, H.E., Gigg, R.H., Law, J.H., Nakayama, T., and Weber, E. (1958). Biochemistry of the sphingolipides. XI. Structure ofphytoglycolipide. The Journal of biological chemistry 233, 1309-1314.


Cheng, H.T., Megha, and London, E. (2009). Preparation and properties of asymmetric vesicles that mimic cell membranes: effectupon lipid raft formation and transmembrane helix orientation. The Journal of biological chemistry 284, 6079-6092.


Deleu, M., Crowet, J.M., Nasir, M.N., and Lins, L. (2014). Complementary biophysical tools to investigate lipid specificity in theinteraction between bioactive molecules and the plasma membrane: A review. Biochimica et biophysica acta 1838, 3171-3190.


Deleu, M., Nott, K., Brasseur, R., Jacques, P., Thonart, P., and Dufrene, Y.F. (2001). Imaging mixed lipid monolayers by dynamicatomic force microscopy. Biochimica et biophysica acta 1513, 55-62.

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Fang, K., Zou, G., and He, P. (2003). Dynamic viscoelastic properties of spread monostearin monolayer in the presence of glycine.Journal of colloid and interface science 266, 407-414.


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http://scholar.google.com/scholar?as_q=Abnormal glycosphingolipid mannosylation triggers salicylic acid-mediated responses in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Mortimer,&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

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http://www.ncbi.nlm.nih.gov/pubmed?term=%28The Plant journal %3A for cell and molecular biology%5BTitle%5D%29 AND 79%5BVolume%5D AND 139%5BPagination%5D AND Voxeur%2C A%2E%2C and Fry%2C S%2EC%2E%5BAuthor%5D&dopt=abstract

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http://scholar.google.com/scholar?as_q=Glycosylinositol phosphorylceramides from Rosa cell cultures are boron-bridged in the plasma membrane and form complexes with rhamnogalacturonan II&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Voxeur,&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

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Corresponding author - plantphysiol.org · 162 and labeled with the environment-sensitive probe di-4-ANEPPDHQ (Grosjean et al., 163 2015). Fluorescence spectroscopy experiments showed