Adiilah Mamode Cassim

Yotam Navon

Yu Gao

Marion Decossas

Laetitia Fouillen

Axelle Grélard

Minoru Nagano

Olivier Lambert

Pierre Van Delft

Delphine Bahammou

Lilly Maneta- Peyret

Françoise Simon-Plas

Laurent Heux

Bruno Jean

Giovanna Fragneto

Jenny C Mortimer

Magali Deleu

Laurence Lins

Sébastien Mongrand
email: sebastien.mongrand@u-bordeaux.fr

Agroécologie

Biophysical analysis of the plant-specific GIPC sphingolipids reveals multiple modes of membrane regulation

Keywords: Plant, plasma membrane, sphingolipids, GIPC, phytosterol, purification, lipidomics, modelling, ζ-Potential, Langmuir monolayer, neutron reflectivity, solid state NMR, cryo-EM

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

The plant plasma membrane (PM) contains three main classes of lipids: phytosterols, sphingolipids and phospholipids, all with a high level of molecular complexity, see [START_REF] Cacas | Revisiting plant plasma membrane lipids in tobacco: A focus on sphingolipids[END_REF][START_REF] Yetukuri | Informatics and computational strategies for the study of lipids[END_REF] . Sphingolipids are part of essential lipids involved in the regulation of cellular signaling, trafficking, growth and stress responses. Ubiquitous to eukaryotes, they are structurally different between the animal, fungi and plant kingdoms [START_REF] Markham | Plant sphingolipids: Function follows form[END_REF] . While some sphingolipid structures such as sphingoid bases are conserved in both plants and animal, others are specific to fungi and plants. In animal, sphingolipids are highly studied for their involvement in human health and pathologies [START_REF] Hannun | Sphingolipids and their metabolism in physiology and disease[END_REF] .

The most abundant sphingolipid in animal is sphingomyelin and gangliosides. In plants and fungi, they are absent, while other complex lipids comprised of sphingoid bases bound to glycan groups are part of the most abundant sphingolipid. The major sphingolipid subclass of sphingolipids in plants is the Glycosyl Inositol Phosphoryl Ceramides (GIPCs). GIPCs were discovered in plants and fungi during the 1950's [START_REF] Carter | Biochemistry of the sphingolipides. XI. Structure of phytoglycolipide[END_REF] . The structural diversity of GIPCs lies in the glycosylation and in the hydroxylation, degree and position of saturation of their fatty acid (FA) chain and long chain base (LCB) [START_REF] Pata | Plant sphingolipids: Decoding the enigma of the Sphinx[END_REF] . Plant GIPCs predominantly consist of a t18:0 or t18:1 LCB (trihydroxylated saturated or monounsaturated) amidified to a Very Long Chain Fatty Acid (VLCFA) or 2-hydroxylated VLCFA (hVLCFA) to form a ceramide [START_REF] Cacas | Revisiting plant plasma membrane lipids in tobacco: A focus on sphingolipids[END_REF][START_REF] Buré | Fast screening of highly glycosylated plant sphingolipids by tandem mass spectrometry[END_REF] .

The GIPC head group linked to the ceramide consists of a phosphate bound to an inositol, forming the inositol phosphoryl ceramide (IPC) backbone, which is then further substituted with further sugar moieties. A broad study of the GIPC polar heads of 23 plant species from algae to monocots showed that polar head structures are largely unknown and vary widely across different biological taxa [START_REF] Cacas | Biochemical survey of the polar head of plant glycosylinositolphosphoceramides unravels broad diversity[END_REF] . GIPCs are classified into series, based on the degree of glycosylation of their polar head group [START_REF] Buré | Fast screening of highly glycosylated plant sphingolipids by tandem mass spectrometry[END_REF] . In plants, all GIPCs characterized to date have a glucuronic acid (GlcA) as the first sugar on the IPC, followed by at least one more sugar unit of varying identity. For example, GIPC series A is defined as one monosaccharide addition to the GlcA-IPC form [START_REF] Buré | Fast screening of highly glycosylated plant sphingolipids by tandem mass spectrometry[END_REF] . In the 1960s, the first characterization of a GIPC structure from Nicotiania tabacum (tobacco) was described .

The GIPC extraction method required hundreds of kilograms of plant material and liters of solvents. From the study, the reported series A GIPC still has the best described structure to date: GlcNAc(α1à4)GlcA(α1à2)inositol-1-Ophosphorylceramide, see Figure 1A. Additional sugar moieties were described, such as glucosamine (GlcN), N-acetyl-glucosamine (GlcNAc), arabinose (Ara), galactose (Gal) and mannose (Man), which may lead to observed glycan patterns of three to seven sugars, the socalled GIPC series B to F. It is noteworthy that Kaul and Lester calculated the ratio between carbohydrate/LCB/inositol in purified polyglycosylated GIPCs and showed that they may contain up to 19-20 sugars [START_REF] Kaul | Characterization of Inositol-containing Phosphosphingolipids from Tobacco Leaves[END_REF] , which opens a very large field of investigation.

Polyglycosylated GIPCs found in Zea mays (corn) seeds and Erodium displays branched polar heads [START_REF] Sperling | Plant sphingolipids: Structural diversity, biosynthesis, first genes and functions[END_REF][START_REF] Buré | Branched glycosylated inositolphosphosphingolipid structures in plants revealed by MS3 analysis[END_REF] . GIPC series are species-and tissue-specific. In Arabidopsis, the GIPC series A headgroup Man-GlcA-IPC is predominant in leaves and callus [START_REF] Mortimer | Abnormal glycosphingolipid mannosylation triggers salicylic acid-mediated responses in Arabidopsis[END_REF]17 , whereas a complex array of N-acetyl-glycosylated with up to three pentose units are present in pollen [START_REF] Luttgeharm | Sphingolipid metabolism is strikingly different between pollen and leaf in Arabidopsis as revealed by compositional and gene expression profiling[END_REF] . Amino-and Nacylated-GIPCs are found in Arabidopsis seeds and oil [START_REF] Tellier | Comparative plant sphingolipidomic reveals specific lipids in seeds and oil[END_REF] . GlcN(Ac)-GlcA-IPC are mainly found in rice and tobacco [START_REF] Buré | Fast screening of highly glycosylated plant sphingolipids by tandem mass spectrometry[END_REF][START_REF] Nagano | Plasma membrane microdomains are essential for Rac1-RbohB/H-mediated immunity in rice[END_REF] . In monocots, the predominant GIPC series is series B [START_REF] Buré | Fast screening of highly glycosylated plant sphingolipids by tandem mass spectrometry[END_REF] , their core

structures are yet to be deciphered.

The GIPC's polar head is responsible for the high polarity of the GIPC, accounting for its insolubility in traditional lipid extraction solvents, such as chloroform/methanol.

Consequently, they are lost in the aqueous phase or at the interface. GIPCs, although one of the fundamental components of the plant PM model have been poorly studied, in part due to the absence of commercial preparations. Recent evidence has demonstrated that a loss of the glycosylation is lethal, [START_REF] Rennie | Identification of a sphingolipid α-glucuronosyltransferase that is essential for pollen function in Arabidopsis[END_REF][START_REF] Ishikawa | GLUCOSAMINE INOSITOLPHOSPHORYLCERAMIDTRANSFERASE1 (GINT1) is a GlcNAc-containing glycosylinositol phosphorylceramide glycosyltransferase[END_REF] , and that misglycosylation affects both abiotic and biotic stress responses, as reviewed in [START_REF] Mortimer | Synthesis and Function of Complex Sphingolipid Glycosylation[END_REF] . This highlighted the importance of investigating and understanding the chemical structures of these molecules and their functions in membrane organization.

Lipids are not homogeneously distributed within the PM bilayers. The lateral partitioning observed in the PM might be due to differential phase behaviours of different lipid species due to specific interactions between their different lipid species [START_REF] Kaiser | Order of lipid phases in model and plasma membranes[END_REF] . This was reported in model membranes, using biophysical approaches and super resolution microscopy [START_REF] Levental | The Continuing Mystery of Lipid Rafts[END_REF] . Lipid domains or liquid-ordered (Lo) phases are formed from saturated phospholipids and sphingolipids in the presence of sterol, while liquid-disordered (Ld)

phases are formed mainly from unsaturated phospholipids [START_REF] Lingwood | Lipid rafts as a membrane-organizing principle[END_REF][START_REF] Baumgart | Fluorescence probe partitioning between Lo/Ld phases in lipid membranes[END_REF] . In Lo phases, the high degree of conformational order is imposed on the acyl tails of lipids by the rigid ring structure of cholesterol. This increases the thickness of the lipid bilayer and lipid packing, although lipids remain laterally mobile [START_REF] Mannock | The effect of variations in phospholipid and sterol structure on the nature of lipid-sterol interactions in lipid bilayer model membranes[END_REF] . Sphingolipid-sterol interactions have recently been reported as important determinants of lipid partitioning and organization within the PM [START_REF] Gerbeau-Pissot | Modification of plasma membrane organization in tobacco cells elicited by cryptogein[END_REF][START_REF] Grosjean | Differential effect of plant lipids on membrane organization specificities of phytosphingolipids and phytosterols[END_REF][START_REF] Beck | Plant sterols in "rafts": a better way to regulate membrane thermal shocks[END_REF]

RESULTS

Extraction and purification of GIPCenriched fractions from different plant species tissues and cell culture

To assist with purifying the milligram amount of GIPCs required for analysis, we first assessed the amount of GIPCs in different plant species and tissues. We chose species/tissues which are easily and abundantly available, and quantified the non-hydroxylated VLCFA and 2hydroxylated hVLCFA, diagnostic of plant GIPC 3 mg/ml and 3.4 mg/ml per fresh weight respectively (Figure 1B). BY2 cells and leek both had a much lower GIPC content, with a mean estimated content of 0.6 mg/ml and 0.4 mg/ml per fresh weight respectively.

To maximize the yield, several trials were performed to test the different published protocols of GIPCs. 12 , 36 13 . Figure 2 shows the extraction and purification processes selected combining the most efficient steps of each of these protocols to obtain GIPC-enriched fractions of cauliflower (Bo-GIPC), tobacco BY-2 (Nt-GIPC), leek (Ap-GIPC) and rice (Os-GIPC). Some fine-tuning was done to maximize the yield such as refluxing in boiling ethanol for 20 min and using large lab-made silica column to process several hundreds of grams of material (see Material and Methods). Crude sphingolipid extracts were directly dried in silica deposited on the top of the column chromatography. The column was then washed with 4 column volumes (cv) of a mix of chloroform/methanol with increasing polarity to remove sterols, glucosylceramide and phospholipids. For the elution of GIPCs, a step gradient of chloroform/methanol/water was used (Figure 2A), so that molecules of increasing polarity were eluted in the last fractions. All washes and elution fractions were collected and analyzed by highperformance thin layer chromatography (HPTLC) as shown in Supplementary data 1.

HPTLC was a quick and reliable way to select fractions enriched with GIPCs, because it allowed the clear separation of sterols, phospholipids, and GIPC series.

Fatty acid content of GIPC-enriched fractions

To estimate the GIPC content, as well as the phospholipid contamination (containing medium chain fatty acid FA of C16-18), samples were trans-esterified to release both fatty acidesterified glycerolipids and fatty acid-amidified sphingolipids. The samples were then analyzed by Gas Chromatograph-Mass Spectrometry (GC-MS). The percentage of fatty acid in the samples with medium chain length (%FA) and hydroxylated and non-hydroxylated very long chain length (%(h)VLCFA) were calculated from 2 to 3 independent experiments. Samples retained after step 1 (raw plant material), step 2 (crude sphingolipid extract) and step 3 (GIPC-enriched fractions) were analyzed for their fatty acid content. As we proceeded through the purification steps, the amount of medium chain FA decreased as the amount of (h)VLCFA increased (Figure 2B). At step 2, the percentage of (h)VLVCFA in the sphingolipid extract was around 50% and at the final step, the amount of (h)VLCFA was at about 80% for all GIPCenriched extracts (Figure 2B A). The detailed FA composition of the GIPC-enriched fractions of all four species is provided in supplementary data 2.

It was estimated that the enrichment in GIPC between the first and last steps of the extraction and purification process was 5-fold for Bo-GIPC, 4.2-fold for Nt-GIPC, 3.6-fold for Ap-GIPC but only 2-fold for Os-GIPC.

The final products were analyzed by HPTLC to verify the lipid composition, and they revealed the predominance of GIPCs (Figure 3). Only traces of sterols and phospholipids were observed, and glucosylceramide (GluCer) was not detected. As reported in [START_REF] Buré | Fast screening of highly glycosylated plant sphingolipids by tandem mass spectrometry[END_REF] , eudicots contained mainly series A, monocots series B and plant cells in liquid culture media a mix of GIPCs with highly glycosylated ones. The Bo-GIPC enriched fraction contained one major band of GIPC series A. The Nt-GIPC fraction contained GIPC series A to F, further separated into three fractions (α, β and γ) of increasing polarity. The less polar fraction α contained two bands of series A GIPC closely packed together, representing PhytoSphingoLipid 1, PSL1 (with N-acetyl glucosamine) and PSL2 (with glucosamine) as described in [START_REF] Kaul | Characterization of Inositol-containing Phosphosphingolipids from Tobacco Leaves[END_REF] (Figure 3), and a band of series B.

The more polar fractions β and γ showed the presence of the highly polyglycosylated D to F series GIPC (Figure 3). As previously published monocots, Ap-GIPC and Os-GIPC enriched fractions contained mainly GIPC series B, some series A and to a lesser extent polyglycosylated GIPCs also present.

Regarding GIPC-derived (h)VLCFA, the predominant species was dependent on the starting material. The Bo-GIPC enriched fraction consisted of h24, h24:1 and h26 as the main fatty acyl chain, Nt-GIPC with h22, h23, h24 and h25 acyl chain, Ap-GIPC with C24, h24, h22 and C22

and Os-GIPC with C24, C22, C20 and h24 (Figure 4A).

We Previous studies have suggested interactions between GIPCs and cell wall components, particularly the pectin Rhamnogalacturonan II (RGII) [START_REF] Voxeur | Glycosylinositol phosphorylceramides from Rosa cell cultures are boronbridged in the plasma membrane and form complexes with rhamnogalacturonan II[END_REF] . However, we did not detect either galacturonic acid (GalA) nor rhamnose (Rha), two main components of pectins, suggesting no major pectin contamination (Figure 4B). We detected, however, a large amount of Ara and Gal (Supplementary data 3). A Yariv reactivity test [START_REF] Kitazawa | β-galactosyl yariv reagent binds to the β-1,3-galactan of arabinogalactan proteins[END_REF] was performed to check for the presence the arabino-galactan (AG) as contaminants in the GIPC-enriched fractions (Figure 4B). No zone of clearance was observed, suggesting no detectable AG in each GIPC sample (50 µg). Gum arabic and saline buffer were used as positive and negative controls respectively (Figure 4B). The potential contamination of the GIPC samples by proteins was also tested using the Bradford method. However, in GIPC samples of up to 30 µg, no protein was detected (data not shown).

The highly purified fractions of Bo-GIPC and Ap-GIPC were analyzed by LC-MS 2 in MRM mode and compared to total sphingolipids extracted from crude cauliflower or crude leek.

Results showed in Supplementary data 4 revealed an absence of CER and gluCER contamination in the purified GIPC sample, and that the LCB and FA content is very similar except a slight loss of h24:0/1-and t18:0/1-containing GIPC (less than 10%), see Supplementary data 5.

Biophysical characterisation of the GIPCsterol interaction

We decided to focus on the Bo-GIPC is characterized by a certain degree of condensing interaction between the molecules at the interface (Figure 5A). The mean interfacial area of Bo-GIPC is 212.9 ± 4.9 Å 2 in its expanded form and at its most condensed form is 60.0 ± 14.6 Å 2 .

These results are in agreement with the results previously obtained with Nicotiana tabaccum-

GIPCs 1 .
The interaction of Bo-GIPC mixed with different sterols was assessed by the thermodynamic analysis of the compression isotherms of mixed GIPC-sterol monolayers. In this comparative study, we adhere to the rule of additivity, which suggests that if two molecules within a mixed monolayer are immiscible, the area occupied by the mixed film will be the sum of the areas of the separated components. The deviation to that rule is attributed to the existence of specific interaction between the two molecules [START_REF] Maget-Dana | The monolayer technique: A potent tool for studying the interfacial properties of antimicrobial and membrane-lytic peptides and their interactions with lipid membranes[END_REF] . The mean molecular area of the mixed monolayer Bo-GIPC: β-sitosterol (80:20) was lower than the calculated theoretical value (using the rule of additivity), at the estimated physiological membrane surface pressure of 30 mN m -1 43 (Figure 5B). This condensing effect of β-sitosterol in presence of Bo-GIPC confirms that previously reported for tobacco GIPCs 1 . This trend was however reversed for the mixed monolayers of Bo-GIPC:SG (80:20) and Bo-GIPC:stigmasterol (80:20), where the mean molecular area is significantly higher than the theoretical value (Figure 5B). For Bo-GIPC:ASG

Modelling the interaction between GIPC and phytosterols

The interaction of one molecule of GIPC series A with t18:0/h24:0 and one molecule of sterol was generated in silico and analyzed using Hypermatrix, a simple docking method used to calculate specific interactions between two amphiphilic molecules (for a review see [START_REF] Deleu | Complementary biophysical tools to investigate lipid specificity in the interaction between bioactive molecules and the plasma membrane: A review[END_REF] ). The sterols used were the four molecules studied by the Langmuir monolayer technique i.e. βsitosterol, stigmasterol, ASG, and SG (Figure 6).

The interacting molecules displayed very different configurations. The differences between the spatial organization of the GIPC/sitosterol and GIPC/stigmasterol were striking: the α-side of the steryl moities of β-sitosterol was directed towards the acyl chains of the GIPC, whereas the steryl rings of stigmasterol was positioned at a perpendicular angle with respect to GIPC hydrocarbon chains (Figure 6A,B). In mammals the interaction of the α face of cholesterol with lipid acyl chains favours its condensing effect [START_REF] Róg | Non-polar interactions between cholesterol and phospholipids: A molecular dynamics simulation study[END_REF] .

This was notably established by comparing the effects of cholesterol and lanosterol, which possesses a methyl group on the α face, on lipid organization [START_REF] Smondyrev | Molecular dynamics simulation of the structure of dimyristoylphosphatidylcholine bilayers with cholesterol, ergosterol, and lanosterol[END_REF][START_REF] Yeagle | Differential effects of cholesterol and lanosterol on artificial membranes[END_REF][START_REF] Róg | Ordering effects of cholesterol and its analogues[END_REF] . For stigmasterol, the structural difference of the unsaturation on C22 seems thus to modify its interaction with GIPC (Figure 6B), and this can be correlated to the non-condensing effect observed experimentally in the monolayer compression experiments (Figure 5). Similarly, the β face of the steryl ring moiety of the steryl glucoside (SG) was oriented towards GIPC acyl chains. It is noteworthy that the bending of the sugar head group of GIPC favours its interaction with the glucose head group of SG (Figure 6C).

In the conformation of the conjugated sterol ASG, the acyl chain is in direct interaction with the α side of the sterol, such that the β-side of the steryl cycle interacts with GIPC acyl chains (Figure 6B). Thus, β-sitosterol is the only sterol tested for which the interaction of its α-face with the GIPC acyl chains is favoured, in good agreement with its condensing effects observed experimentally.

Effect of GIPC on membrane organization and thickness

To further investigate the properties of GIPC in influencing plant PM organization, we used Bo-GIPC to make large unilamellar vesicles (LUV) by freeze/thawing method. GIPC alone made aggregates but not vesicles (Figure 7A). Phase contrast microscopy observations of Nt-GIPC containing LUV in water at RT, pH 7 led to similar results (Supplementary data 6A).

However, by adding phospholipids we could observe the formation of LUV (Supplementary data 5A). To closer mimic the outer leaflet of the PM enriched in GIPCs, we generated LUV with a ternary system of GIPC:phospholipid:β-sitosterol

(1:1:1). For all phospholipids used i.e. 1palmitoyl-2-linoleoyl-sn-glycero-3phosphocholine (PLPC), 1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine (POPC) or dioleoyl-sn-glycero-3-phosphocholine (DOPC), the ternary system yielded liposomes using the freeze-thaw method (Figure 7A) (Supplementary data 6B). Giant unilamellar vesicles (GUVs) were also made using the Teflon method [START_REF] Kubsch | Phase behavior of charged vesicles under symmetric and asymmetric solution conditions monitored with fluorescence microscopy[END_REF] with a ternary mix of GIPC/DOPC/sitosterol (Figure 7B). GIPC cannot organize alone in bilayer, but need phospholipids and sterols.

The incorporation of GIPCs into the liposomes were analyzed by dynamic light scattering (DLS) which gives the hydrodynamic diameter of the liposomes. The addition of GIPC did not seem to affect the hydrodynamic diameter of liposomes, which was about 100 nm.

The structure of the LUVs made by the freeze/thaw method was further investigated by cryo-EM. GIPC/POPC-2 H31/ β-sitosterol-and GIPC/POPC-2 H31/stigmasterol-containing LUVs formed not only regular-shaped bilayer vesicles, but also planar bilayer structures that seem more rigid and not able to bend and make proper vesicles (arrow in Figure 8A).

Comparison of the bilayer thickness of these GIPC-containing LUVs with LUVs containing only POPC and sterols, showed a significant difference of thickness from 4.5 nm for the ternary LUV to 3.5 for the binary LUV (Figure 8B). We also investigated the influence of GIPCs on membrane thickness by neutron reflectivity (Figure 9A). Supported Lipid Bilayers (SLB)

were formed by vesicle fusion of liposomes containing POPC, GIPC and constant β-sitosterol concentration. Three different membrane compositions were tested with increasing (0, 15 and 30 % mol.) GIPC concentration. The reflectivity profile was analyzed, and following model fitting, the scattering length density profile and the thickness of the polar head and acyl tail in the bilayer were obtained. The results showed that liposomes containing 30 % mol of GIPC did not form a continuous bilayer on the surface, as indicated by a high solvent content in the hydrophobic tail region. This implies that the high GIPC content modified the bilayer properties, such that it did not adhere to the support. However, 0 and 15 % mol containing GIPC liposomes did form continuous bilayers.

The addition of GIPCs increased the bilayer thickness by 8 Å, as compared to GIPC-free SLBs, due to the 4 Å of sugar head group in each layer (Figure 9A). Refer to the tables of Figure 9B for more details of the structural parameters that were generated. Figure 9C shows an overlay of the estimated SLB dimensions on the scattering length density profile of the SLBs.

The ζ-potential of the GIPC-containing liposomes was measured to be around -26 mV, while DOPC/ β-sitosterol alone had a ζ-potential of -5 mV (Figure 7C). The difference in ζpotential between GIPC and GIPC-free liposomes is attributed to the fact that GIPCs are negatively charged because of the presence of the glucuronic acid, and furthermore, confirms that GIPC was indeed incorporated into the lipid membrane. [START_REF] Jiang | Plant cell-surface GIPC sphingolipids sense salt to trigger Ca2+ influx[END_REF] showed that the ζ-potential of the surface of wild-type Arabidopsis mesophyll cells are at -20mV which is quite close to that of our liposomes. It seems that GIPC contributes significantly in the negative potential of the plant PM outer leaflet, which might influence, for example, its interaction with cell wall components.

Effect of GIPC on membrane biophysical properties

Finally, we asked whether GIPCs have an effect on the gel-to-fluid phase transition of a fully hydrated binary and ternary lipid mix system using solid-state 2 H-NMR spectroscopy as a nonintrusive method giving structural and dynamic information about lipid bilayers [START_REF] Seelig | Deuterium magnetic resonance: Theory and application to lipid membranes[END_REF][START_REF] Davis | The description of membrane lipid conformation, order and dynamics by 2H-NMR[END_REF] . Here, we aimed to find the nature of the membrane phases, their dynamics, and how GIPCs and phytosterols are regulating the membrane phase transition, such as the well-described effect of cholesterol on the membrane [START_REF] Kang | Spectroscopic Studies of Specifically Deuterium Labeled Membrane Systems. Nuclear Magnetic Resonance Investigation of Protein-Lipid Interactions in Escherichia coli Membranes[END_REF] .

We used Bo-GIPC enriched fractions to make membrane systems using deuterated palmitoyloleoyl phosphatidylcholine containing 31 atoms of deuterium on the palmitoyl chain (POPC-2 H31) as a probe for solid-state 2 H-NMR. We chose to use POPC as it is a phospholipid with a long chain fatty acid with an unsaturation found in plant PM

1 and a gel-to-fluid transition temperature of -2.5°C ± 2.4 [START_REF] Seneviratne | Phases and phase transitions of P(EO)6LiSbF6[END_REF] . We generated liposomes using the freeze/thaw method as previously described.

DISCUSSION

Fine-tuning GIPC purification

GIPCs are essential components of plant plasma membranes [START_REF] Cacas | Biochemical survey of the polar head of plant glycosylinositolphosphoceramides unravels broad diversity[END_REF][START_REF] Mortimer | Abnormal glycosphingolipid mannosylation triggers salicylic acid-mediated responses in Arabidopsis[END_REF] . As there are no commercially available GIPCs, reasonable quantities of these molecules must be isolated with good purity to study their biophysical properties. This paper describes a protocol for efficient purification of milligram amount of GIPCs. It has been inspired by three publications [START_REF] Carter | Sphingolipids in bean leaves (Phaseolus vulgaris)[END_REF][START_REF] Kaul | Characterization of Inositol-containing Phosphosphingolipids from Tobacco Leaves[END_REF][START_REF] Markham | Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry[END_REF] , whereby the steps were rearranged in order to get rid of contamination such as sterols and glycerolipids during the extraction procedure. This is simpler and more convenient than the one used in previous publications where large amounts of solvents were used. It is also time-efficient and achieve a reasonable yield since we were able to obtain 30 mg of GIPC of up to 85% purity from 4 distinct plant materials of 600-800 g fresh weight.

In the near future, to improve yield and purity, we still have to fine-tune the purification and extraction process of GIPCs, and it will also be important to purify different GIPC series individually, so as to decipher the number, bonds and types of sugar residues which make up the different plant GIPC polar head groups, as was done for fungi GIPC by NMR [START_REF] Simenel | Glycosylinositolphosphoceramides in Aspergillus Fumigatus[END_REF][START_REF] Gutierrez | Characterization of glycoinositolphosphoryl ceramide structure mutant strains of Cryptococcus neoformans[END_REF] . To do so, ad hoc preparative chromatography needs to be developed with elution solvents with the right polarity.

This study demonstrated that the fractions purified using the newly designed protocol,

GIPCs specifically modulate the properties of model membranes

Since GIPCs have large polar heads and VLCFAs, they tend to agglomerate, we used them in a binary mixture with phospholipids, or a ternary mixture with phospholipids and sterols .

As expected for lipids with (h)VLCFAs, GIPCs increase the thickness of the model membrane by a few nm, as shown by neutron reflectivity on a supported bilayer (Figures 8,9). The bilayer thickness of liposomes containing GIPCs as observed using cryo-EM are around 6-7 nm for the ternary mix, which corresponds well with the observed thickness of purified PM from Medicago truncatula and tobacco [START_REF] Lefebvre | Characterization of lipid rafts from Medicago truncatula root plasma membranes: A proteomic study reveals the presence of a raft-associated redox system[END_REF][START_REF] Mongrand | Lipid rafts in higher plant cells: Purification and characterization of triton X-100-insoluble microdomains from tobacco plasma membrane[END_REF] .

One important feature of the PM is its electrostatic charge. PM purification using polymers phase separation PEG/Dextran relies on the fact that PMs are highly negatively charged and that the PM right-side-out (RSO) fraction is attracted to the positively charged PEG phase [START_REF] Morré | Aqueous two-phase partition applied to the isolation of plasma membranes and Golgi apparatus from cultured mammalian cells[END_REF] .

The membrane surface charge (MSC) is regulated by lipids and post transcriptional modification of proteins such as phosphorylation [START_REF] Goldenberg | Surface charge: A key determinant of protein localization and function[END_REF] . The ζpotential of GIPC-containing liposomes is -26 mV, five times higher than a PC/ β-sitosterolcontaining bilayer, likely due to the large negativity of GIPC conferred by its phosphate group and the GlcA residue of the polar head.

Therefore, since GIPCs are expected to be mostly located in the outer leaflet of the PM 1 , we conclude here that GIPCs contribute strongly to the negative charge of the PM outer leaflet.

GIPCs affect membrane order through specific interactions with different molecular species of phytosterols

Using the biophysical techniques of Langmuir compression isotherms and molecular modelling, we showed that GIPCs interact differentially with different phytosterols. We confirmed that GIPCs with β-sitosterol has a condensation effect as described in 1 whereas non condensing interactions occur between stigmasterol, SG or ASG and GIPCs (Figures 5, 10 Recent studies provide new insight on the importance role of GIPC structure in plants through genetic approach [START_REF] Mortimer | Abnormal glycosphingolipid mannosylation triggers salicylic acid-mediated responses in Arabidopsis[END_REF]17,[START_REF] Ishikawa | GLUCOSAMINE INOSITOLPHOSPHORYLCERAMIDTRANSFERASE1 (GINT1) is a GlcNAc-containing glycosylinositol phosphorylceramide glycosyltransferase[END_REF][START_REF] Jiang | Plant cell-surface GIPC sphingolipids sense salt to trigger Ca2+ influx[END_REF] . By generating mutants combined to the multidisciplinary approaches, we can uncover more about GIPC intricate structure and its biological implications.

EXPERIMENTAL PROCEDURES Plant Material

Cauliflower and leek were store-bought. Wildtype tobacco (cv. Bright Yellow) cell culture and rice cell culture were obtained as previously described in 8 and 20 respectively.

Extraction and purification of GIPCs

The green parts of the cauliflower and leek were

Molecular modelling Approaches

The Hypermatrix docking procedure was used to study the interaction of GIPC with the different sterols, as already described in 1 . Briefly, one GIPC molecule is positioned and fixed for the whole calculation at the centre of the system, oriented at the hydrophobic /hydrophilic interface. The interacting molecule is also oriented at the hydrophobic/hydrophilic interface, and by rotations and translations, more than 10 million positions of the interacting molecule around the central molecule are calculated. The lowest energy matching is considered as the most stable interaction. Refer to 1 for more details.

Liposomes preparation (Freeze and thaw method)

The lipid solution of 1 mg/ml (GIPC/PLPC or POPC or DOPC/Stigmasterol or β-sitosterol) at different molar ratio, was dried and resuspended in water. Several cycles of freeze and thaw were done with freezing occurring in liquid nitrogen for 5 min and thawing at 50°C for 15 min.

LUV preparation for DLS and ζ-Potential

LUVs

GUV preparation (Teflon method)

GUV were prepared as previously described by Dimova [START_REF] Kubsch | Phase behavior of charged vesicles under symmetric and asymmetric solution conditions monitored with fluorescence microscopy[END_REF] . Briefly, 50 µL of lipid mixture (1 mg mL -1 ) dissolved in organic solvent mixture were deposited on a pre-cleaned Teflon disk and the solvent was evaporated with vacuum for 2 hr. The disk was then placed in a 4 mL sealed glass vial with 200 mM sucrose and 50 mM NaCl at 60°C for 12 hours, until a cloudy deposit was formed.

For microscopy observation, one volume of the vesicle suspension was mixed with 4 volumes of iso-osmolar glucose/NaCl solution for better contrast.

Cryogenic Electronic Microscopy (Cryo-EM)

Lacey carbon formvar 300 mesh copper grids were used. They were first submitted to a First moments were calculated using a C 2+ homemade routine (Buchoux S., unpublished).

LC-MS analysis

For the analysis of sphingolipids by LC-MS/MS, lipids extracts were then incubated 1h at 50°C in

1 .

 1 Four species were selected to get different GIPC series.: 2 eudicot plants: cauliflower (Brassica oleacera, Bo) head and tobacco (Nicotiana tabacum, Nt) cell culture Bright-Yellow 2 (BY-2) and 2 monocot plants: the white leaves of leek (Allium porrum, Ap) and rice (Oryza sativa, Oz) cell culture. The white part of plant tissues and cell cultures were used to avoid contamination by the abundant plastidial lipids and pigments. Cauliflower and rice cell culture have the highest GIPC content with an estimated 4.

  preparation to perform various biophysical analyses in order to obtain elements of understanding on how GIPCs molecular characteristics contribute to the organization of the plant plasma membrane. We first characterized the lipid-lipid interactions at the micrometric level by the Langmuir trough compression technique applied on a monolayer model at the air-water interface 40 . The interaction of Bo-GIPC with free and conjugated sterols (βsitosteryl Glucoside, SG and Acyl (18:2) βsitosteryl Glucoside, ASG). The ratio of GIPC:sterol (80:20 mol ratio) is consistent with the estimated ratio of the lipids in the outer leaflet of the PM 1,41 . The compression isotherm of Bo-GIPC (green line) (Figure 5A) shows a low and relatively constant surface pressure in large molecular areas, corresponding to a 'gaseous' state. Compression of the monolayer induced a progressive increase in surface pressure, indicating the appearance of a liquid-expanded state (in agreement with the two-dimensional compressibility modulus, Cs -1 , of 38.3 mN m -1 in the 160-to 110-Å 2 per molecular region), which

(80: 20 )

 20 , the effect is intermediate. The most significant difference between the experimental and theoretical mean molecular area was obtained for the mixed monolayer Bo-GIPC:stigmasterol (80:20). Interestingly, the only structural difference between β-sitosterol and stigmasterol is the presence of a double bond at C22 in stigmasterol. The mixed monolayer GIPC:ASG (80:20) had a comparable mean molecular area to GIPC molecule at low surface area (Figure5A) and the average difference between the mean molecular area and its theoretical value is 30 Å 2 per molecule for all three surface pressures (Figure5B).In order to thermodynamically analyse the interaction of the two components and the stability of the mixed monolayer, the excess free energy of the mixing (ΔGex) and the free energy of mixing (ΔGM) were respectively calculated for all four mixed monolayers (Figure5C). The negative value of ΔGex for the mixed monolayer GIPC: β-sitosterol (80:20) suggested a strong attractive interaction between the two components and the negative value of ΔGM indicated thermodynamic stability of the mixed monolayer (Figure 5C) as suggested by 1 . The values of ΔGex and of ΔGM for the mixed monolayers GIPC:SG (80:20), GIPC:ASG (80:20) and GIPC/Stigmasterol (80:20) were both positive in all three mixed monolayers (Figure 5C) showing repulsion between the molecules within the monolayer and thermodynamic instability of the mixed monolayers.

Figure 10A showed 2 H

 2 Figure 10A showed 2 H NMR spectra of two lipid mix systems containing GIPC (GIPC/ POPC-2 H31/β-sitosterol (1:1:1, mol ratio) and GIPC/ POPC-2 H31/stigmasterol (1:1:1, mol ratio) and two control samples without GIPC (POPC-2 H31/β-sitosterol (1:1, mol ratio) and POPC-2 H31/stigmasterol (1:1, mol ratio). Spectra were acquired by varying the temperature from -10°C to 40°C, corresponding to plausible thermal variations that plants may experience in nature.The obtained 2 H NMR spectra exhibit the typical powder pattern line shape with a spectral width decreasing as the temperatures increase. This qualitative observation can be supplemented by a quantitative characterization using the first spectral moment[START_REF] Davis | The description of membrane lipid conformation, order and dynamics by 2H-NMR[END_REF] . Figure10Bshows the temperature plots of first moments (M1) calculated from 2 H-NMR powder spectra of liposomes with or without Bo-GIPCs, as well as pure POPC-2 H31. On figure 10B left, we can hence appreciate the phase transition of a pure POPC-2 H31 membrane such that the low M1 corresponds to the fluid (Ld) phase and the high M1 to the rigid (Lo) phase. The thermal variation showed an abolished phase transition upon adding phytosterols to POPC-2 H31 (Figure10Bleft). This abolition is more pronounced for βsitosterol with a higher ordering effect above the phase transition temperature compared to stigmasterol. These conclusions can be transposed to ternary systems with the difference

1 (

 1 displayed the signature hVLCFA attributed to sphingolipids with little to no presence of phospholipids (C16 to C18 FA) or sterols(Figures 2, 3, 4). It further established the presence of GIPC with LC-MS experiments, confirming the high purity of our purified GIPC fractions using this particular protocol with no ceramide and glucosylceramide (Supplemental data 4). The calculated yield for the different GIPC series was enough to perform all the necessary biophysics experiments for structural analysis and interactant assays.In all enriched GIPC fractions purified in this study, GalA and Rha are absent (Figure4B), suggesting no contamination by pectins, particularly RGII, which is reported to bind to GIPCs[START_REF] Voxeur | Glycosylinositol phosphorylceramides from Rosa cell cultures are boronbridged in the plasma membrane and form complexes with rhamnogalacturonan II[END_REF] . Previous work in Arabidopsis thaliana (At, also a Brassicaceae, like cauliflower), identified a mannose as the first sugar attached to the GlcA-Ins-P-Cer core in vegetative tissues, a reaction catalysed by the glycosyltransferase AtGMT1(Fang et al., 2016). We hypothesized that this also might be the case in cauliflower. In the seed tissue of At, Glucosamine InositolPhosphorylCeramide Transferase AtGINT1), another glycosyltransferase, adds a GlcNAc instead of a Man to the core structure of GIPC[START_REF] Ishikawa | GLUCOSAMINE INOSITOLPHOSPHORYLCERAMIDTRANSFERASE1 (GINT1) is a GlcNAc-containing glycosylinositol phosphorylceramide glycosyltransferase[END_REF] . However, in both cases, Ara and Gal were detected as part of the sugar composition of their GIPC enrichments, which might be cell wall contaminant or inherent sugar moieties of GIPC.The polar head sugar diversity of GIPCs are clearly species-dependent[START_REF] Buré | Fast screening of highly glycosylated plant sphingolipids by tandem mass spectrometry[END_REF] . For instance, the increasingly large amounts of Gal and Ara in the Nt-GIPC fractions has been described in[START_REF] Kaul | Isolation of Six Novel Phosphoinositol-Containing Sphingolipids from Tobacco Leaves[END_REF] where GIPCs of tobacco leaves contain up to 4 Ara and 2 Gals attached to the core GIPC structures of GlcN/GlcNAc-GlcA-Ins-P-Cer. Hence, it is correct to assume that the large amount of Ara and Gal of Nt-GIPC(fraction γ) derives from the polyglycosylated GIPCs of up to GIPC series E of Ara-Ara-Gal-Gal-Man-GlcN/GlcNAc-GlcA-Ins-P-Cer.The full structure and diversity of sugar moieties in the GIPCs polar head remains to be understood and investigated. The diversity seems to be important for example in plant/pathogen interactions. A recent study showed that the GIPC polar head may be receptor for oomycete necrotic toxins called Necrosis and ethylene-inducing peptide 1-like (NLPs). Plants enriched in GIPC series A are sensitive to NLPs while those enriched in GIPC series B are insensitive to NLPs, hence conferring resistance against pathogens secreting NLPs 57 . The efficiency of the designed purification protocol as well as the structural differences of sugar moieties listed in the different plant species in this paper, might expand new possibilities of further studying the complexity of GIPC's intriguing role in membrane organization and in plant immunity. As the most abundant sphingolipid in plants, GIPCs have arguably fundamental roles in molecular and cellular responses that are yet to be discovered. This redesigned protocol allows access to a readily adequate amount of pure GIPCs at the bench from any type of plant tissues.

  ). These differential interactions appear to be structure dependent. Just adding a glucose head group (SG) and an acyl chain (ASG) or an unsaturation (C22 in stigmasterol) to the β-sitosterol steryl moieties changes the interaction with the GIPCs and modifies the properties of the model membranes (Figure5, 6, 7). Interestingly, GIPCs, ASG and SG all accumulate after drought stress[START_REF] Tarazona | An enhanced plant lipidomics method based on multiplexed liquid chromatography-mass spectrometry reveals additional insights into cold-and droughtinduced membrane remodeling[END_REF] . The differential interaction of GIPCs with the different kind of sterols could explain how plants cope with such stress.As mentioned, stigmasterol also displayed a noncondensation effect. The structural difference between sitosterol and stigmasterol is only an unsaturation on C22. This has a dramatic effect on membrane fluidity as discussed in[START_REF] Grosjean | Differential effect of plant lipids on membrane organization specificities of phytosphingolipids and phytosterols[END_REF] . Using model membranes and environment-sensitive probes, they showed that plant lipids promote various spatial organization of membrane and that β-sitosterol promotes Lo phases while stigmasterol has a low ordering effect and is correlated with low level of Lo phases. Plant sterols and sphingolipids form lipid rafts which are signalling platforms[START_REF] Mongrand | Lipid rafts in higher plant cells: Purification and characterization of triton X-100-insoluble microdomains from tobacco plasma membrane[END_REF][START_REF] Gronnier | Divide and Rule: Plant Plasma Membrane Organization[END_REF] . These structures can be clearly seen as lipid domains in model monolayers containing β-sitosterol and GIPC that interact with each other (Figure7). This interaction might translate into Lo phases.Stigmasterol, on the other hand, tends to sequester small structures containing GIPC which might contribute to membrane fluidity.Potential functions ofGIPCs in plant physiology based on these structural information Plants are poikilothermic and have to adapt the viscosity of their membrane to temperature changes, a process called homeoviscosity. By modulating the fluidity of their membrane to be functionally viable, plants can adapt to temperature fluctuations. For example, plants can readily convert β-sitosterol to stigmasterol by expressing the C22 desaturase CYP710 during temperature acclimation 64 . Specific plant membrane components like β-sitosterol, stigmasterol and glucosylcerebrosides are synthesized as part of temperature adaptations to make membrane-associated biological processes possible 31 . Here, we showed that GIPCs are more conducive to enable homeoviscosity. It will be interesting to further investigate how GIPCs are involved in modulating PM fluidity in thermal adaptation in synergy with other PM lipid components.

  The modification of the ceramide length and hydroxylation of GIPC might alter the organization of the membrane as does sphingomyelin (SM) in animal cell, which is responsible for interdigitating between the bilayers and domain formation with cholesterol[START_REF] Róg | Interdigitation of long-chain sphingomyelin induces coupling of membrane leaflets in a cholesterol dependent manner[END_REF][START_REF] London | Insolubility of lipids in Triton X-100: Physical origin and relationship to sphingolipid/cholesterol membrane domains (rafts)[END_REF] . The closest biological molecule in terms of membrane structuring role of plant GIPC series A and B could be SM, even if the latter -absent in plant PM-is made up of a phosphocholine head group. The theoretical model of plant PM showed GIPC as the major sphingolipid in the outer leaflet, just like sphingomyelin, and inducing a lateral segregation to form Lo phases with phytosterols 1,41 . Interestingly, the exact distribution of sterols in the two layers of the PM is still a matter of debate, including in animal biology research fields 67 . To know where sitosterol or stigmasterol are located and how they regulate the fluidity of one or both of the PM leaflets is of great interest. Unfortunately, the tools to study phytosterol distribution remain to be developed. Plant GIPCs are structurally homologous to the animal gangliosides that are absent in plants. Gangliosides are acidic glycolipids containing sialic acid in their polar head that play an important role in immunity, signal transduction in the PM that essential for brain and retinal functions in animal cells 68,69 . It is possible that polyglycosylated GIPCs have a similar role to gangliosides. Further investigation will require a better understanding of the GIPC glycosylation pattern, and the enzymes involved in GIPC biosynthesis. The present study paves the way for tackling the function of plant glycosylated sphingolipids in membrane organization and function.

  removed to prevent contamination by galactolipids, which are mainly present in chloroplasts. Plant material (800 g fresh weight)was blended with 5 liters of cold 0.1 N aqueous acetic acid in a chilled stainless-steel Waring Blender at low, medium and high speed for 30s each. The slurry was filtered through 16 layers of acid-washed Miracloth. The residue was reextracted once (twice for leek) again in the same manner. The aqueous acetic acid filtrate was discarded. The residue was air-dried overnight under a fume hood and was then refluxed with 2 liters of hot 70% ethanol (0.1 N in HCl) for 20 min. The slurry was filtered hot through 16 layers of Miracloth pre-washed with acidic ethanol (pressed well to remove all liquid). This process was repeated twice more using a total of 5 liters of acidic ethanol. The combined filtrates were chilled at -20°C for 48h. The precipitate was removed by centrifugation at 30,000 g (14000 rpm at using a Sorvall SLA-1500 rotor) at 4°C for 15 min. Sphingolipids were then extracted from the precipitates in hot isopropanol/hexane/water (55:20:25, v/v). The solution was homogenized using an Ultra-Turrax for 20 s and incubated at 60°C for 20 min. After centrifugation at 3000 g for 10 min, the supernatant was decanted to another tube and the residue extracted twice more with the hot solvent. A total of 100 ml of solvent was used at this step. The supernatants were combined and its lipid content was analyzed by TLC and GC-MS to evaluate the amount of GIPC content. Porous silica beads (Silica gel for chromatography 60 Å, 75-125 um, Acros Organics), were used throughout for packing the column chromatography. The column consists of 70 ml of silica beads, sand of Fontainebleau, followed by the sphingolipid sample dried in 20 ml of silica beads (see Figure 2). The column was washed and equilibrated with chloroform. Apolar lipids were washed with a mix of chloroform/methanol of different volume ratios of increasing polarity (4:1 then 3:1 and 2:1). The volume used was equivalent to 4-fold the volume of the column. The column was then eluted with a step gradient of chloroform:methanol:water. Solvent A was chloroform:methanol:water (59:37.5:3.5, v/v) and the solvent B chloroform:methanol:water (46:42:12, v/v). The step gradient elution started with 100% A to end with 100% B, with 10% intervals. The volume of elution corresponds to 2-fold the volume of the column. 1/100 th of each elution fractions were collected and dried for GC-MS and TLC analysis to test the purity of the fractions. Fractions containing the same type of GIPCs were pooled and dried. The estimated quantity of GIPC is assessed by calculating the amount of (h)VLCFA ((hydroxylated-) Very Long Chain Fatty Acid). (h)VLCFA represents 1/3 of total GIPC molecular mass. High performance thin-layer chromatography analysis High-performance thin-layer chromatography (HP-TLC) plates were Silicagel 60 F254 (Merck, Rahway, NJ). HP-TLC plates were impregnated for 3 min with freshly prepared 0.2 M ammonium acetate in methanol, and further dried at 110C for 15 min. Purified lipids as well as crude extracts were chromatographed in Chloroform/Methanol/ 4N NH4OH (9:7:2, v/v) on. Lipids were located under UV after staining with Primuline in acetone/water 80/20. Carbohydrate Analysis Samples (0.2 mg) were hydrolysed with fresh 2 M TFA at 120°C for either 1 h, 3 h, or 4 h. The supernatants were retained, dried in a vacuum concentrator, redissolved in 2 mL of water and filtered through 0.22 µm filters. Samples were analyzed by High-performance anion-exchange chromatography (HPAEC) on an ICS-5000 instrument (Thermo Fisher Scientific) equipped with a CarboPac PA20 analytical anion exchange column (3 mm × 150 mm; Thermo Fisher Scientific), a PA20 guard column (3 mm × 30 mm; Thermo Fisher Scientific), a borate trap, and a pulsed amperometric detector. The column was equilibrated with 40 mM NaOH for 5 min before injection of the sample. Monosaccharides were separated using the following methods: a linear gradient from 4mM NaOH to 3mM NaOH in the first 6 min, followed by a linear gradient of 3mM NaOH to 1mM NaOH from 6 to 8 min. An isocratic gradient was held at 1 mM NaOH from 8 to 23 min, and then increased to 450 mM NaOH to elute the acidic sugars from 23.1 min to 45 min. Monosaccharide standards were used for quantification.Fatty Acid AnalysisEach sample was transmethylated at 110°C overnight in methanol containing 5% (v/v) sulfuric acid and spiked with 10 mg of heptadecanoic acid (c17:0) and 10 mg of 2hydroxy-tetradecanoic acid (h14:0) as internal standards. After cooling, 3 mL of NaCl (2.5%, w/v) was added, and the released fatty acyl chains were extracted in hexane. Extracts were washed with 3 mL of saline solution (200 mM NaCl and 200 mM Tris, pH 8), dried under a gentle stream of nitrogen, and dissolved in 150 mL of BSTFA and trimethylchlorosilane. Free hydroxyl groups were derivatized at 110°C for 30min, surplus BSTFA-trimethylchlorosilane was evaporated under nitrogen, and samples were dissolved in hexane for analysis using GC-MS under the same conditions as described[START_REF] Buré | Fast screening of highly glycosylated plant sphingolipids by tandem mass spectrometry[END_REF] . Quantification of fatty acids and hydroxyl acids was based on peak areas, which were derived from total ion current, and using the respective internal standards.Langmuir monolayer troughPurified GIPC-enriched fractions were used in this study. A solution at 0.4 mM in chloroform:methanol:water (30:60:8) was prepared. Sterols and PLPC were purchased from Avanti Polar Lipids. They were dissolved at 0.4 mM in chloroform:methanol (2:1). The surface pressure-area (π-A) isotherms were recorded by means of an automated Langmuir trough (KSV Minitrough [width, 75 mm; area, 24.225 mm 2 ]; KSV Instruments) equipped with a platinum plate attached to a Wilhelmy-type balance. The GIPC sample was heated to 60°C for 15 min for a better solubilization. Pure solutions and lipid mixtures were spread (fixed volume of 30 µL) as tiny droplets to form a uniform monolayer on a Tris:NaCl 10:150 mM (Millipore) subphase adjusted to pH 7 with HCl. After evaporation of the solvent (15 min), monolayers were compressed at a rate of 5 mm/min and at a temperature of 22°C ± 1°C. Before each experiment, the cleanliness of the system was confirmed by checking the surface pressure over the surface compression of the pure subphase. At least two independent experiments (distinct sample preparations) with three repetitions for each of them (distinct deposition of the same sample) were performed. The variation coefficient in surface pressure and area was found to be 10% or less.

  Navon et al., 2017) with small modifications. Briefly, the lipid solution (GIPC/DOPC/Sterol) in 3/1 v/v THF/H2O methanol mixture was transferred into a roundbottom flask and the organic solvent was removed by evaporation under high vacuum pumping for 5 h, until complete evaporation of the solvent. The lipid film was then hydrated in an appropriate amount of buffer solution and subjected to 3-5 freeze thaw cycles, yielding multilamellar vesicles. The resulting suspensions (1 g L -1 ) were then successively extruded 20 times through 200 and 100 nm polycarbonate membranes using a mini-extruder (Avanti Polar Lipids). DLS and ζ-potential values Dynamic Light Scattering (DLS) and ζ-Potential. DLS measurements were performed with a Malvern NanoZS instrument operating with a 2 mW HeNe laser at a wavelength of 632.8 nm and detection at an angle of 173°. All measurements were performed in a temperature-controlled chamber at 20 °C (±0.05 °C). Three measurements of 15 runs each were usually averaged. The intensity size distribution was obtained from the analysis of the correlation function using the multiple narrow mode algorithm of the Malvern DTS software. The electrophoretic mobility of the vesicles and CNCs was measured by using the same Malvern NanoZS apparatus performing at 17° from which the ζ-potential values are determined by applying the Henry equation. The ζ-potential values and the ζ-deviation were averaged over at least three measurements with at least 30 runs per measurement. They were expressed as mean ± SD (n ≥ 3).

  standard glow discharged procedure (3 mbar, 3 mA for 40 sec). Plunge freezing was realized using the EM-GP apparatus (Leica). Four microliters of the sample was deposited on the grid and immediately blotted for 2 sec with a whatmann paper grade 5 before plunging into a liquid ethane bath cooled with liquid nitrogen (-184°C). The settings of the chamber were fixed at 70% humidity and 20°C. Total lipid concentration was 0.3 mg/ml Lipids molar ratio were as followed: POPC-2 H31 / sterol (2:1), and GIPC/ POPC-2 H31/Sitosterol (1:1:1). Specimens were observed at -170 °C using a cryo holder (626, Gatan, USA), with a ThermoFisher FEI Tecnai F20 electron microscope operating at 200 kV under low-dose conditions. Images were acquired with an Eagle 4k x 4k camera (ThermoFisher FEI) and processed in ImageJ. Deuterated POPC (POPC-2 H31) were bought from Avanti and used as a marker for NMR measurements, GIPC were prepared from cauliflower. Sitosterol and stigmasterol were store bought from Avanti.Neutron ReflectivityNeutron reflectivity experiments were performed at the ILL, on the FIGARO reflectometer[START_REF] Campbell | FIGARO: The new horizontal neutron reflectometer at the ILL[END_REF] , on SLBs formed through vesicle fusion on silicon crystals[START_REF] Montis | Nucleolipid bilayers: A quartz crystal microbalance and neutron reflectometry study[END_REF][START_REF] Richter | Formation of solid-supported lipid bilayers: An integrated view[END_REF] . The crystals (dimensions l × w × h of 80 × 50 × 10 mm 3 ) were polished through bath sonication in different solvents (5 min in chloroform; 5 min in acetone; 5 min in ethanol) followed by plasma cleaning. The substrates were then extensively rinsed with milliQ water and stored in milliQ water prior to use. The specular reflectivity (R) is defined as the ratio of reflected intensity over incident intensity of a neutron beam, when the angle of reflection is equal to the angle of incidence. It is measured from a flat surface using a highly collimated neutron beam as a function of momentum transfer 𝑄, where 𝑄 = 4𝜋𝑠𝑖𝑛𝜃/𝜆, with 𝜃 glancing angle and 𝜆 wavelength. The measured reflectivity depends on the variation in the scattering length density profile, ρ(z), perpendicular to the surface. The scattering length density profile over the zaxis was modeled as a sum of discrete contributions from separate layers, each characterized by a defined scattering length density, with a gaussian roughness contribution for each interface and a solvent penetration degree. The MOTOFIT software 74 which runs in the IGOR Pro environment (http://www.wavemetrics.com), was used for the analysis of the NR curves. A multilayer model was used to analyze the reflectivity profiles of the SLBs, with fixed scattering length density values calculated for each layer: (i) a first layer of a bulk subphase of Si (ρ = 2.07 × 10 -6 Å -2 ) and a superficial layer of SiO2 (ρ= 3.41 × 10 -6 Å -2 ), were introduced. Their thickness and interfacial roughness were characterized in control NR measurements in D2O and H2O before vesicle injection. (ii) The polar headgroups of the SLB of the inner and outer leaflet (ρ = 1.86 × 10 -6 Å -2 ) (iii) the bilayer lipid chains (ρ = -0.30 × 10 -6 Å -2 ) 75 (iv) the sugar heads of the GIPC were represented as additional layer to the phosphate polar head group in the inner and outer leaflets (ρ = 1.9 × 10 -6 Å -2 ). (v) finally, a bulk super phase of solvent was introduced to the model. All measurements were performed in four contrast solvents, namely H2O (ρ =-0.56 × 10 -6 Å -2 ), D2O (ρ = 6.34 × 10 -6 Å -2 ), 4MW (34% H2O and 66% D2O, ρ= 4.0 × 10

Figure 1 .

 1 Figure 1. Structure and amount of GIPC in plants

Figure 6 .

 6 Figure 6. Modelling of the interaction between GIPC and sterols.

Figure 7 .

 7 Figure 7. Study of GIPC containing-liposomes in binary and ternary

Figure 8 .Figure 9 .

 89 Figure 8. Membrane thickness vary with the presence of GIPC.

Figure 10 .

 10 Figure 10. Solid state NMR studies of GIPC-containing liposomes

  -6 Å -2 ), or SMW (62% H2O and 38% D2O,ρ= 2.07 × 10 -6 Å -2 ).

	NMR analyses. 2 H-ssNMR experiments were
	performed at 76.77 MHz with a phase-cycled
	quadrupolar echo pulse sequence (90°x-t-90°y-t-
	acq) 76 and using a Bruker Avance III 500 MHz
	WB (11.75 T) spectrometer equipped with a solid
	state CPMAS 4mm H/F/X probe (IECB structural
	biophysics platform, Bordeaux, France).
	Acquisition parameters were as follows: spectral
	window of 500 kHz, π/2 pulse width of 3.5 µs,
	interpulse delays of 40 µs, recycling delay of 2 s;
	number of scans from 1K to 6K. Spectra were
	processed using a Lorentzian line broadening of
	300 Hz before Fourier transformation from the
	top of the echo. Samples were equilibrated for 20
	min at a given temperature before data
	acquisition. All spectra were processed and
	analyzed using Bruker Topspin 4.0.6 software.
	Solid State 2 H-NMR
	Samples were prepared by co-solubilizing the
	appropriate amount of Bo-GIPC, POPC 2 H31,
	sitosterol and stigmasterol in chloroform. Solvent
	was evaporated under a flow of nitrogen to obtain
	a thin lipid film, rehydrated with ultra-pure water
	before one-night lyophilization. The lipid powder
	was finally hydrated with 100µl of deuterium-
	depleted water (hydration of 97%). Samples were
	transferred into 100µl 4-mm zirconia rotors for

ACKNOWLEDGEMENTS

We thank Jean-Paul Douliez, Catherine Sarazin and Sébastien Buchoux for critical reading and advice and Laure Beven for bright field microscopy. We thank Claire Bréhélin for the help in electronic microscopy observations. We thank Yuri Gerelli for the neutron analysis.

DATA AVAILABILITY STATEMENT

All data contained within the manuscript in form of main figures, table and supplementary materials.

AUTHOR CONTRIBUTIONS

A. Mamode Cassim designed and performed GIPC purification; A. Mamode Cassim and A. Grélard between Lawrence Berkeley National Laboratory and the U. S. Department of Energy. We thank Bordeaux-Metabolome platform for lipid analysis supported by ANR PlayMobil (grant no. ANR-19-CE20-0016-02 to S.M., L.F., F.S.-P. and Bordeaux Metabolome Facility-MetaboHUB (grant no. ANR-11-INBS-0010 to S.M. and L.F.). We thank the Bordeaux Metabolomics and Lipidomics Platform (https://www.biomemb.cnrs.fr/en/)

CONFLICT OF INTEREST

The authors declare no conflict of interest based on Cacas et al., 2016 1 . Three to five independent samples were processed. Means +/-SD are shown.

Figure 2. Extraction and purification protocol of GIPCs

A, GIPC purification scheme, adapted from [START_REF] Carter | Sphingolipids in bean leaves (Phaseolus vulgaris)[END_REF][START_REF] Kaul | Characterization of Inositol-containing Phosphosphingolipids from Tobacco Leaves[END_REF][START_REF] Markham | Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry[END_REF]

SUPPLEMENTARY DATA FIGURE LEGENDS

Supplementary data 1

High performance thin layer chromatography (HPTLC) assay of eluted fractions collected during the GIPC purification process (described in Figure 2). #2 refers to the crude extract deposited on the silica column.

Fractions containing GIPC without visible contamination of sterols and phospholipids were selected and pooled to make up fraction #3.

Supplementary data 2

High performance anion exchange chromatography coupled with Pulsed Amperometric Detection analysis (HPAEC-PAD) of GIPC-enriched samples shows the monosaccharide content after TFA hydrolysis.

Abbreviations are as follows: GlcA: glucuronic acid; Glc: glucose; GlcN: glucosamine; Man: mannose; Gal: galactose; Ara: arabinose; Xyl: xylose; Fuc: fucose; Rha: rhamnose; GalA: galacturonic acid. C, Yariv reactivity test of GIPC-enriched samples to detect arabino-galactan content. No arabino-galactan were detected. 50 ug of each sample (1mg/ml) was deposited in each well, the picture was taken 48 h after initiating the reaction. The data shows of one representative analysis from purification of GIPCs, previously checked by HPTLC and LC-MS

Supplementary data 3

Determining glycan content by HPAE analysis of GIPC-enriched samples. A slight change in sugar amount was seen after 1h, 3h and 4h of TFA hydrolysis. (GlcA: glucuronic acid; Glc: glucose; GlcN: glucosamine; Man: mannose; Gal: galactose; Ara: arabinose; Xyl: xylose; Fuc: fucose; Rha: rhamnose; GalA: galacturonic acid).

Supplementary data 4

LC-MS-based sphingolidomic showing the LCB, gluCER, and GIPC content found in crude extract of cauliflower and leek, and purified Bo-GIPC and Ap-GIPC. Analyses were performed on three independent purifications and expressed as the mean +/-SD.

Supplementary data 5

LC-MS-based sphingolidomic showing the different molecular species found in crude extract of cauliflower and leek, and purified Bo-GIPC and Ap-GIPC according to the LCB or (very long chain) fatty acid (VLC)FA content. Analysis were performed on triplicate of three independent purifications. The mean relative amount of each species was calculated and expressed in percentage of total fatty acid.

Supplementary data 6

Phase-contrast microscopy observations of Nt-GIPC containing liposomes in water at RT, pH 7. A, Liposomes obtained after 3 cycles of freeze in liquid nitrogen and thaw (water bath at 60°C) containing (I) Nt-GIPCs 2mg/ml; (II) Nt-GIPC/ 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) (4:1 mol/mol) at 2mg/ml; (III) Nt-GIPC/ DMPC (1:1 mol/mol) at 2mg/ml shows crystals; (IV) Nt-GIPC/DMPC molar ratio (1:4 mol/mol) at 2mg/ml forms liposomes of 10µm. (V) HPTLC analysis of lipid mixture confirms the presence of GIPC and DMPC in the liposome mix observed. The higher the GIPC content, the higher the occurrence of crystal formation. GIPCs can form liposomes with phospholipids of short acyl chains when the latter is four-folds more abundant in the mix. Scale bar, 5µm. B, Liposomes of Bo-cauliflower GIPC/POPC/ β-sitosterol (1:1:1 mol/mol) at 1mg/ml in TBS 1X. Clusters as shown in (I) were obtained after three cycles of 20min freezing at -20°C and thawing in a water bath at 60°C. Liposomes in (II) were formed after three cycles of freezing in liquid N2 and 20 min heating in a water bath at 60°C; no cluster was observed. The type of freeze/thaw determines the size and shape of liposomes formed. Scale bar, 5 µm.

Supplementary table 1

Molecular and fragmentation ions of plant sphingolipids monitored by LC/MS 2 .

Step 1

Step 2

Step 3

B. GC-MS analysis of FAMES

A. Protocol of extraction of GIPCs

Steps Steps

Steps

Steps BO-GIPC (cauliflower)