Revisiting Plant Plasma Membrane Lipids in Tobacco: A Focus on Sphingolipids

Abstract

The lipid composition of plasma membrane (PM) and the corresponding detergent-insoluble membrane (DIM) fraction were analyzed with a specific focus on highly polar sphingolipids, so-called glycosyl inositol phosphorylceramides (GIPCs). Using tobacco (Nicotiana tabacum) 'Bright Yellow 2' cell suspension and leaves, evidence is provided that GIPCs represent up to 40 mol % of the PM lipids. Comparative analysis of DIMs with the PM showed an enrichment of 2-hydroxylated very-long-chain fatty acid-containing GIPCs and polyglycosylated GIPCs in the DIMs. Purified antibodies raised against these GIPCs were further used for immunogold-electron microscopy strategy, revealing the distribution of polyglycosylated GIPCs in domains of 35 ± 7 nm in the plane of the PM. Biophysical studies also showed strong interactions between GIPCs and sterols and suggested a role for very-long-chain fatty acids in the interdigitation between the two PM-composing monolayers. The ins and outs of lipid asymmetry, raft formation, and interdigitation in plant membrane biology are finally discussed.

Figure 1.

Long-chain fatty acid (LCFA), VLCFA, and hVLCFA contents of tissue, microsomal, PM, and DIM fractions from tobacco leaf or BY-2 cell culture. A, Fatty acids were released from biological samples by acid methanolysis; the resulting fatty acid methyl esters were subsequently derivatized with N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) before GC-MS analysis. The data are expressed as means of three independent experiments. Long-chain fatty acids have 16, 18, or 20 carbon atoms, VLCFAs have 22 to 26 carbon atoms, and hVLCFAs have 22 to 26 carbon atoms hydroxylated in position 2. B, Histograms show the comparison between VLCFA and hVLCFA contents of DIM and GIPCs purified from tobacco leaves or BY-2 cell culture. The data are expressed as means of four independent experiments ± sd.

Figure 2.

Analysis of GIPCs extracted from tobacco leaf and BY-2 cells by MALDI-TOF mass spectrometry and high-performance thin-layer chromatography (HP-TLC). Polyglycosylated GIPCs are enriched in DIMs. A, MALDI-TOF mass spectrometry analysis of GIPC extracts from BY-2 cells and tobacco leaf. Spectra were acquired in the negative ion mode using 2,6-dihydroxyacetophenone as a matrix. GIPCs are grouped in series according to their number of saccharide units, from two (series A) to six (series E); for detailed analysis of the peaks, see Supplemental Figure S7. B, HP-TLC was used to separate the different series of GIPCs. Note that series A shows two bands called by Kaul and Lester (1975) phytosphingolipid: PSL1 and PSL2, corresponding to GlcNAc-GlcA-inositol phosphorylceramide and GlcN-GlcA-inositol phosphorylceramide, respectively. GIPCs extracted from Arabidopsis (Arabidopsis thaliana; At.) and leek (Allium porrum; Ap.) were used as HP-TLC standards for series A and B, respectively, according to Cacas et al. (2013).

Figure 3.

Quantification of polyglycosylated GIPCs found in PM and DIMs of BY-2 cells. Quantification was by HP-TLC coupled to GC-MS of polyglycosylated GIPCs found in PM and DIMs of BY-2 cells. The data are expressed as means of three independent experiments (percentage of total GIPCs found in PM and DIMs, respectively) ± sd.

Figure 4.

Lipid contents of tobacco leaf (A) and BY-2 cell (B) PM and DIMs. Left, from the results presented in Figures 1 and 2 and those obtained from phospholipids and sterols on the same plant materials (Furt et al., 2010), we were able to determine the lipid contents 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, i.e. sum for glycerolipids (PE, PC, PA, PI, PI4P, PI4,5P2, and DGDG), sterols (free sterols, SG, and ASG), and sphingolipids (gluCER and GIPCs). The data are expressed as means of three independent experiments ± sd. Abbreviations are as follows: phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PI4P), phosphatidylinositol 4,5-bisphosphate (PI4,5P2), digalactosyldiacylglycerol (DGDG), and free sterols (sterols).

Figure 5.

Test for the specificity of antibodies to polyglycosylated GIPCs. A, Home-made polyvinylidene difluoride (PVDF) dot blots were used with DEAE fractions for GIPC purification (Supplemental Fig. S11). PVDF membranes were blotted with preimmune serum (1:100) or with 53-d serum (1:100) immunized against polyglycosylated GIPCs (see “Materials and Methods”). B, Lipids from BY-2 cell PM were separated by HP-TLC. The plates were blotted with preimmune serum (1:100) or with 53-d serum (1:100) immunized against polyglycosylated GIPCs (see “Materials and Methods”). PL, Phospholipids.

Figure 6.

Polyglycosylated GIPCs locate in nanoscale membrane domains on 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 secondary antibodies. Circles indicate obvious membrane domains. Bars = 20 nm. B, A total of 49 independent gold-labeled PM vesicles were analyzed for statistics. C, Ripley’s K-function analysis of GIPC distribution on the surface of PM vesicles: K(r) (y axis) is the average number of particles lying at a distance less than r (x axis), normalized by the mean particle density. This Ripley’s analysis of the labeled PM vesicles (black line) indicates a clustering of the gold particles when compared with a theoretical simulation for a completely random (Poisson) point pattern (red dotted line).

Figure 7.

Effect on membrane order level of tobacco leaf or BY-2 cell purified GIPCs, in combination with phospholipids and free sterols. The RGM of 1 µm diameter of LUVs of different composition labeled with di-4-ANEPPDHQ (3 µm) was measured by spectrofluorimetry in the presence of GIPCs isolated from tobacco BY-2 cells or tobacco leaves. Data shown are means ± sd (n = 5 or more independent repetitions). The different letters indicate significantly different values (P < 0.05). DOPC, Dioleoylphosphatidylcholine; DPPC, dipalmitoylphosphatidylcholine; TM, tobacco mix.

Figure 8.

Surface pressure-area (Π-A) isotherms, at the air-aqueous phase interface, of pure GIPC (circles) and sitosterol (squares) monolayers and of mixed GIPC/sitosterol monolayer (triangles) prepared at a molar ratio of 0.85. A, The isotherms were recorded at 22°C ± 1°C with an aqueous subphase composed by 10 mm Tris buffer at pH 7. 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, 20, and 30 mN m⁻¹ for a GIPC/sitosterol molar ratio of 0.85. 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 (ΔG^ex; white bars) and free energy of mixing (ΔG^M; black bars) of the mixed monolayer GIPC/sitosterol at a molar ratio of 0.85 for various surface pressures. ΔG^ex and ΔG^M 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 to pure components 1 and 2, respectively, and subscript refers 12 to their mixtures; and, where ΔG^id is the free energy for ideal mixing and can be calculated from the following equation: , where R is the universal gas constant and T is the absolute temperature.

Figure 9.

Modeling approaches. A, Theoretical interactions between eight GIPC and eight sitosterol molecules calculated by the Hypermatrix docking method. Sitosterol molecules are in green, and GIPCs are colored with carbon atoms in gray, 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 stands for the water/lipid polar head interface. C, Interaction energies calculated for GIPC and GIPC/sitosterol (from A) monolayers. E^polar corresponds to polar and electrostatic interactions, and E^pho and E^vdw correspond 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.

Figure 10.

Model for the organization of lipids in tobacco PM. To build this model, we took the molar composition of the BY-2 PM obtained in Figure 4 and used the data obtained by Tjellstrom et al. (2010), who were able to calculate the distribution of cytosolic/apoplastic lipids. We hypothesize that GIPCs are located exclusively in the apoplastic face. DGDG, Digalactosyldiacylglycerol; PS, phosphatidylserine.