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First published online November 17, 2006; 10.1104/pp.106.091496 Plant Physiology 143:461-472 (2007) © 2007 American Society of Plant Biologists Insights into the Role of Specific Lipids in the Formation and Delivery of Lipid Microdomains to the Plasma Membrane of Plant Cells1,[W]Laboratoire de Biogenèse Membranaire, Unité Mixte de Recherche 5200-Centre National de la Recherche Scientifique-Université Victor Segalen Bordeaux 2, BP 33076 Bordeaux cedex, France (M.L., A.M.P., L.C., S.M., M.V., R.L., P.M.); Laboratoire de Physiologie Cellulaire et Moléculaire des Plantes, Unité Mixte de Recherche 2846-Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, 94200 Ivry-sur-Seine, France (C.C., M.N.V., A.Z.); Institut Européen de Chimie et Biologie, 33600 Pessac, France (K.B., J.M.S.); and Institut de Biologie Moléculaire des Plantes, Unité Propre de Recherche-Centre National de la Recherche Scientifique 2357, 67084 Strasbourg cedex, France (M.A.H.)
The existence of sphingolipid- and sterol-enriched microdomains, known as lipid rafts, in the plasma membrane (PM) of eukaryotic cells is well documented. To obtain more insight into the lipid molecular species required for the formation of microdomains in plants, we have isolated detergent (Triton X-100)-resistant membranes (DRMs) from the PM of Arabidopsis (Arabidopsis thaliana) and leek (Allium porrum) seedlings as well as from Arabidopsis cell cultures. Here, we show that all DRM preparations are enriched in sterols, sterylglucosides, and glucosylceramides (GluCer) and depleted in glycerophospholipids. The GluCer of DRMs from leek seedlings contain hydroxypalmitic acid. We investigated the role of sterols in DRM formation along the secretory pathway in leek seedlings. We present evidence for the presence of DRMs in both the PM and the Golgi apparatus but not in the endoplasmic reticulum. In leek seedlings treated with fenpropimorph, a sterol biosynthesis inhibitor, the usual  5-sterols are replaced by 9 ,19-cyclopropylsterols. In these plants, sterols and hydroxypalmitic acid-containing GluCer do not reach the PM, and most DRMs are recovered from the Golgi apparatus, indicating that 5-sterols and GluCer play a crucial role in lipid microdomain formation and delivery to the PM. In addition, DRM formation in Arabidopsis cells is shown to depend on the unsaturation degree of fatty acyl chains as evidenced by the dramatic decrease in the amount of DRMs prepared from the Arabidopsis mutants, fad2 and Fad3+, affected in their fatty acid desaturases.
Despite the ongoing debate on the size, lifespan, and dynamics of lipid microdomains (Munro, 2003  ; Pike, 2004; Nichols, 2005; Hancock, 2006), the existence of sterol- and sphingolipid-enriched membrane microdomains in the plasma membrane (PM) of eukaryotic cells is now well recognized (Simons and Ikonen, 1997; Brown and London, 2000; Simons and Toomre, 2000; Bagnat and Simons, 2002; Simons and Vaz, 2004; Hancock, 2006). Evidence for microdomains comes in part from examination of membrane constituents that are resistant to solubilization by nonionic detergents at low temperature, giving rise to the concept of lipid rafts (Simons and Ikonen, 1997; Brown and London, 1998; Röper et al., 2000; Hancock, 2006). Such sterol- and sphingolipid-enriched microdomains can be isolated as detergent-resistant membranes (DRMs). In the following, the terms of DRMs and microdomains will be assigned to respectively indicate the isolated entities and the corresponding membrane entities. Lipid properties of microdomains are similar to liquid-ordered domains, which are characterized by tightly packed hydrocarbon tails but with a high degree of lateral mobility (Brown and London, 2000; Simons and Vaz, 2004). Cholesterol is thought to contribute to the tight packing of lipids in liquid-ordered domains by filling interstitial spaces between lipid molecules (Brown, 1998). Thus, a new model for the organization of the PM of animal cells has emerged, in which cholesterol and sphingolipid-rich microdomains (rafts) coexist with more fluid domains containing phospholipids with unsaturated hydrocarbon chains (Simons and Ikonen, 1997). It has been suggested that these microdomains, to which specific classes of proteins would be associated, play a role in a wide range of biological processes, including cell polarity, protein trafficking, and signal transduction (Simons and Ikonen, 1997; Brown and London, 1998; Keller and Simons, 1998; Simons and Toomre, 2000; Simons and Vaz, 2004; Hancock, 2006). Similar microdomains in the PM of yeast (Saccharomyces cerevisiae) cells, enriched in ergosterol, have also been reported (Bagnat et al., 2000, 2001). Recent reports clearly indicate that microdomains enriched in sterols and sphingolipids also exist in the PM of plant cells (Peskan et al., 2000; Mongrand et al., 2004; Bhat and Panstruga, 2005; Borner et al., 2005). However, the much greater structural diversity of plant sterols and sphingolipids, compared to their animal and fungal counterparts (Hartmann, 1998; Sperling and Heinz, 2003), suggests that plant DRMs might exhibit some specific structural features. In particular, plant DRMs have been shown to contain several sterol molecules, as represented by 24-methylcholesterol, sitosterol, and stigmasterol, instead of one major sterol (cholesterol or ergosterol) and two distinct classes of sphingolipids, inositolphosphorylceramides and glucosylceramides (GluCer; Mongrand et al., 2004; Markham et al., 2006). To obtain more insight into the lipid molecular species required for the formation of microdomains in plant cells, we isolated and characterized DRMs from different plant materials.
The first objective of this article was to investigate the distribution of microdomains along the secretory pathway of plant cells and the involvement of sterols in this process. In addition to their role at the cell surface, lipid microdomains might indeed govern protein and lipid sorting and trafficking toward the PM and other membrane compartments. In that context, the occurrence of lipid microdomains in the Golgi apparatus (GA; Hansen et al., 2000
We have determined the intracellular distribution of lipid microdomains in leek (Allium porrum) seedlings by preparing DRMs from ER-, GA-, and PM-enriched fractions and probed the involvement of sterols in microdomain formation by treatment with fenpropimorph (FEN), a sterol biosynthesis inhibitor that inhibits the cycloeucalenol-obtusifoliol isomerase (Rahier et al., 1986 The second objective of this article was to check the impact of the unsaturation degree of phospholipid fatty acyl chains on the segregation of lipids into DRMs. For this purpose, we took advantage of two Arabidopsis (Arabidopsis thaliana) cultured cell lines, fad2 and Fad3+, which are affected in fatty acid desaturases. Our results indicate that the amount of DRMs recovered from Arabidopsis cells is closely dependent on the unsaturation degree of phospholipid fatty acyl chains.
Characterization of DRMs from the PM of Leek and Arabidopsis Seedlings
PM, ER, and GA fractions from leek seedlings were purified and characterized as previously published (Moreau et al., 1998b
To determine the best conditions to isolate DRMs from the membranes of leek and Arabidopsis seedlings, we first tested different Triton X-100-to-protein ratios varying from 2:16, at 1% Triton final concentration. We obtained the maximum yields and the more constant lipid composition of DRMs at a detergent to protein ratio of 8, and this ratio was used in the following experiments. Under these conditions, the amount of DRMs recovered from the PM of leek seedlings was about 10% to 15% of the PM on a protein basis, whereas DRMs from the PM of Arabidopsis seedlings represented only 2% to 3%, indicating that the PM arising from different plants may not contain similar proportions of microdomains, at least according to the present method of isolation.
To investigate the protein content of PM DRMs, we used antibodies raised against three putative protein markers of DRMs (Mongrand et al., 2004
These DRM preparations were also analyzed for their lipid content. As shown in Figure 1B, DRMs isolated from the PM of leek and Arabidopsis seedlings were clearly enriched in sterols, sterylglucosides (SG), and GluCer and depleted of phospholipids and neutral lipids such as diacylglycerol and free fatty acids. Inositolphosphorylceramides species (Markham et al., 2006 ) were not determined, as previous reports have already demonstrated that DRMs from Arabidopsis membranes are enriched in these lipids (Borner et al., 2005).
In PM DRMs recovered from leek seedlings, sterols constituted 71% of neutral lipids, compared to 47% in the PM, thus representing 35% of total lipids. The sterol composition of leek membranes and of corresponding DRMs is given in Table II
. In microsomes (including the PM), sitosterol was by far the major sterol (63%). The other sterols were represented by 24-methylcholesterol (11%), cholesterol (19%), and stigmasterol (6%). Consistent with previous reports (Mongrand et al., 2004
A new observation was the occurrence of SG in DRM preparations from both the PM of leek and Arabidopsis seedlings. SG amounted to about 7% of total lipids in DRMs isolated from leek seedlings compared to less than 2% in the PM. In DRMs from Arabidopsis seedlings, SG constituted up to 15% to 20% of the total lipids and was even more abundant than GluCer.
To determine where the formation of microdomains could take place along the secretory pathway of plant cells, we took advantage of the model of leek seedlings, which allow the isolation of ER- and GA-enriched fractions (Sturbois-Balcerzak et al., 1999 GA and GA DRMs were analyzed for their respective lipid composition (Fig. 2 ). GA DRMs were found to contain the same lipids as PM DRMs. GA DRMs were enriched in free sterols, which represented 48% of total lipids compared to 20% in GA membranes. They were also enriched in GluCer (11% of total lipids compared to only 2% in GA membranes) and in SG (Fig. 2). In contrast, the levels of phospholipids, especially phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and other lipids (free fatty acids, diacylglycerols, and triacylglycerols) were reduced compared to those of GA membranes.
We also analyzed the fatty acid composition of lipids in GA DRMs and PM DRMs. The glycerolipids of DRMs contained preferentially saturated palmitic acid compared to the glycerolipids of the PM and GA membranes (Fig. 3, A–C ). Gas chromatography (GC) analyses ran on the individual lipid species clearly indicated that 16-OH fatty acids exclusively arose from GluCer.
Thus, our results demonstrate that DRMs can be isolated from GA membranes with a lipid composition similar to that of PM DRMs, suggesting that microdomain formation starts at the level of the GA membranes in the plant secretory pathway.
To further investigate the role of sterols in DRM formation along the secretory pathway, we deregulated the sterol biosynthesis in leek and Arabidopsis seedlings. Seedlings from both leeks and Arabidopsis were grown in the presence of 2 mg/L of FEN, a fungicide that inhibits the sterol pathway at the level of the cycloeucalenol-obtusifoliol-isomerase. In leek seedlings, such a treatment was found to trigger an almost complete replacement of the usual sterols (
In leek treated with FEN, the modification of the sterol profile consisted of a significant decrease in the amount of
Therefore, FEN treatment induced both an accumulation of sterols and 16-OH GluCer in the GA membranes. Because sterols and GluCer (and especially 16-OH containing GluCer) can be considered as critical lipid species required for the formation of lipid microdomains (Mongrand et al., 2004 ), we determined the effect of FEN treatment on the distribution of DRMs in the secretory pathway (Fig. 5A
). In agreement with the sterol and GluCer analyses, we observed a shift of the DRMs from the PM to the GA membranes in FEN-treated leek seedlings.
In untreated plants, 86.4% of total purified DRMs were isolated from the PM (145 µg protein/mg PM protein). In contrast, in FEN-treated plants, 87.2% of the total amount of purified DRMs was recovered from the GA membranes (73.4 ± 23.9 µg protein/mg GA protein). It must be pointed out that these results cannot be attributed to differences of recoveries between the various membrane fractions. The corresponding recoveries were as follows: for ER, control: 26.3 ± 11.7 µg protein/g seedlings and FEN: 28.1 ± 1.8 µg protein/g seedlings; for GA, control: 77.4 ± 2.2 µg protein/g seedlings and FEN: 54.2 ± 6.4 µg protein/g seedlings; for PM, control: 47.3 ± 7.9 µg protein/g seedlings and FEN: 39.7 ± 7.2 µg protein/g seedlings. Thus, on a protein basis, the PM fraction represented 31.3% and 32.5% of the sum of the three membrane fractions in control and treated plants, respectively. In addition, no increase in the recovery of GA membranes was observed in FEN-treated plants (44.5% versus 51.2% in control plants). Therefore, our results clearly demonstrate that inhibition of sterol biosynthesis by FEN induces a preferential formation of DRMs in the GA and that these DRMs are not delivered to the PM with the same efficiency. Such a finding is supported by the results presented in Figure 5B, which shows respective recoveries of DRM protein markers in the PM fractions from control and FEN-treated leek and Arabidopsis seedlings, as detected by their respective antibodies. Although the amount of remorin in the PM from Arabidopsis seedlings was less affected by FEN treatment than expected, the levels of the other marker proteins were clearly decreased after FEN treatment.
For this study, we took advantage of two Arabidopsis cultured cell lines, fad2 and Fad3+, which are affected in fatty acid desaturases. Because of their deficit in 18:1 desaturase (Okuley et al., 1994
DRMs were isolated directly from suspension cultures of Arabidopsis cells. In this case, we applied a methodology already used with success with animal cell cultures (Legembre et al., 2002 The lipid profile of DRMs isolated from microsomes of Arabidopsis cells was found to be very similar to that of DRMs isolated from the PM of Arabidopsis seedlings. The results in the wt column of Figure 6A correspond, therefore, to the mean values determined from both analyses run separately.
Using the same methodology as for the wild-type cells, we isolated DRMs from Arabidopsis fad2 and Fad3+ cells, two mutants characterized, respectively, by the accumulation of 18:1 and 18:3 fatty acids. In both cases, the amount of DRMs recovered dramatically decreased and represented only 3.3 µg protein ± 0.6 and 2.1 µg protein ± 0.3/mg microsomal membrane proteins for fad2 and Fad3+ cells, respectively (i.e. about 20% of the amount of DRMs recovered from Arabidopsis wild-type cells). The lipid composition of DRMs isolated from both mutant cell lines was compared to that of DRMs isolated from wild-type cells (Fig. 6A). DRMs from fad2 and Fad3+ cells were highly enriched in GluCer, SG, and free sterols, indicating that these DRMs likely represented lipid microdomains. However, it should be noted that phosphatidyl-Ser and phosphatidylinositol contents in DRMs from both mutants were strongly reduced, and an unexpected high amount of PC was observed in DRMs isolated from fad2 cells (Fig. 6A). These results prompted us to investigate in more details the fatty acid composition of the different lipid molecular species of DRMs. Figure 6B shows the total fatty acid composition of microsomal membranes from wild-type cells and of DRMs from wild-type and mutant cell lines. As expected, the major fatty acid found in wild-type microsomal membranes was 18:3, but this fatty acid was barely detectable in the DRMs (Fig. 6B). The 18:3 fatty acids also disappeared from the DRMs isolated from mutant cells. Compared to the wild-type microsomal fraction, we observed a high increase in 16:0 in all DRMs (Fig. 6B) and higher amounts of 16:1 and 18:0 fatty acids in DRMs isolated from fad2 cell membranes (Fig. 6B). Thus, the saturated-to-unsaturated fatty acid ratios of DRM lipids from Arabidopsis wild-type and mutant cells were found to be 1.24 ± 0.07, compared to only 0.43 ± 0.06 for wild-type microsomal membranes. We then compared the fatty acid composition of PC and PE in the microsomes and DRMs recovered from wild-type and mutant cells. An interesting observation was that 16:0 and 18:1 were the two major fatty acids of PC and PE in all DRM preparations, regardless of the initial abundance of these fatty acids in the microsomal fractions from where each DRM fraction was recovered (Fig. 7 ). This result strongly suggests that the fatty acid composition of these phospholipids governs their recovery into DRMs. In addition, we observed that DRMs from fad2 cells contained a higher amount of PC species with 18:0 and 18:1 and lower amounts of PC with 18:2 compared to wild-type DRMs. The significant increase in 16:1 fatty acids found in the DRMs from fad2 cell membranes (Fig. 6B) was due to the segregation of 16:1-PE into DRMs (Fig. 7). In DRMs from Fad3+ cells, we observed an increase of 18:1-PC, a decrease of 18:0-PC, and an increase of 16:0- and 16:1-PE compared to wild-type DRMs (Fig. 7).
Thus, the important changes in the fatty acid composition of fad2 and Fad3+ membranes (Shah et al., 1997 ; Caiveau et al., 2001), and therefore their different fluidity and dynamic properties (Vaultier et al., 2006), have an impact not only on the amount of DRMs recovered from the membranes but also on the nature of the lipid species present in these DRMs.
Evidence for the existence of lipid microdomains within the PM of higher plant cells has been given only recently (Peskan et al., 2000 ; Mongrand et al., 2004; Borner et al., 2005). To gain more insight into the role of microdomains in the regulation of the lipid (and protein) composition of the PM in plant cells, we addressed two major questions in this article: (1) What are the lipid species playing a critical role in lipid microdomain organization? and (2) Where are lipid microdomains formed along the secretory pathway?
To investigate where lipid microdomain formation may take place, we isolated DRMs from ER-, GA-, and PM-enriched fractions from leek seedlings. This article clearly demonstrates that DRMs can be isolated from the GA of plant cells, a result in line with the presence of lipid microdomains in the GA of animal cells (Hansen et al., 2000
To further examine the involvement of lipid microdomains in membrane trafficking in higher plant cells, our approach has been to manipulate the PM sterol content by using FEN, a sterol biosynthetic inhibitor. This compound is known to mainly inhibit the cycloeucalenol-obtusifoliol isomerase (Rahier et al., 1986
These results raise several comments. In all DRMs, sterols were present as a mixture of the same sterol molecules as that of the PM or microsomal fractions and in similar proportions. Such a result confirms that no specific enrichment in a given sterol occurs in plant DRMs, indicating either that all sterol molecules may contribute to microdomain formation with the same efficiency, as recently pointed out for sitosterol and stigmasterol (Xu et al., 2001
The fact that the highest amount of DRMs was found in the GA instead of the PM after treatment of leek seedlings by FEN indicates that lipid microdomain delivery to or formation at the PM was perturbed. A concomitant decrease of sterols and hydroxy-palmitic acid-containing GluCer in the PM may provide new evidence that close relationships exist between sterol and sphingolipid biosynthetic pathways (Hartmann et al., 2002
In mammalian cells, cholesterol levels can influence both the domain association and biological activity of associated proteins (Friedrichson and Kurzchalia, 1998
In addition, cholesterol was proposed to regulate protein sorting by a bilayer-mediated mechanism, in which proteins are targeted to bilayers whose hydrophobic thickness matches the length of their transmembrane domain (Bretscher and Munro, 1993
Another critical point that has been investigated in this article was whether the degree of unsaturation of phospholipid fatty acyl chains may have an impact on the formation of lipid microdomains in plant cells. We took advantage of two mutant Arabidopsis cell lines, fad2 and Fad3+, which are affected in their fatty acid desaturases. The 18:1 desaturase is shut down in the fad2 mutant (Okuley et al., 1994 Taken together, these results suggest that the unsaturation degree of lipid acyl chains of plant cell membranes plays a crucial role in the ability of lipids to partition into lipid microdomains, and two aspects may be highlighted: (1) it is more the fatty acyl moieties than the polar heads that drive lipid segregation in plant cell membranes; and (2) the overall degree of unsaturation of the membrane and therefore its fluidity has an impact on the amount of lipids that can be segregated into microdomains.
In conclusion, we have given evidence that sterols, GluCer, and the unsaturation degree of lipid acyl chains play critical roles in the formation of lipid microdomains in the PM and GA of plant cells. Recent evidence suggests that the wide functional diversity in which lipid microdomains may be involved is provided by the coexistence of distinct lipid microdomain populations with different protein and lipid compositions (Pike, 2004
Plant and Cell Growth Conditions
Leek (Allium porrum) seeds were purchased from Vilmorin and stored overnight at 4°C before being hydrated with distilled water for 2 h. The seeds were allowed to germinate and grow in the dark for 7 d at 22°C to 24°C on a culture medium containing 0.8 mM NaH2PO4, 1 mM MgSO4, 7 mM NaNO3, 0.8 mM CaCl2, 10 mM KCl, and 5% (w/v) agar. Arabidopsis (Arabidopsis thaliana) ecotype Columbia seedlings were grown according to Krysan et al. (1996) FEN, which was kindly supplied by BASF, was added to the culture medium at a final concentration of 2 mg/L (6.6 µM).
ER-, GA-, and PM-rich fractions from leek seedlings were isolated and characterized as described earlier (Vincent et al., 1999 PM-rich fractions from Arabidopsis seedlings were isolated by the same procedure and under similar conditions as described above.
Protein concentrations were determined according to either Lowry et al. (1951)
The various membrane fractions isolated from leek and Arabidopsis seedlings were treated at 4°C for 30 min with 1% Triton X-100 (1% final concentration) using detergent-to-protein ratios varying from 2 to 16. Membranes were then brought to a final concentration of 48% (w/w) Suc, overlaid with 2 mL of 40%, 35%, 30%, and 5% (w/w) Suc in the washing buffer, and spun for 16 h at 150,000g at 4°C. DRMs were recovered from below the 30% to 35% interface, washed with water to remove residual Suc, and recentrifuged at 200,000g for 15 min.
Membrane microdomains from Arabidopsis cell cultures were isolated according to the method of Ko et al. (1999)
SDS-PAGE was carried out on 12% polyacrylamide gels. Membrane protein samples were mixed with an equal volume of Laemmli sample buffer (Sigma) and heated at 90°C for 3 min before being loaded on the gels. After electrophoresis, the bands were blotted on polyvinylidene difluoride membranes (Perkin Elmer) by transverse electrophoresis. Three different antibodies were used: the anti-PMA2 antibody was generously provided by M. Boutry (University of Louvain, Belgium), the anti-PIP1 antibody was kindly provided by V. Santoni and C. Maurel (Centre National de la Recherche Scientifique/Institut National de la Recherche Agronomique/Université Montpellier 2, France), and the anti-remorin antibody, which was generously provided by E.E. Farmer (University of Lausanne, Switzerland). The anti-PMA2 antibody may recognize PMA4 as well (Lefebvre et al., 2004
Lipids were extracted by chloroform:methanol (2:1, v/v) for 30 min at room temperature and then washed three times with 9% NaCl. The solvent was evaporated and lipids were dissolved in an appropriate volume of chloroform/methanol (1:1, v/v).
Polar lipids were analyzed by loading total lipids onto HPTLC plates (60F254, Merck), which were developed in methyl acetate/n-propanol/chloroform/methanol/0.25% aqueous KCl (25:25:25:10:9, v/v) according to Heape et al. (1985)
Fatty acids of individual lipids were determined and quantified by GC (using a Hewlett-Packard 5890 series II chromatograph) after conversion to the corresponding methyl esters by hot methanolic H2SO4 according to Browse et al. (1986)
Free sterols from leek membrane fractions and DRMs were analyzed as previously reported (Hartmann et al., 2002 Mass spectrometry of GluCer was performed as follows: ceramide extracts from DRMs were evaporated to dryness and dissolved in a methanol/dichloromethane 3:1 mixture prior to addition of aqueous lithium iodide (5 mM final concentration). Exact mass measurements of lipids were made in the electrospray mode using a Waters LCT Premier mass spectrometer fitted with a dual sprayer (Lockspray) source. Lockmass correction used the doubly charged ion of a peptide reference compound (Glufibrinopeptide from Sigma). MS2 and MS3 mass measurements were made with an ion trap mass spectrometer (Thermo Instruments LCQ Advantage) operated in the electrospray ionization mode. A 4-D isolation width was applied for precursor selection.
The following materials are available in the online version of this article.
We thank Professor M. Boutry (University of Louvain, Belgium), Drs. V. Santoni and C. Maurel (Centre National de la Recherche Scientifique/Institut National de la Recherche Agronomique/Université Montpellier 2, France), and Professor E.E. Farmer (University of Lausanne, Switzerland) for providing us with anti-PMA2, anti-PIP1, and anti-remorin antibodies, respectively. Received October 18, 2006; accepted November 10, 2006; published November 17, 2006.
1 This work was supported by the Centre National de la Recherche Scientifique (postdoctoral fellowship to M.L.), by the Victor Segalen University of Bordeaux 2, by the Conseil Régional d'Aquitaine (M.L., A.M.P., L.C., M.V., R.L., P.M.; mass spectrometry equipment to J.M.S.), and by the Pierre and Marie Curie University of Paris (C.C., M.N.V., A.Z.). 
2 Present address: Laboratoire de Physiologie, Biochimie et Biologie Végétales, Génétique des Levures, UMR 6161-CNRS-Université de Poitiers, 40 Avenue du Recteur Pineau, 86022 Poitiers-cedex, France.
3 Present address: Department of Biology, 112 Science Place, University of Saskatchewan, Saskatoon, SK S7N 5E2, Canada. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Patrick Moreau (pmoreau{at}biomemb.u-bordeaux2.fr).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.106.091496 * Corresponding author; e-mail pmoreau{at}biomemb.u-bordeaux2.fr; fax 33–5–56–51–83–61.
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-hydroxypalmitic acid (16-OH GluCer), or dihydroxypalmitic acid (Sullards et al., 2000
