Insights into the role of specific lipids in the formation and delivery of lipid microdomains to the plasma membrane of plant cells.

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 Delta(5)-sterols are replaced by 9beta,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 Delta(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.

Evidence for a different metabolism of PC and PE in shoots and roots.

We investigated phosphatidylcholine (PC) and phosphatidylethanolamine (PE) labelling in shoots and roots from leek plantlets, maize seedlings and Arabidopsis thaliana through the incorporation of radiolabelled acetate. Regardless of the pathway followed in shoots, PC labelling was always higher than PE labelling. However, we obtained an opposite situation in leek and A. thaliana roots since PC labelling was much lower than PE labelling. Several hypotheses to explain the origin(s) of these discrepancies between roots and shoots were tested. Among them, neither the level of the respective AAPT activities, nor specific regulations of PC biosynthesis through the mRNA levels of several enzymes (choline citidylyltransferase (CCT), ethanolamine citidylyltransferase (ECT), phosphoethanolamine methyltransferase (PEAMT)), nor the fatty acyl chain composition of PC, PE, and diacylglycerol, were responsible for the differences observed between PC and PE metabolism in roots and shoots. Finally, we investigated the acylation of PC and PE in vitro in both shoots and roots of A. thaliana seedlings, and demonstrated that some specific remodelling of PC and PE by acylation was responsible for the differences in labelling observed in vivo.

Anti-phospholipid antibodies in diabetes mellitus.

The presence of autoantibodies to phospholipids may be associated with various pathological disorders; diabetes could be one of them because of the changes occurring in lipid metabolism but there are only few reports examining this question, and they are not always leading to the same conclusions because of the differences in the procedures or in the phospholipids tested. We carried out a systematic comparative study of diabetic serum antibody binding to all phospholipids, anionic and zwitterionic, by a quantitative ELISA. The implication of the hydrophobic moiety of the lipids was also studied: the presence of autoantibodies to the fatty acyl chains was investigated. Our results show the presence of anti-phospholipid antibodies in diabetic sera, particularly anti-phosphatidylinositol and anti-phosphatidylcholine which have never been tested before, and appear to be associated with macroangiopathic complications. The antigenic epitopes are mainly the polar heads as no antibody binding to the hydrophobic moiety was observed. We discuss the relation of those antibodies to the angiopathic complications and to the direct effects of hyperglycemia on lipid antigenicity.

Inhibition of the sterol pathway in leek seedlings impairs phosphatidylserine and glucosylceramide synthesis but triggers an accumulation of triacylglycerols.

Like most higher plants, leek seedlings (Allium porrum L.) contain a mixture of Delta(5)-sterols in which sitosterol largely predominates. As previously reported (Plant Physiol., 117 (1998) 931), these compounds, which are synthesized at the endoplasmic reticulum level, were shown to be actively transported to the plasma membrane via a membrane-mediated process, together with phosphatidylserine (PS). In the present work, leek seedlings were allowed to germinate for 7 days in the presence of fenpropimorph, a sterol biosynthesis inhibitor. Such a treatment was found to trigger an almost complete replacement of the usual sterols by 9beta,19-cyclopropylsterols (mainly cycloeucalenol and 29-norcycloartenol). Extensive lipid analyses and labeling experiments with sodium [14C]acetate were performed to examine potential changes in the content and the rate of synthesis of the other lipid molecular species. The results indicate that the inhibition of the sterol pathway was accompanied by a severe decrease in PS and glucosylceramide synthesis as well as by a redirection of fatty acids toward the storage triacylglycerol pathway. Triacyglycerols are shown to accumulate concomitantly with a significant increase in intracellular lipid droplets in both aerial parts and roots of leek seedlings. Taken together, the present data emphasize that a coordinated regulation of the biosynthetic pathways of sterols and some specific lipid molecular species could take place during plant membrane biogenesis.

Effects of brefeldin A and nordihydroguaiaretic acid on endomembrane dynamics and lipid synthesis in plant cells.

Effects of brefeldin A (BFA) and nordihydroguaiaretic acid (NDGA) on endomembrane structures and lipid synthesis were compared in maize root cells and tobacco Bright Yellow-2 cells. Immunofluorescence and electron microscopy studies showed that NDGA altered the structure and distribution of the endoplasmic reticulum (ER) within 1 h but not of the Golgi apparatus whereas, as shown previously, BFA altered that organization of the Golgi apparatus and, only subsequently, of the ER. Biochemical studies revealed that both drugs and especially BFA led to a strong inhibition of the phytosterol biosynthetic pathway: BFA led to accumulation of sterol precursors. The importance of phytosterols in membrane architecture and membrane trafficking is discussed.