Chantal Demandre

Analysis of molecular species of plant polar lipids by high-performance and gas liquid chromatography

Abstract Total lipid extracts from potato tubers and tobacco leaves are separated into lipid classes by two step HPLC using a silicic column. Elution is first performed for 20 min with a programmed linear gradient of two mixed solvents running from 100% of solution A (isopropanol-hexane, 4:3) to 100% of solution B (isopropanol-hexane-water, 8:6:1.5); the column is then eluted with pure solution B in an isocratic mode for 20 min more. The main polar lipids (MGDG, DGDG, PC, PE, PG) from both plant tissues can be collected and further separated into component molecular species on a simplified HPLC system with a C18 column eluted in an isocratic mode with a polar solvent. Molecular species separations are achieved within 35 min; quantifications are made through GLC analysis of attached fatty acids. Three to five main molecular species are thus clearly identified in each lipid class. In potato tuber, phospholipids (PC, PE) 18:2/18:2 species are predominant. In tobacco leaf, six double bond species (18:3/18:3 and 16:3/18:3) are predominant in galactolipids, whereas PC contains a greater number of molecular species varying by their degree of unsaturation (from 18:3/18:3 to 16:0/18:2). Only certain molecular species of PG contain Δ3-trans-hexadecenoic acid.

No discrimination by choline- and ethanolamine phosphotransferases from potato tuber microsomes in molecular species of endogenous diacylglycerols

Abstract Molecular species of phosphatidylcholine, phosphatidylethanolamine and diacylglycerols extracted from potato tuber microsomal membranes were separated by high-performance liquid chromatography. The same quantitative distribution of molecular species was found in those three lipid classes. The molecular species of diacylglycerols resulting from the hydrolysis of phosphatidylcholine molecules by phospholipase C were identical to molecular species of endogenous free diacylglycerols. Kinetics of labelling in vitro of the molecular species of microsomal phospholipids (from CDP[14C]choline and CDP[14C]ethanolamine) showed that choline- and ethanolaminephosphotransferases do not show any selectivity towards the molecular species of the endogenous pool of diacylglycerols when phosphatidylcholine or phosphatidylethanolamine are synthesized.

Oleate desaturation in six phosphatidylcholine molecular species from potato tuber microsomes

Abstract When microsomes prepared from aged potato tuber slices, were incubated for 10 min with [14C]oleoyl-CoA, in the absence of NADH, three labelled molecular species of phosphatidylcholine (PC) were mainly formed: 1-palmitoyl-2-[14C]oleoyIPC (50.1%), 1-linoleoyl-2-[14C]oleoyIPC (31%) and 1-[14C]oleoyl-2-linoleoyIPC (10%). Three other molecular species were less heavily labelled: 1-[14C]oleoyl-2-palmitoyIPC (4.9%), 1-linolenoyl-2-[14C]oleoyIPC (2.4%) and 1-[14C]oleoyl-2-linolenoylPC (1.6%). Acylation of oleoyl residues occurred preferentially in position 2 of sn-glycerol. When NADH was present in the incubation medium the following labelled species ( 18:3 18:1− PC , 18:2 18:1−PC and 16:0 18:1−PC ) accumulated for the first 10 min of incubation. These species were then progressively desaturated by the oleate desaturase of microsomes and clear precursor-product relationships could be observed between 18:2 18:1−PC and 18:2 18:2−PC on one side, and 16:0 18:1− PC and 16:0 18:2−PC on the other. The following molecular species accumulated as final desaturation products in microsomes: 1-palmitoyl-2-[14C]linoleoylPC (38.8%), 1-linoleoyl-2[14C]linoleoylPC (36.8%), 1-[14C]Iinoleoyl-2-linoleoylPC (12.3%), 1-[14C]linoleoyl-2-palmitoylPC (6.8%), 1-linolenoyl-2-[14C]linoleoylPC (3.5%) and 1-[14C]linoleoyl-2-linolenoylPC (1.8%). No selection among oleoylPC molecular species was realized by potato oleate desaturase.

Linolenic acid biosynthesis via glycerolipid molecular species in pea and spinach leaves

Abstract Only molecular species of the eukaryotic type (18:3/18:3; 18:2/18:3; 18:2/18:2, 16:0/18:3, etc…) are present in the phosphatidylcholine (PC) of pea or spinach leaves. In pea leaves, total monogalactosyldiacylglycerol (MGDG) is mostly represented by two eukaryotic molecular species: 18:3/18:3 (79 mol%) and 18:2/18:3 (9%). In spinach leaves, the prokaryotic molecular species 18:3/16:3 forms 39 mol% of total MGDG; however the eukaryotic species 18:3/18:3 (60 mol%) is dominant. At the end of a 30 min pulse with 14 C-oleate, four molecular species of PC were intensively labelled in pea leaves: 18:3/ 14 C-18: 1; 18:2/ 14 C-18: 1; 16:0/ 14 C-18: 1 and 18: 1/ 14 C-18: 1. During the following 48 hr chase period, clear precursor-product relationships could be observed between 14 C-18: 1 PC and 14 C-18:2 PC molecular species on one side and between 14 C-18:2 PC and 14 C-18:3 PC molecular species on the other side. In spinach leaves the same 14 C-18: 1 PC molecular species were labelled more slowly and desaturation was not observed beyond 14 C-18:2 PC molecular species. In MGDG from pea leaves, labelled linolenic acid accumulated steadily in 18:3/18:3 MGDG, after a lag time of 1 hr. In MGDG from spinach leaves labelled 18:3/18:2 MGDG was a precursor for labelled 18:3/18:3 MGDG. It is concluded that linolenic acid is synthesized via PC molecular species in the eukaryotic pathway of pea leaves and via MGDG molecular species in the eukaryotic pathway of spinach leaves.

Molecular species synthesized by phosphatidylinositol synthases from potato tuber, pea leaf and soya bean

5–9 different molecular species of phosphatidylinositol (PI) were separated, combining HPLC and GLC analyses, from microsomes extracted from potato tuber, pea leaf and germinated soya beans. In microsomes from the three plant tissues, 16:0/18:2-PI was the most abundant molecular species. The second most abundant species was 16:0/18:3-PI in pea leaf and potato tuber, whereas 18:0/18:2-PI was more abundant than 16:0/18:3-PI in soya bean microsomes. Labelling PI molecular species from [ 14 C] myo -inositol, in the presence of EDTA to avoid any exchange reaction, showed that only two molecular species (16:0/18:2-PI and 16:0/18:3-PI) were prominently produced by PI synthases from the three tissues. The molecular species 18:0/18:2-PI and 18:2/18:3-PI were slightly labelled (less than 10% of total PI radioactivity) in soya bean and pea leaf, respectively. A good correspondence could be observed between the quantitative distributions of PI molecular species and the relative labellings of the same species in the three tissues. Those results suggest that de novo synthesis of PI molecular species is not followed by subsequent alterations (acyl exchanges or desaturations) of these molecules.

Biosynthesis of triacylglycerols by developing sunflower seed microsomes

Abstract In order to elucidate the biochemical pathway involved in the formation of plant triacylglycerols rich in C18 polyunsaturated fatty acids, microsomes from developing sunflower cotyledons were incubated with [1- 14 C ]oleate or [1- 14 C ]linoleate. Total lipids were extracted and separated into lipid classes. The molecular species of phosphatidylcholine, diacylglycerols and triacylglycerols were analyzed. After [ 14 C ]oleate feeding differences were found in the labelling of phosphatidylcholine and diacylglycerols labelled molecular species, whereas distribution of these molecules was the same when [ 14 C ]linoleate was supplied. The role of phosphatidylcholine-diacylglycerol interconversion that may be catalysed by CDP choline:diacylglycerol phosphotransferase (EC 2.7.8.2) in generating triacylglycerols rich in linoleate is discussed. All the results obtained corroborate previous studies involving phosphatidylcholine in the biosynthesis of such triacylglycerols and allowed us to suggest reactional mechanisms for the formation of the different triacylglycerols molecular species in sunflower seeds.

Choline- and ethanolaminephosphotransferases from pea leaf and soya beans discriminate 1-palmitoyl-2-linoleoyldiacylglycerol as a preferred substrate

Abstract Combining HPLC and GLC analyses, 7–13 different molecular species were found in phosphatidylcholine (PC), phosphophatidylethanolamine (PE) and diacylglycerols from pea leaves and germinating soya beans. In soya bean microsomes, the quantitative distributions of molecular species are nearly similar in the three lipid classes, except for the 16:0 18:3 species, which are more abundant in diacylglycerols (20 mol%) than in PC (5.9 mol%) or PE (4.6 mol%). In pea leaf microsomes, the 18:3 18:2 species is predominant (44.5 mol%) in diacylglycerols whereas 16:0 18:2 PC and 16:0 18:2 PE are the major molecular species in phospholipids. Labelling of PC molecular species from CDP[14C]choline reveals that in both pea leaf and soya bean microsomes, cholinephosphotransferase selected actively 1-palmitoyl-2-linoleoyldiacylglycerol as a preferred substrate. The same molecular species was selected by ethanolaminephosphotransferase, as shown by kinetics of labelling from CDP[14C]ethanolamine. However, in both tissues, PE was relatively poorly labelled in its 18:2 18:2 plus 16:0 18:3 fraction as compared with PC.

What is the origin of the lipids of potato tuber plasmalemma

Abstract Plasmalemma isolated from potato tuber cells was prepared according to a flotation method in a sucrose density gradient. When incubated in vitro with various radioactive lipid precursors, the plasmalemma fragments were unable to synthesize any lipid. When labelled lipid precursors ([ 14 C]acetate of [ 32 P]phosphate) were furnished to potato slices, radioactive fatty acids or phospholipids were integrated into plasmalemma lipids. When incubated in a suspension of [ 32 P]liposomes, plasmalemma fragments accepted labelled lipids from the suspension; this exchange process was markedly stimulated by the phospholipid exchange protein prepared from the potato cytoplasmic supernatant. These results suggest that potato tuber plasmalemma is dependent on other cellular fractions for lipid synthesis and exchange.

Effects of a Heat Shock on the Molecular Species of Rape Polar Lipids

Rape (Brassica napus L. var Bienvenue) is an oil seed plant of great potential economical interest for Algeria. The main problem for growing rape in this country is the high environmental temperature encountered during summer. Growth at high temperature results in a decrease in the abundance of polyunsaturated fatty acids. Such a decrease is associated with reduced membrane fluidity [1].

In Vivo Manipulation of Lipid Composition in Mutants of Chlamydomonas ReinhardtII: A Tool to Study the Roles of Lipids in the Biogenesis of the Photosynthetic Membrane

Photosynthesis in eucaryotic green organism uses solar energy collected by the light-harvesting chlorophyll a/b protein complex associated with photosystem II (LHCII) which is the most abondant membrane protein in chloroplasts binding half of the pigments involved in plant photosynthesis. Each polypeptide organizes a minimum of 12 chlorophylls, 2 carotenoids (Nusberger et al. 1993)(1) and at least two galactolipids (one mono- and one di-galactosyldiacylglycerol) and one phosphatidylglyce-rol (Tremolieres et al. 1994)(2).