Paul Mazliak

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.

Biosynthetic pathway of α-linolenic acid in developing pea leaves in vivo and in vitro study

Abstract In vivo and in vitro incorporations of [1- 14 C]acetate, [1- 14 C]oleate and [U- 14 C]linoleate into the lipids of developing pea leaves show that the following pathway is involved: Evidence points to the desaturation of oleate to linoleate in microsomes, whereas the desaturation of linoleate to linolenate takes place in the plastids of the cells. A cooperation between microsomes and plastids seems to be necessary for the biosynthesis of linolenic acid in leaves.

Lipid composition of Euglena gracilis in relation to carbon-nitrogen balance

Abstract The lipid class and fatty acid compositions of Euglena gracilis were analysed after growth under various conditions of autotrophy or photoheterotrophy, in order to identify the contribution of lactate (a carbon source) and ammonium phosphate (a nitrogen source) to the metabolism of these compounds. When ammonium and lactate were both present in the medium, in concentrations that allowed growth, the lipid composition of algae appeared to be independent of the ammonium concentration. The effects of increasing ammonium phosphate concentration on lipid metabolism were observed only when lactate was depleted. With increasing nitrogen concentration in the medium, an increase in the content of galactolipids rich in polyunsaturated C 16 and C 18 fatty acids (FA) was noted, as well as an increase in the monogalactosyldiacylglycerol: digalactosyldiacylglycerol ratio; these changes could be related to an increase in the number of the stacked lamellae present in chloroplasts. Nitrogen concentration had no influence on the C 12 and C 14 FA but induced a loss of C 22 FA. In the absence of ammonium phosphate in the culture medium, increasing lactate concentrations were accompanied by a decrease in all plastid lipids, whereas the content of storage lipids (enriched in 14 : 0 and 16 : 0) increased. Partial inhibition of the polydesaturation of C 18 FA was observed and was accompanied by an accumulation of 18 : 1.

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.

Plant Membrane Lipids : Changes and Alterations during Aging and Senescence

Some types of lipid molecules (phospholipids or galactolipids), because of their amphiphilic properties, are essential constituents of all biomembranes. Changes occurring in the lipid composition of these membranes will certainly modify their permeability, their energy transduction capacity, the activities of their membranebound enzymes, before and after harvest, when plants of economical value are concerned. Furthermore, after harvest, many alterations are likely to affect membrane lipids, thus playing a major role in the senescence or the post-harvest physiology of plant crops. It seems therefore reasonable to pay some attention to plant membrane lipids when discussing the factors allowing good crop preservation.

Etude comparee des lipides et de la fixation passive du calcium dans les racines et les fractions subcellulaires du Lupinus luteus et de la Vicia faba

Abstract In both lupin and broad bean, the root lipids contain paraffins, triglycerides, diglycerides, free fatty acids and polar lipids (phospholipids and galactolipids). The polar lipids and the triglycerides are the more abundant classes. The root galactolipids are mono- and di-galactosyldiglycerides; two steryl glycosides are also present. The phospholipids in both species are: phosphatidylinositol, phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, diphosphatidylglycerol and phosphatidic acid. This last phospholipid represents 8·3% of total lipid phosphorus in Lupinus against 2·3% in Vicia . The other acidic phospholipids represent 30·4% in Lupinus against 20·9% in Vicia . The lipids of Lupinus are rich in linolenic acid whereas those found in Vicia are richer in linoleic acid. The various subcellular fractions prepared from the roots of both species have an homogeneous lipid composition, reflecting exactly that of entire cells. The calcium passive fixation capacity in microsomes and mitochondria of Lupinus roots is more important than that in the same organelles of Vicia faba roots. Thus a relationship is suggested between the amount of phospholipids in membranes and the passive fixation of calcium.

Effect of drought on photodynamic peroxidation of leaf total lipophilic extracts

Abstract In vitro lipid peroxidation, induced by photochemically generated reactive oxygen species, was carried out either on purified α-linolenic acid (18:3n — 3) (1.32 mM) or on total lipophilic extracts ofleaf(standardized at 1.32 mM for 18:3n — 3 content). Three cultivars of plants differing in their sensitivity to water deficit were used: Vigna unguiculata L. cv. EPACE (drought-tolerant), Vigna unguiculata L. cv. IT-83D (moderately drought-tolerant) and Phaseolus vulgaris L. cv. Carioquinha (drought-sensitive). The time-course of 18:3n — 3 photoperoxidation was comprised of three phases: (i) monohydroperoxidation, characterized by formation of conjugated diene patterns, (ii) secondary oxidation, characterized by formation of conjugated triene patterns, (iii) oxidative fragmentation of peroxidized compounds, characterized both by the disappearance of conjugated polyenic patterns and by the formation of thiobarbituric acid reacting substances. Only the first and second phases were observed on photoperoxidation of total lipophilic extracts from leaves of well-hydrated plants. When each cultivar was subjected to drought treatment, the conjugated diene pattern maximum was attained earlier than for well-hydrated plants, suggesting that drought affects the resistance capacity of total lipophilic extracts from leaves to photoperoxidation. The more severe the drought, the more susceptible the leaf extracts were to lipid peroxidation induced by reactive oxygen species. The more resistant to drought the plant was, the slower the rate of formation of conjugated polyenic patterns. These results are discussed in relation with the chlorophyll a/b and carotenoids/18:3n — 3 molar ratios in the photoperoxidation apparatus. This work is the first attempt to correlate plant drought tolerance and the sensitivity of their leaf total lipophilic extracts to lipid peroxidation.

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.