Waldemar Eichenberger

Lipids of Pavlova lutheri: Cellular site and metabolic role of DGCC

Pavlova lutheri contains triacylglycerols (TAG), monogalactosyldiacylglycerols (MGDG), galactosyldiacylglycerols (DGDG), sulfoquivonosyldiacylglycerols (SQDG), diacylglyceryl hydroxymethyl-N,N,N-trimethyl-β-alanine (DGTA), diacylglyceryl carboxyhydroxymethylcholine (DGCC) and diacylglyceryl glucuronide (DGGA) as major lipid components. Prominent fatty acids are 14:0, 16:0, 16:1, 18:4, 20:5, 22:5 and 22:6. In MGDG and DGDG, 18:4 and 20:5 predominate, while SQDG contains high proportions of 14:0 and 16:0. DGCC is enriched in 16:0 and 20:5, DGGA in 18:1, 22:5 and 22:6, and DGTA in 22:6. Analysis of subcellular membrane fractions demonstrated an accumulation of DGCC, DGTA and DGGA in non-plastid membranes. On incubation of cells with [1-14C] oleic acid, after 60 min of pulse, 30% of the incorporated label was found in TAG and 70% in polar lipids. DGCC (52%) and DGTA (12%) were the most strongly labelled polar lipids. Within 72 h of chase, in DGCC and in TAG the label rapidly decreased to 11 % and 17%, respectively, but in MGDG it increased in the same time up to 35% of the total. Only minor changes were observed in DGTA and no significant label was recorded in DAG during the chase-period. The shift of label from 18:1 to 18:4, 20:5 and 22:6 reflected further elongation and/or desaturation of the substrate. Very similar results obtained using [2-14C] acetate as precursor suggested that DGCC acts as a primary acceptor of de novo-formed or exogenous fatty acids which subsequently undergo processing and redistribution. It is suggested that C18 and C20 fatty acids are transferred individually from the cytoplasm to the chloroplast allowing the synthesis of eukaryotic MGDG without the import of DAG.

Distribution of diacylglyceryl-O-4′-N,N,N-trimethyl)- homoserine in different algae

Abstract Diacyglyceryl- O -4′-( N , N , N -trimethyl) homoserine (DGTS), a lipid which is typical of Ochromonas (Chrysophyceae) and Chlamydomonas (Chlorophyceae), has also been detected in Polytoma and Fritschiella (Chlorophyceae), but not in Chlorella and Prototheca (Chlorophyceae), Euglena (Euglenophyceae) and Fucus (Phaeophyceae). The large contents of DGTS in Ochromonas and Chlamydomonas parallel low contents of phosphatidyl choline (PC). From this it is suggested that DGTS may substitute for PC in these algae. DGTS is formed by algae of different taxonomic positions and by both photosynthesizing and non-photosynthesizing species. A correlation between the presence of DGTS and cellular organization has not been detected.

Betaine lipids and phospholipids in brown algae

Abstract The lipids of more than 100 species representing all of the 16 orders of brown algae (Phaeophyceae) have been analysed by TLC. The glycolipids, monogal

Lipids of Chlamydomonas reinhardtii. Incorporation of [14C]Acetate, [14C]Palmitate and [14C]Oleate into Different Lipids and Evidence for Lipid-Linked Desaturation of Fatty Acids

The lipids of Chlamydomonas reinhardtii mainly consist of MGDG, DGDG, SQDG, PG, PE, PI and the betaine lipid DGTS. PC, however, was shown to be strictly absent from this alga (Giroud et al. 1988). The chloroplast lipids are of the prokaryotic, DGTS and PE of the eukaryotic type. Each lipid class is characterized by an individual pattern of molecular species suggesting that fatty acids are desaturated in a lipid-linked process in which the different lipids individually act as substrates for the desaturase(s). In order to provide additional evidence for this process, cells were labelled with 14C-acetate, 14C-palmitate or 14C-oleate and the radioactivity was measured after different times in each lipid class and in their fatty acids and molecular species.

Lipids of Chlamydomonas reinhardi under different growth conditions

Abstract Photo-, mixo- and heterotrophically grown cultures of Chlamydomonas reinhardi (wild type ss and 2 streptomycin-resistant mutants sr 3 and sr 35 ) have been analyzed for lipids and fatty acids. Ether-soluble lipids, chlorophyll, monogalactosyl diglyceride, digalactosyl diglyceride, sulfolipid, phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl glycerol and the relative amounts of fatty acids in total and individual lipids have been determined. The lipid and fatty acid compositions are very similar in the 3 strains and are not affected by the mutations. Fatty acids belong exclusively to the C 16 and C 18 series, 16:0, 16:4, 18:1, 18:2, 18:3 (6,9,12) and 18:3 (9,12,15) comprising about 90% of the total. 18:3 (6,9,12) is concentrated in phosphatidyl ethanolamine. In streptomycin-bleached sr 3 cells, ether-soluble lipids increase from 7 to 11% of dry weight on greening, mostly due to synthesis of monogalactosyl diglyceride and chlorophyll. Monogalactosyl diglyceride of bleached cells exhibits the same fatty acid pattern before and after greening.

1(3),2-Diacylglyceryl-3(1)-O-2′-(hydroxymethyl)(N,N,N,-trimethyl)-β-alanine (DGTA): A novel betaine lipid from Ochoromonas danica (Chrysophyceae)

Abstract The structure of lipid B, another betaine lipid from Ochromonas danica (Crysophyceae) has been elucidated by NMR, MS and IR data from the intact lipid and from derivatives obtained by chemical degradation. The structure of 1(3),2-diacylglyceryl-3(1)- O -2′- (hydroxymethyl)( N , N , N -trimethyl)-β-alanine (DGTA) has been assigned to the intact lipid which is a structural isomer of the already known homoserine lipid or 1(3),2-diacylglyceryl-3(1)- O -4′-( N , N , N -trimethyl)-homoserine (DGTS). 1(3),2- Diacylglyceryl-3(1)- O -2′-(hydroxymethyl)-propenoic acid was obtained from DGTA by deamination in chloroform with traces of formic acid. 1(3),2-diacylglycerol-3(1)- O -2′-(hydroxymethyl)-propanoic acid methyl ester was formed by treatment of DGTA with diazomethane in the presence of formic acid and by subsequent hydrogenation. From this compound, the corresponding diacetyl derivative was obtained by trans -esterification and acetylation. The diacetate was identical with synthetic 1(3),2-diacetylglyceryl-3(1)- O - 2′-(hydroxymethyl)-propanoic acid methyl ester, the preparation of which, starting from isopropylideneglycerol and 2-(bromomethyl)-acrylic acid methyl ester, is described. DGTA accounts for approximately 5% of the total lipids of O. danica . The major fatty acids of DGTA are 22:5 (42%), 18:0 (15%), 20:4 (13%); 20:2 (6%), 18:1 (5%), 20:3 (4%) 18:2 (4%), 22:4 (3%) and 16:0 (2%). From DGTA, trimethylamine is produced by a spontaneous deamination.

Evidence for the involvement of diacylglyceryl (N,N,N,-trimethy homoserine in the desaturation of Oleic and linoleic acids in Chlamydomonas reinhardi (chlorophyceae

Abstract [1-14C]Oleic (18:1) acid was incorporated by cells of Chlamydomonas reinhardi primarily into diacylglyceryl(N,N,N-trimethylhomorserine (aDGTS). This lipid consists mainly of 16:0/γ18:3 acyl pairings, but also contains 16:0/18:1, 16:0/18: 2 and 18:2 /γ18:3 species. Within 3 h about 80δ h about 80°of the labelled monoene acid incorporated in DGTS was desaturated to linoleic (18:2) and γ-linolenic (18.3n^) acid. The same time, the label was displ from 16:0/18:1 specie.8 Cells also incorporated di-18:-DGSTS which had seen 3H-La labelled in the polar part and 14C-labelled in the acyl part. A major part of the substrate was taken up in intact fomn. In part its oleoyl moieties were desaturated as shown by the consecutive labelling of the 18:1/18:1 and 18:2/γ18:3 species. From these results it is concluded that in this alga DGTS serves as an acyl carrier in the desaturation of oleic and linoleic acids.

Isolation and characterization of a new phospholipid from brown algae. Intracellular localization and site of biosynthesis

Summary In various orders of brown algae a novel lipid constituent PX was detected and suggested to be a phosphoglyceride bearing an amino-containing polar head group. We discuss its tentative structure based on IR, NMR, MS and chemical data. PX accounted for 2–4% of total lipids and always exhibited a fatty acid composition rich in 20:4 (arachidonic) and 20:5 (eicosapentaenoic) acids. High proportions of these long-chain polyunsaturated acids implied a predominantly cytoplasmic biosynthesis of its diglyceride part. In Ectocarpus siliculosus (Phaeophyceae) PE has a fatty acid pattern similar to PX. In its gametes PX accumulates in the plasma membrane and, in addition, shows a characteristic sex-specific composition of its acyl moieties. In female plasma membranes the content of 20:5 acid is significantly higher than in male plasma membranes or whole plants. This is possibly due to 20:5 acid from PX acting as a direct precursor for pheromone biosynthesis. PX is suggested to be of special physiological importance in terms of an acyl donor in this process. The 20:5 pool in PX was estimated to be sufficient to synthesize 70% of the pheromone ectocarpene.

Sterol mutants of Chlamydomonas reinhardtii: Characterisation of three strains deficient in C24(28) reductase

Abstract Three mutants of Chlamydomonas reinhardtii (strain arg7cw15 ) were obtained using the strategy of insertional mutagenesis by random plasmid integration with subsequent selection for resistance against the polyene antibiotic nystatin. Sterols were isolated by precipitation with digitonin, fractionated by both normal and argentation TLC, and then analysed by GLC and GC-MS. All the mutants accumulated ergosta-5,7,22,24(28)-tetraenol, ergosta-5,7,24(28)-trienol, ergosta-7,24(28)-dienol, stigmasta-5,7,22,24(28)-tetraenol, stigmasta-5,7,24(28)-trienol, stigmasta-8,24(28)-dienol and stigmasta-7,24(28)-dienol, while ergosterol and 7-dehydroporiferasterol which are the only major sterol components of the original strain were absent in the mutants. It is concluded that all these mutants are impaired in this C24(28) reductase which catalyses the reduction of the C24(28) tetraenol to the corresponding 24-alkyl sterol. There is strong evidence that the same enzyme acts on both the C 28 and C 29 sterol series. This view is also supported by Southern blot hybridisation analysis revealing that in all three mutants, plasmid insertion occurred at the same site indicating the disruption of the same gene. Due to the insertional nature of the mutations, the strains can be used for cloning the corresponding gene.

CROSSING EXPERIMENTS, LIPID COMPOSITION, AND THE SPECIES CONCEPT IN ECTOCARPUS SILICULOSUS AND E. FASCICULATUS (PHAEOPHYCEAE, ECTOCARPALES)1

Sporophytes of Ectocarpus siliculosus (Dillwyn) Lyngbye and E. fasciculatus Harvey were collected in the vicinity of Roscoff Brittany, France. Gametophytes derived from meiospores were used for intra‐ and interspecific crosses. Intraspecific gamete combinations gave viable zygotes, which developed into fertile sporophytes. Interspecific crosses were unsuccessful. Gamete fusions did not occur between female gametes of E. fasciculatus and male gametes of E. siliculosus. Hybrid zygotes were formed in the reciprocal combination but died soon after germination. We conclude that the two species of Ectocarpus at Roscoff represent distinct taxonomic entities, which are separated by pre‐ and postzygotic compatibility barriers. These biological findings are confirmed by the differential occurrence of the chemotaxonomic marker betaine‐lipid diacetylglycerylhydroxymethyltrimethyl‐β‐alanine, which is present in our cultures of E. fasciculatus but absent in E. siliculosus.