Highlights of recent progress in plant lipid research.

Raw fossil material reserves are not inexhaustible and as prices continue to raise it is necessary to find new sources of alternative and renewable energy. Oils from oleaginous field crops (sunflower and rape) with properties close to those of fossil fuel could constitute an alternative source of energy for the production of raw materials. This is the context in which the 18th International Symposium on Plant lipids (ISPL) was held in Bordeaux from 20th to 25th July 2008 at "La Cité Mondiale". The 18th ISPL gathered 270 researchers from 33 countries. Sixty nine oral communications and 136 posters were presented during the 12 sessions of the Symposium. The sessions have covered all the different aspects of the Plant Lipid field including: Surface lipids: suberin, cutin and waxes, Fatty acids, Glycerolipids, Plant lipids as renewable sources of energy, Seed oils and bioengineering of metabolic pathways, Lipid catabolism, Models for lipid studies: lower plants, micro-organisms and others, Modifications of proteins by lipids, Sphingolipids, sterols and isoprenoids, Lipid signaling and plant stress responses, Lipid trafficking and membrane dynamics, New methods and technologies: functional lipidomics, fluxome, modelling. During the ISPL 2008 Bordeaux, important and new information was reported in the different fields. A selection of these results is presented here.

Acyl-CoA elongase expression during seed development in Brassica napus.

The Bn-FAE1.1 and Bn-FAE1.2 genes encode the 3-ketoacyl-CoA synthase, a component of the elongation complex responsible for the synthesis of very long chain monounsaturated fatty acids (VLCMFA) in the seeds of Brassica napus. Bn-FAE1 gene expression was studied during seed development using two different cultivars: Gaspard, a high erucic acid rapeseed (HEAR), and ISLR4, a low erucic acid rapeseed (LEAR). The mRNA developmental profiles were similar for the two cultivars, the maximal expression levels being measured at 8 weeks after pollination (WAP) in HEAR and at 9 WAP in LEAR. Differential expression of Bn-FAE1.1 and Bn-FAE1.2 genes was also studied. In each cultivar the same expression profile was observed for both genes, but Bn-FAE1.2 was expressed at a lower level than Bn-FAE1.1. Secondly, VLCMFA synthesis was measured using particulate fractions prepared from maturating seeds harvested weekly after pollination. The oleoyl-CoA and ATP-dependent elongase activities increased from the 4th WAP in HEAR and reached the maximal level at 8 WAP, whereas both activities were absent in LEAR. In contrast, the 3-hydroxy dehydratase, a subunit of the elongase complex, had a similar activity in both cultivars and reached a maximum from 7 to 9 WAP. Finally, antibodies against the 3-ketoacyl-CoA synthase revealed a protein of 57 kDa present only in HEAR. Our results show: (i) that both genes are transcribed in HEAR and LEAR cultivars; (ii) that they are coordinately regulated; (iii) that Bn-FAE1.1 is quantitatively the major isoform expressed in seeds; (iv) that the Bn-FAE1 gene encodes a protein of 57 kDa responsible for the 3-ketoacyl-CoA synthase activity.

Mutations in the fatty acid elongation 1 gene are associated with a loss of beta-ketoacyl-CoA synthase activity in low erucic acid rapeseed.

Low erucic acid rapeseed (LEAR) is characterised by a near absence of very long chain fatty acids in the seed oil which has been correlated with a lack of acyl-CoA elongation activity. Here we show that the absence of acyl-CoA and ATP-dependent elongation activities in microsomes isolated from LEAR embryos is associated with an absence of beta-ketoacyl-CoA synthase activity encoded by the Bn-fatty acid elongation 1 (FAE1) genes. Size exclusion chromatography of solubilised microsomes revealed the presence of a high molecular mass acyl-CoA elongase complex in high erucic acid rapeseed which was absent in microsomes isolated from LEAR seeds. Although transcripts for the Bn-FAE1 genes were detected in LEAR embryos, immunoblots using antisera raised against the beta-ketoacyl-CoA synthase indicated an absence of this protein. Comparison of the deduced amino acid sequences of immature embryo cDNAs reveals that LEAR alleles of Bn-FAE1 encode variant beta-ketoacyl-CoA synthase proteins.

Purification of the acyl-CoA elongase complex from developing rapeseed and characterization of the 3-ketoacyl-CoA synthase and the 3-hydroxyacyl-CoA dehydratase.

Oleoyl-CoA elongase catalyzes four successive reactions: condensation of malonyl-CoA to oleoyl-CoA, reduction, dehydration, and another reduction. Evidence supporting this mechanism and the multienzymatic nature of the elongation complex are reported. A particulate membrane fraction from rapeseed is able to elongate intermediates (R,S) 3-hydroxy-20:0-CoA and (E) 2,3-20:1-CoA to very long chain fatty acids in the presence of malonyl-CoA. Studies of the 3-ketoacyl-CoA synthase activities showed that maximal activity could be measured by using 15 to 30 microM 18:1-CoA and 30 microM malonyl-CoA, and that 18:0-CoA and 18:1-CoA were the best substrates. Comparison of the condensation and the overall elongation activities indicated that condensation is the rate-limiting step of the elongation process. The 3-hydroxyacyl-CoA dehydratase activity was maximal in the presence of 75 microM Triton X-100 and 25 microg of proteins. Finally, the acyl-CoA elongase complex was solubilized and purified. During the purification process, the 3-hydroxyacyl-CoA dehydratase copurified with the elongase complex, strongly suggesting that this enzyme belongs to the elongase complex. The apparent molecular mass of 700 kDa determined for the elongase complex, and the fact that four different polypeptide bands were detected after sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the purified fraction, further suggest that the acyl-CoA elongase is a multienzymatic complex.

Enzymic activities and gene expression of enzymes of the acyl-CoA elongase during rapeseed development.

Enzymic activities and gene expression of oleoyl-CoA elongase were studied during seed development using two different rapeseed cultivars, high-erucic-acid rapeseed (HEAR) and low-erucic-acid rapeseed (LEAR). The overall elongase activities were maximal in HEAR between the fourth and eighth weeks after pollination (WAP) and absent in LEAR. The 3-ketoacyl-CoA synthase (condensing enzyme, CE) mRNA levels and the developmental profiles in the two cultivars were different since maximal expression levels were detected in HEAR and LEAR at WAP 4 and WAP 6, respectively. Anti-CE antibodies revealed two proteins of 60 and 67 kDa in both cultivars and an additional reacting protein of 57 kDa in HEAR.

Biochemical and molecular characterization of corn (Zea mays L.) root elongases.

Root surfaces are protected against the soil environment by the deposition of lignin and suberin. In order to obtain more insight into the regulation of root suberin biosynthesis, elongases from primary roots of corn (Zea mays L.) seedlings were characterized. Elongase activities (acyl-CoA and ATP-dependent) were located in the microsomal fraction of the root cells. C(20), C(22) and C(24) fatty acids were detected as primary products of elongases. Preferred substrates of the acyl-CoA elongases were C(18:0)-CoA and C(20:0)-CoA. Applying a molecular approach, using PCR and degenerate primers derived from the sequences of known leaf and seed 3-ketoacyl-CoA synthases (KCSs), catalysing the first step of very-long-chain fatty acid synthesis, the cDNA of a putative root KCS was obtained showing high homology to known leaf and seed KCSs at the DNA and amino acid levels. Thus, our approach provides the first direct evidence for the presence and the activity of root elongases in Z. mays. Ongoing research is focusing on the molecular analysis and the regulation of KCS expression in roots in reaction to different environmental stimuli.

Mutagenesis of a plastidial lysophosphatidic acid acyltransferase.

A combination of site-directed and random mutagenesis generated sequence variants of a plastidial lysophosphatidic acid acyltransferase. Alanine substitutions of residues present within two conserved motifs including the putative catalytic histidine resulted in a loss of acyltransferase activity assessed as complementation competence. Substitutions at five sites within the central core resulted in reduced or loss of activity. Truncation mutants reveal that sequences in the C-terminal moiety are essential for function.

Evidence that oleoyl-CoA and ATP-dependent elongations coexist in rapeseed (Brassica napus L.).

The elongation of different substrates was studied using several subcellular fractions from Brassica napus rapeseed. In the presence of malonyl-CoA, NADH and NADPH, very-long-chain fatty acid (VLCFA) synthesis was observed from either oleoyl-CoA (acyl-CoA elongation) or endogenous primers (ATP-dependent elongation). No activity was detected using oleic acid as precursor. Acyl-CoA and ATP-dependent elongation activities were mainly associated with the 15 000 g/25 min membrane fraction. Reverse-phase TLC analysis showed that the proportions of fatty acids synthesized by these activities were different. Acyl-CoA elongation increased up to 60 microM oleoyl-CoA, and ATP-dependent elongation was maximum at 1 mM ATP. Both activities increased with malonyl-CoA concentration (up to 200 microM). Under all conditions tested, acyl-CoA elongation was higher than ATP-dependent elongation, and, in the presence of both ATP and oleoyl-CoA, the elongation activity was always lower. ATP strongly inhibited acyl-CoA elongation, whereas ATP-dependent elongation was slightly stimulated by low oleoyl-CoA concentrations (up to 15 microM) and decreased in the presence of higher concentrations. CoA (up to 150 microM) had no effect on the ATP-dependent elongation, whereas it inhibited the acyl-CoA elongation. These marked differences strongly support the presence in maturing rapeseed of two different elongating activities differently modulated by ATP and oleoyl-CoA.

Study of the 3-hydroxy eicosanoyl-coenzyme A dehydratase and (E)-2,3 enoyl-coenzyme A reductase involved in acyl-coenzyme A elongation in etiolated leek seedlings

(R,S)-[1-14C]3-Hydroxy eicosanoyl-coenzyme A (CoA) has been chemically synthesized to study the 3-hydroxy acyl-CoA dehydratase involved in the acyl-CoA elongase of etiolated leek (Allium porrum L. ) seedling microsomes. 3-Hydroxy eicosanoyl-CoA (3-OH C20:0-CoA) dehydration led to the formation of (E)-2,3 eicosanoyl-CoA, which has been characterized. Our kinetic studies have determined the optimal conditions of the dehydration and also resolved the stereospecificity requirement of the dehydratase for (R)-3-OH C20:0-CoA. Isotopic dilution experiments showed that 3-hydroxy acyl-CoA dehydratase had a marked preference for (R)-3-OH C20:0-CoA. Moreover, the very-long-chain synthesis using (R)-3-OH C20:0-CoA isomer and [2-14C]malonyl-CoA was higher than that using the (S) isomer, whatever the malonyl-CoA and the 3-OH C20:0-CoA concentrations. We have also used [1-14C]3-OH C20:0-CoA to investigate the reductant requirement of the enoyl-CoA reductase of the acyl-CoA elongase complex. In the presence of NADPH, [1-14C]3-OH C20:0-CoA conversion was stimulated. Aside from the product of dehydration, i.e. (E)-2,3 eicosanoyl-CoA, we detected eicosanoyl-CoA resulting from the reduction of (E)-2,3 eicosanoyl-CoA. When we replaced NADPH with NADH, the eicosanoyl-CoA was 8- to 10-fold less abundant. Finally, in the presence of malonyl-CoA and NADPH or NADH, [1-14C]3-OH C20:0-CoA led to the synthesis of very-long-chain fatty acids. This synthesis was measured using [1-14C]3-OH C20:0-CoA and malonyl-CoA or (E)-2,3 eicosanoyl-CoA and [2-14C]malonyl-CoA. In both conditions and in the presence of NADPH, the acyl-CoA elongation activity was about 60 nmol mg-1 h-1, which is the highest ever reported for a plant system.

Arginyl residues are involved in acyl-CoA binding to the elongase from etiolated leek seedlings.

The C18:0-CoA elongase from etiolated leek seedling microsomes was inactivated by treatment with phenylglyoxal, a reagent which specifically modifies arginyl residues. In the presence of 20 mM phenylglyoxal, 95% of the C18:0-CoA elongation was inhibited. The condensation and dehydration reactions of the overall elongation were totally inhibited, whereas enoyl-CoA reductase activity was diminished by 75%, but the nature of the final elongation product was unchanged. Phenylglyoxal did not modify the C18:0-CoA partition between membrane and aqueous compartments; moreover, [1-14C]phenylglyoxal labeling experiments showed a covalent binding of the inhibitor to membrane proteins. The ability of several substrates to prevent the inactivation by phenylglyoxal was investigated. NADH and NADPH had no effect. CoA led to a 75% protection, and the incorporation of [14C]phenylglyoxal was strongly affected by 10 mM CoA. The acyl chain length of the acyl-CoAs played also a crucial role in preventing the binding of phenylglyoxal. The maximal prevention of phenylglyoxal inhibition was obtained with C18:0-CoA. This suggests that arginyl residues could be present in the vicinity of the acyl-CoA binding site of the subunits of C18:0-CoA elongase.

Effect of cerulenin on the synthesis of very-long-chain fatty acids in microsomes from leek seedlings.

Cerulenin inhibits the elongation of stearoyl-CoA and eicosanoyl-CoA by microsomes from leek seedlings. The inhibition depends on the cerulenin concentration and affects the biosynthesis of docosanoic and tetracosanoic acids only slightly more than that of eicosanoic acid. A 30-min preincubation of the microsomes with cerulenin allows a quantitative inhibition of the elongation at 50 microM cerulenin (50% inhibition at 15 microM cerulenin). A kinetic study of the elongating activity in the presence or in the absence of the inhibitor suggests that the inhibition is non-competitive. Analysis of the products of the reaction suggests that 3-ketoacyl-CoA synthase is the target of cerulenin. A study of the partial reactions demonstrates that the inhibition affects almost exclusively the condensation step.

Occurrence and characterization of a dehydratase enzyme in the leek icosanoyl-CoA synthase complex.

The presence of a beta-hydroxyacyl-CoA dehydratase involved in the icosanoyl-CoA synthase (EC 2.3.1.119) complex of leek epidermis has been demonstrated using antibodies raised against the purified beta-hydroxyacyl-CoA dehydratase from rat liver. In a first step the leek icosanoyl-CoA synthase activity was measured in the presence of different amounts of this antibody, the results obtained showed a 75% inhibition of the activity using a 8:1 IgG/microsomal protein ratio, whereas only a weak diminution of the activity occurred using pre-immune IgG. The analysis of the reaction products after incubation in the presence of increasing IgG amounts showed a decrease of the fatty acids (the final product) and an accumulation of beta-hydroxy fatty acids using immune IgG, whereas no change occurred in the presence of pre-immune IgG. Moreover, the beta-hydroxyacyl-CoA dehydratase activity was strongly inhibited, whereas in the same conditions, the beta-ketoacyl-CoA reductase and the (trans-2-3) enoyl-CoA reductase activities were not affected. The protein fractions that eluted from the DEAE and Ultrogel columns containing the leek icosanoyl-CoA synthase activity were able to specifically bind the anti beta-hydroxyacyl-CoA dehydratase from rat liver. The cross-reactivity was demonstrated. In immunoblotting experiments using the same antiserum after SDS-PAGE of the purified leek icosanoyl-CoA synthase, only one of the four protein bands constituting the leek icosanoyl-CoA synthase was detected. This protein, having an apparent molecular mass of 65 kDa, could be the dehydratase component of the elongation complex.

Evaluation of the amount of acyl-CoA elongases in leek (Allium porrum L) leaves.

Polyclonal antibodies have been raised against the acyl-CoA elongase purified from leek epidermal cells. The antibodies recognize the fractions containing the elongating activity after DEAE or Ultrogel chromatography and their response with the other fractions is very low. The immune complex is immunoprecipitable with Protein A-Sepharose. 1% of the solubilized proteins from leek epidermis microsomes are immunoprecipitated. The immunoprecipitate contains an elongating activity which is 86 +/- 20-times that of the unbound fraction.

Solubilization and partial purification of constituents of acyl-CoA elongase from Lunaria annua.

All the constituent enzymes of acyl-CoA elongase, i.e., beta-ketoacyl-CoA synthase, beta-ketoacyl-CoA reductase, beta-hydroxyacyl-CoA dehydrase and trans-2-enoyl-CoA reductase, have been solubilized from a 15,000 x g particulate fraction from developing seeds of honesty (Lunaria annua) using Triton X-100. All these activities were retained upon subsequent precipitation of the solubilized protein with polyethylene glycol and resuspension of the precipitate followed by ion exchange chromatography of the resulting protein on DEAE-cellulose. A 4.2-fold enrichment of the acyl-CoA elongase was thus obtained. Further chromatography of the DEAE fraction containing all the constituents of acyl-CoA elongase on Ultrogel yielded a major protein fraction exhibiting the activities of beta-ketoacyl-CoA synthase and beta-ketoacyl-CoA reductase only. Almost 30-fold purification of the beta-ketoacyl-CoA synthase was thus achieved. The beta-ketoacyl-CoA synthase was inhibited only at high concentrations of cerulenin, but at very low concentrations of iodoacetamide. Inhibition could be reduced by preincubation with thioesters, indicating that an enzyme thioester intermediate is involved in the condensation reaction of the acyl-CoA elongation.

Partial purification of the acyl-CoA elongase of Allium porrum leaves.

Acyl-CoA elongase has been partially purified from leek (Allium porrum L.) epidermal cells. The microsomal elongase is first solubilized by Triton X-100. The solubilized proteins are then submitted to anion exchange chromatography on DEAE-cellulose and, finally, to gel filtration on Ultrogel 34 AcA. The purification of the elongase activity is accompanied by the enrichment in three major protein bands of 59, 61, and 65 kDa. The partially purified elongase is highly delipidated (about 10 mol lipid/mol of 60- to 65-kDa protein) and phosphatidylserine and phosphatidylethanolamine account respectively for 60 and 40% of the remaining phospholipids. The partially purified elongase retains some activities associated with fatty acid biosynthesis. The overall activity is strongly stimulated by the addition of exogenous lipids. In the presence of a mixture of PS, PE, and PC the C18-CoA elongase activity is increased more than sixfold. The Km value of stearoyl-CoA, in the presence of lipid vesicles, was determined to be 1.7 microM.

Localization of the synthesis of very-long-chain fatty acid in mitochondria from Saccharomyces cerevisiae.

The localization of the mitochondrial elongation activities ('elongases') from Saccharomyces cerevisiae has been investigated. It was shown, using carboxyatractyloside in the incubation mixture, that synthesis of very-long-chain fatty acids probably occurred outside the matrix and, by fractionation experiments, that elongases are membrane-bound enzymes. The solubilization of the outer membrane by digitonin showed that three elongating activities are correlated with a marker of the outer membrane and not with an inner membrane marker. A further partial purification of the outer membrane showed that elongases are present in the outer membrane of mitochondria.

Fatty acid synthesis in mitochondria from Saccharomyces cerevisiae.

The ability of purified mitochondria isolated from S. cerevisiae to synthesize fatty acids and especially very long chain fatty acids (VLCFA) has been investigated. The VLCFA synthesis requires malonyl-CoA as the C2 unit donor and NADPH as the reducing agent. Moreover the yeast mitochondrial elongase is able to accept either exogenous long chain fatty acyl-CoAs as substrates or elongate endogenous substrates. In the latter case, ATP is required for full activity. Besides this important VLCFA formation, the mitochondria from S. cerevisiae were also able to synthesize C16 and C18.

Nature of the reaction product of [1-14C]stearoyl-CoA elongation by etiolated leek seeding microsomes.

The elongation of [1-14C]stearoyl-CoA by microsomes from etiolated leek seedlings, in the presence of malonyl-CoA and NADPH, has been studied at different substrate and enzyme concentrations. The HPTLC analysis of the whole reaction mixture, followed by the analysis of the label in the fatty acid methyl esters of long-chain acyl-CoAs, phosphatidylcholine (PC), and neutral lipids, showed that the acyl-CoA fraction contained most of the labeled very-long-chain fatty acids. The very-long-chain fatty acids were rapidly formed and released from the elongase(s) as acyl-CoAs. The label of long-chain acyl-CoAs increased for 20 min and then decreased, whereas it increased in PC. Labeled very-long-chain fatty acids appeared in the neutral lipid + free fatty acid fraction after a 20-min lag.