Biochemical characterization of acyl-CoA:diacylglycerol acyltransferase2 from the diatom Phaeodactylum tricornutum and its potential effect on LC-PUFAs biosynthesis in planta.

BACKGROUND: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), belonging to ω-3 long-chain polyunsaturated fatty acids (ω3-LC-PUFAs), are essential components of human diet. They are mainly supplemented by marine fish consumption, although their native producers are oleaginous microalgae. Currently, increasing demand for fish oils is insufficient to meet the entire global needs, which puts pressure on searching for the alternative solutions. One possibility may be metabolic engineering of plants with an introduced enzymatic pathway producing ω3-LC-PUFAs. RESULT: In this study we focused on the acyl-CoA:diacylglycerol acyltransferase2b (PtDGAT2b) from the diatom Phaeodactylum tricornutum, an enzyme responsible for triacylglycerol (TAG) biosynthesis via acyl-CoA-dependent pathway. Gene encoding PtDGAT2b, incorporated into TAG-deficient yeast strain H1246, was used to confirm its activity and conduct biochemical characterization. PtDGAT2b exhibited a broad acyl-CoA preference with both di-16:0-DAG and di-18:1-DAG, whereas di-18:1-DAG was favored. The highest preference for acyl donors was observed for 16:1-, 10:0- and 12:0-CoA. PtDGAT2b also very efficiently utilized CoA-conjugated ω-3 LC-PUFAs (stearidonic acid, eicosatetraenoic acid and EPA). Additionally, verification of the potential role of PtDGAT2b in planta, through its transient expression in tobacco leaves, indicated increased TAG production with its relative amount increasing to 8%. Its co-expression with the gene combinations aimed at EPA biosynthesis led to, beside elevated TAG accumulation, efficient accumulation of EPA which constituted even 25.1% of synthesized non-native fatty acids (9.2% of all fatty acids in TAG pool). CONCLUSIONS: This set of experiments provides a comprehensive biochemical characterization of DGAT enzyme from marine microalgae. Additionally, this study elucidates that PtDGAT2b can be used successfully in metabolic engineering of plants designed to obtain a boosted TAG level, enriched not only in ω-3 LC-PUFAs but also in medium-chain and ω-7 fatty acids.

Phospholipid:diacylglycerol acyltransferase1-overexpression stimulates lipid turnover, oil production and fitness in cold-grown plants.

BACKGROUND: Extensive population growth and climate change accelerate the search for alternative ways of plant-based biomass, biofuel and feed production. Here, we focus on hitherto unknow, new promising cold-stimulated function of phospholipid:diacylglycerol acyltransferase1 (PDAT1) - an enzyme catalyzing the last step of triacylglycerol (TAG) biosynthesis. RESULT: Overexpression of AtPDAT1 boosted seed yield by 160% in Arabidopsis plants exposed to long-term cold compared to standard conditions. Such seeds increased both their weight and acyl-lipids content. This work also elucidates PDAT1's role in leaves, which was previously unclear. Aerial parts of AtPDAT1-overexpressing plants were characterized by accelerated growth at early and vegetative stages of development and by biomass weighing three times more than control. Overexpression of PDAT1 increased the expression of SUGAR-DEPENDENT1 (SDP1) TAG lipase and enhanced lipid remodeling, driving lipid turnover and influencing biomass increment. This effect was especially pronounced in cold conditions, where the elevated synergistic expression of PDAT1 and SDP1 resulted in double biomass increase compared to standard conditions. Elevated phospholipid remodeling also enhanced autophagy flux in AtPDAT1-overexpresing lines subjected to cold, despite the overall diminished autophagy intensity in cold conditions. CONCLUSIONS: Our data suggest that PDAT1 promotes greater vitality in cold-exposed plants, stimulates their longevity and boosts oilseed oil production at low temperature.

The utilization of the acyl-CoA and the involvement PDAT and DGAT in the biosynthesis of erucic acid-rich triacylglycerols in Crambe seed oil.

The triacylglycerol of Crambe abyssinica seeds consist of 95% very long chain (>18 carbon) fatty acids (86% erucic acid; 22:1∆13) in the sn-1 and sn-3 positions. This would suggest that C. abyssinica triacylglycerols are not formed by the action of the phospholipid:diacylglycerol acyltransferase (PDAT), but are rather the results of acyl-CoA:diacylglycerol acyltransferase (DGAT) activity. However, measurements of PDAT and DGAT activities in microsomal membranes showed that C. abyssinica has significant PDAT activity, corresponding to about 10% of the DGAT activity during periods of rapid seed oil accumulation. The specific activity of DGAT for erucoyl-CoA had doubled at 19 days after flowering compared to earlier developmental stages, and was, at that stage, the preferred acyl donor, whereas the activities for 16:0-CoA and 18:1-CoA remained constant. This indicates that an expression of an isoform of DGAT with high specificity for erucoyl-CoA is induced at the onset of rapid erucic acid and oil accumulation in the C. abyssinica seeds. Analysis of the composition of the acyl-CoA pool during different stages of seed development showed that the percentage of erucoyl groups in acyl-CoA was much higher than in complex lipids at all stages of seed development except in the desiccation phase. These results are in accordance with published results showing that the rate limiting step in erucic acid accumulation in C. abyssinica oil is the utilization of erucoyl-CoA by the acyltransferases in the glycerol-3-phosphate pathway.

Fusion of oil bodies in endosperm of oat grains.

Few microscopical studies have been made on lipid storage in oat grains, with variable results as to the extent of lipid accumulation in the starchy endosperm. Grains of medium- and high-lipid oat (Avena sativa L.) were studied at two developmental stages and at maturity, by light microscopy using different staining methods, and by scanning and transmission electron microscopy. Discrete oil bodies occurred in the aleurone layer, scutellum and embryo. In contrast, oil bodies in the starchy endosperm often had diffuse boundaries and fused with each other and with protein vacuoles during grain development, forming a continuous oil matrix between the protein and starch components. The different microscopical methods were confirmative to each other regarding the coalescence of oil bodies, a phenomenon probably correlated with the reduced amount of oil-body associated proteins in the endosperm. This was supported experimentally by SDS-PAGE separation of oil-body proteins and immunoblotting and immunolocalization with antibodies against a 16 kD oil-body protein. Much more oil-body proteins per amount of oil occurred in the embryo and scutellum than in the endosperm. Immunolocalization of 14 and 16 kD oil-body associated proteins on sectioned grains resulted in more heavy labeling of the embryo, scutellum and aleurone layer than the rest of the endosperm. Observations on the appearance of oil bodies at an early stage of development pertain to the prevailing hypotheses of oil-body biogenesis.

Effects of different cultivation temperatures on plasma membrane ATPase activity and lipid composition of sugar beet roots.

Sugar beet seedlings (Beta vulgaris L. cv. Monohill) were cultivated for 3 weeks at different root and shoot temperatures and the plasma membranes (PM) from roots were purified by aqueous two-phase partitioning and analyzed for lipid composition and ATPase activities. Lipid analyses, undertaken immediately after PM purification from the roots, showed that a low root zone temperature (10 degrees C) decreased the ratio between the major lipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). A low temperature in the root environment increased the mol% of PE and decreased the mol% of phosphatidic acid (PA), independent on the shoot growth temperature. A low temperature also decreased the mol% of linoleic acid (18:2) and increased mol% of linolenic acid (18:3) in the analyzed lipid classes, especially in PC and PE. The ratio between acyl chain lipids and protein generally increased in PM from roots grown at 10 degrees C, compared with higher temperature. The changes in lipid composition correlated with changes in ATPase activities, detected as hydrolyses of MgATP. The kinetic parameters, K(m) and V of the PM H(+)ATPase in roots increased at a low cultivation temperature, independent on shoot temperature. Moreover, Arrhenius analyses showed that the transition temperature was independent of both root or shoot growth temperature at 10-24 degrees C, whereas the activation energy of the ATPase was dependent on the growth temperature of the root, and independent on shoot temperature. Thus, acclimation processes can take place in roots, irrespective of the shoot temperature.

Expression in yeast of a novel phospholipase A1 cDNA from Arabidopsis thaliana.

During a search for cDNAs encoding plant sterol acyltransferases, we isolated four full-length cDNAs from Arabidopsis thaliana that encode proteins with substantial identity with animal lecithin : cholesterol acyltransferases (LCATs). The expression of one of these cDNAs, AtLCAT3 (At3g03310), in various yeast strains resulted in the doubling of the triacylglycerol content. Furthermore, a complete lipid analysis of the transformed wild-type yeast showed that its phospholipid content was lower than that of the control (void plasmid-transformed) yeast whereas lysophospholipids and free fatty acids increased. When microsomes from the AtLCAT3-transformed yeast were incubated with di-[1-14C]oleyl phosphatidylcholine, both the lysophospholipid and free fatty acid fractions were highly and similarly labelled, whereas the same incubation with microsomes from the control yeast produced a negligible labelling of these fractions. Moreover when microsomes from AtLCAT3-transformed yeast were incubated with either sn-1- or sn-2-[1-14C]acyl phosphatidylcholine, the distribution of the labelling between the free fatty acid and the lysophosphatidylcholine fractions strongly suggested a phospholipase A1 activity for AtLCAT3. The sn-1 specificity of this phospholipase was confirmed by gas chromatography analysis of the hydrolysis of 1-myristoyl, 2-oleyl phosphatidylcholine. Phosphatidylethanolamine and phosphatidic acid were shown to be also hydrolysed by AtLCAT3, although less efficiently than phosphatidylcholine. Lysophospatidylcholine was a weak substrate whereas tripalmitoylglycerol and cholesteryl oleate were not hydrolysed at all. This novel A. thaliana phospholipase A1 shows optimal activity at pH 6-6.5 and 60-65 degrees C and appears to be unaffected by Ca2+. Its sequence is unrelated to all other known phospholipases. Further studies are in progress to elucidate its physiological role.