Storage Lipid Accumulation Research Articles

Biotechnological conversions of bio‐diesel‐derived crude glycerol by Yarrowia lipolytica strains

AbstractIn the present report, crude glycerol, waste discharged from bio‐diesel production, was used as carbon substrate for three natural Yarrowia lipolytica strains (LFMB 19, LFMB 20 and ACA‐YC 5033) during growth in nitrogen‐limited submerged shake‐flask experiments. In media with initial glycerol concentration of 30 g/L, all strains presented satisfactory microbial growth and complete glycerol uptake. Although culture conditions favored the secretion of citric acid (and potentially the accumulation of storage lipid), for the strains LFMB 19 and LFMB 20, polyol mannitol was the principal metabolic product synthesized (maximum quantity 6.0 g/L, yield 0.20–0.26 g per g of glycerol consumed). The above strains produced small quantities of lipids and citric acid. In contrast, Y. lipolytica ACA‐YC 5033 produced simultaneously higher quantities of lipid and citric acid and was further grown on crude glycerol in nitrogen‐limited experiments, with constant nitrogen and increasing glycerol concentrations (70–120 g/L). Citric acid and lipid concentrations increased with increment of glycerol; maximum total citric acid 50.1 g/L was produced (yield 0.44 g per g of glycerol) while simultaneously 2.0 g/L of fat were accumulated inside the cells (0.31 g of lipid per g of dry weight). Cellular lipids were mainly composed of neutral fraction, the concentration of which substantially increased with time. Moreover, in any case, the phospholipid fraction was more unsaturated compared with total and neutral lipids, while at the early growth step, microbial lipid was more rich in saturated fatty acids (e.g. C16:0 and C18:0) compared with the stationary phase.

A Novel In Vitro Multiple-Stress Dormancy Model for Mycobacterium tuberculosis Generates a Lipid-Loaded, Drug-Tolerant, Dormant Pathogen

Background Mycobacterium tuberculosis (Mtb) becomes dormant and phenotypically drug resistant when it encounters multiple stresses within the host. Inability of currently available drugs to kill latent Mtb is a major impediment to curing and possibly eradicating tuberculosis (TB). Most in vitro dormancy models, using single stress factors, fail to generate a truly dormant Mtb population. An in vitro model that generates truly dormant Mtb cells is needed to elucidate the metabolic requirements that allow Mtb to successfully go through dormancy, identify new drug targets, and to screen drug candidates to discover novel drugs that can kill dormant pathogen.Methodology/Principal FindingsWe developed a novel in vitro multiple-stress dormancy model for Mtb by applying combined stresses of low oxygen (5%), high CO2 (10%), low nutrient (10% Dubos medium) and acidic pH (5.0), conditions Mtb is thought to encounter in the host. Under this condition, Mtb stopped replicating, lost acid-fastness, accumulated triacylglycerol (TG) and wax ester (WE), and concomitantly acquired phenotypic antibiotic-resistance. Putative neutral lipid biosynthetic genes were up-regulated. These genes may serve as potential targets for new antilatency drugs. The triacylglycerol synthase1 (tgs1) deletion mutant, with impaired ability to accumulate TG, exhibited a lesser degree of antibiotic tolerance and complementation restored antibiotic tolerance. Transcriptome analysis with microarray revealed the achievement of dormant state showing repression of energy generation, transcription and translation machineries and induction of stress-responsive genes. We adapted this model for drug screening using the Alamar Blue dye to quantify the antibiotic tolerant dormant cells.Conclusions/SignificanceThe new in vitro multiple stress dormancy model efficiently generates Mtb cells meeting all criteria of dormancy, and this method is adaptable to high-throughput screening for drugs that can kill dormant Mtb. A critical link between storage-lipid accumulation and development of phenotypic drug-resistance in Mtb was established. Storage lipid biosynthetic genes may be appropriate targets for novel drugs that can kill latent Mtb.

Dietary supplementation of salmon roe phospholipid enhances the growth and survival of Pacific bluefin tuna Thunnus orientalis larvae and juveniles

Previous studies have shown that feeding enriched Artemia induces growth failure in Pacific bluefin tuna (PBT) Thunnus orientalis larvae. This growth failure cannot be improved even if the docosahexaenoic acid (DHA) content in enriched Artemia is elevated to the same level as that in yolk-sac larvae, an ideal live feed for PBT larviculture. This might be caused by the differences in the DHA in the live feeds; i.e., yolk-sac larvae of marine fish have a high level of DHA in the larval phospholipids (PLs) but enriched Artemia store DHA in their neutral lipids (NLs). To test this hypothesis two experiments were conducted to evaluate the effect of dietary PL rich in DHA on growth and survival of PBT larvae (Experiment 1) and juveniles (Experiment 2). Three isoproteic and isolipidic artificial test diets (Diets 1, 2 and 3) and two reference live feeds (Diets 4 and 5) were prepared. Diets 1 and 3 were supplemented with NL and PL fractions of commercial salmon Oncorhynchus gorbuscha roe lipid (SRL) as the lipid source, respectively, whereas Diet 2 was supplemented with a mixture of both lipid fractions. Diets 4 and 5 were enriched Artemia and yolk-sac larvae of Japanese parrot fish Oplegnathus fasciatus, respectively. Diets 1, 3, 4 and 5 were given to PBT larvae for 10 days, whereas Diets 1, 2 and 3 were fed to PBT juveniles for 10 days. In Experiment 1, the growth and survival of PBT larvae fed Diet 3 (high PL fraction) were significantly improved when compared with larvae fed Diets 1 and 4, although the best growth and survival were obtained in larvae fed Diet 5. Similar results were obtained in Experiment 2; i.e., growth and survival were significantly improved in PBT juveniles fed Diets 2 and 3. In both experiments, fish fed Diets 2, 3 and/or 5 had higher total lipid contents, TAG levels, n-3 HUFA and/or DHA levels in NL fraction of the body when compared with fish fed Diets 1 or 4, while such a difference was not observed in the PL fraction. In juveniles over 90% of deaths were caused by collisions with the tank walls and the significant difference in mortality between treatments implies diet also affects behavior. SRL PL is concluded to enhance the growth and survival of PBT larvae and juveniles along with the accumulation of storage lipid and DHA in the body.

Biodegradation of phytane (2,6,10,14-tetramethylhexadecane) and accumulation of related isoprenoid wax esters byMycobacterium ratisbonensestrain SD4 under nitrogen-starved conditions

The accumulation of storage lipids during the biodegradation of 2,6,10,14-tetramethylhexadecane (phytane) by Mycobacterium ratisbonense strain SD4 grown under nitrogen-starved conditions was investigated. Detailed chemical analysis of intracellular metabolites revealed the existence of (at least) three different pathways for the catabolism of phytane, and the accumulation of significant proportions (39% of the total lipids) of several isoprenoid wax esters formed by condensation of oxidation products of the hydrocarbon. In contrast, triacylglycerols but no wax esters were accumulated by strain SD4 grown on hexadecane, the unbranched homologue of phytane.

The SebHLH transcription factor mediates trans-activation of the SeFAD2 gene promoter through binding to E- and G-box elements

Microsomal oleic acid desaturase (FAD2) catalyzes the first extra-plastidial desaturation in plants, converting oleic acid to linoleic acid, which is a major constituent in all cellular membranes as well as in seed storage oils. Seed-specific FAD2 (SeFAD2) produced 40% of linoleic acids in the total fatty acids of sesame (Sesamum indicum) seeds. The expression of SeFAD2 transcripts was spatially and temporally controlled during seed development. To investigate the regulatory mechanism controlling seed-specific SeFAD2 expression, we isolated a well-matched sequence homologous to the basic region/helix-loop-helix proteins by yeast one-hybrid screening and named it SebHLH. SebHLH transcripts were expressed in developing seeds and roots of sesame. SebHLH:GFP fusion protein localized in the nucleus. Recombinant SebHLH protein bound E-box (CANNTG) and G-box (CACGTG) elements in the region from -179 to -53 of the SeFAD2 gene promoter, and the external C and G nucleotides in the E- and G-box motifs were essential for SebHLH protein binding. The SebHLH gene, under the CaMV35S promoter, and the GUS reporter gene driven by E- and G-box motifs were co-expressed in developing sesame seeds and Arabidopsis transgenic leaves. This co-expression demonstrated that SebHLH protein mediates transactivation of the SeFAD2 gene promoter through binding to E- and G-box elements. E- or G-box elements frequently occur in the 5'-flanking region of genes that are involved in triacylglycerol biosynthesis and that exhibit seed-specific expression in Arabidopsis and other plants, suggesting that bHLH transcription factors play a key role in the transcriptional regulation of genes related to storage lipid biosynthesis and accumulation during seed development.

Analysis of Storage Lipid Accumulation in Alcanivorax borkumensis : Evidence for Alternative Triacylglycerol Biosynthesis Routes in Bacteria

Marine hydrocarbonoclastic bacteria, like Alcanivorax borkumensis, play a globally important role in bioremediation of petroleum oil contamination in marine ecosystems. Accumulation of storage lipids, serving as endogenous carbon and energy sources during starvation periods, might be a potential adaptation mechanism for coping with nutrient limitation, which is a frequent stress factor challenging those bacteria in their natural marine habitats. Here we report on the analysis of storage lipid biosynthesis in A. borkumensis strain SK2. Triacylglycerols (TAGs) and wax esters (WEs), but not poly(hydroxyalkanoic acids), are the principal storage lipids present in this and other hydrocarbonoclastic bacterial species. Although so far assumed to be a characteristic restricted to gram-positive actinomycetes, substantial accumulation of TAGs corresponding to a fatty acid content of more than 23% of the cellular dry weight is the first characteristic of large-scale de novo TAG biosynthesis in a gram-negative bacterium. The acyltransferase AtfA1 (ABO_2742) exhibiting wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT) activity plays a key role in both TAG and WE biosynthesis, whereas AtfA2 (ABO_1804) was dispensable for storage lipid formation. However, reduced but still substantial residual TAG levels in atfA1 and atfA2 knockout mutants compellingly indicate the existence of a yet unknown WS/DGAT-independent alternative TAG biosynthesis route. Storage lipids of A. borkumensis were enriched in saturated fatty acids and accumulated as insoluble intracytoplasmic inclusions exhibiting great structural variety. Storage lipid accumulation provided only a slight growth advantage during short-term starvation periods but was not required for maintaining viability and long-term persistence during extended starvation phases.

Effect of essential oil of Citrus cinensis cv new hall – Citrus aurantium (indigenous in Greece) upon growth of Yarrowia lipolytica

The effect of essential oil from Citrus cinensis cv new hall – Citrus aurantium (indigenous in Greece) upon growth of the dimorphic non-conventional yeast Yarrowia lipolytica strain ACA-DC 50109 was studied. The microorganism was aerobically cultivated in batch mode in carbon-limited media. The essential oil was added into the culture medium in different quantities while the control experiment was carried out without addition. The essential oil caused a relatively important decrease of the highest concentration of biomass produced. Additionally, biomass yield on glucose consumed was significantly decreased with the addition of the oil on the cultivation medium. Moreover, the addition of the essential oil considerably increased the lag time of the culture. In all trials, a remarkable drop the pH value of the medium was observed due to the biosynthesis of small amounts of organic acids. Given that one principal component of this membrane is the one of cellular lipids, it was assumed that the extraction and the analysis of cellular lipids could provide information about the microbial behaviour. Total lipids were extracted, methanolyzed and analyzed with the aid of G.L.C. In the control experiment, the culture conditions did not favour accumulation of storage lipid inside the yeast cells and, hence, lipid produced corresponded to 5–9% (wt/wt) in dry cell mass. Similar concentrations of cellular lipids were produced when essential oil was added in various amounts. When essential oils were added, an increase of lower aliphatic chain saturated fatty acids was observed, suggesting an alteration in the membrane function.

Delayed embryo development in the ARABIDOPSIS TREHALOSE‐6‐PHOSPHATE SYNTHASE 1 mutant is associated with altered cell wall structure, decreased cell division and starch accumulation

The tps1 mutant, which is disrupted in the TREHALOSE-6-PHOSPHATE SYNTHASE 1 gene, has been previously characterized as a recessive embryo lethal. tps1 embryos do not develop past late torpedo or early cotyledon stage. We report here that at the ultrastructural, biochemical, and transcriptional levels tps1 exhibits many features typically associated with the maturation phase. The appearance of storage reserve transcripts and organelles follows the same temporal pattern in tps1 and wild-type (WT) embryos in the same silique as does accumulation of storage lipid and protein. The mutant plastids accumulate large starch granules that persist until the end of seed development, in contrast with WT plastids where starch accumulation is transient. The transcriptome of tps1 embryos shows a coordinate downregulation of genes involved in starch and sucrose degradation. Interestingly, genes involved in lipid mobilization and gluconeogenesis are induced in tps1 embryos. The cell walls of tps1 embryos show a remarkable degree of thickening at the ultrastructural level and immunodetection of cell wall components shows that altered deposition of pectins accounts for this altered morphology. Consistent with this at the transcriptome level, genes involved in sugar nucleotide and pectin metabolism are altered in the mutant. The frequency of cell division in tps1 embryos is half that of the wild type at the heart and torpedo stages. These results suggest that TPS1 may play a major role in coordinating cell wall biosynthesis and cell division with cellular metabolism during embryo development.

Storage lipid accumulation and acyltransferase action in developing flaxseed

Investigations of storage lipid synthesis in developing flaxseed (Linum usitatissimum) provide useful information for designing strategies to enhance the oil content and nutritional value of this crop. Lipid content and changes in the FA composition during seed development were examined in two cultivars of flax (AC Emerson and Vimy). The oil content on a dry weight basis increased steadily until about 20 d after flowering (DAF). The proportion of alpha-linolenic acid (alpha-18:3, 18:3cisDelta9,12,15) in TAG increased during seed development in both cultivars while the proportions of linoleic acid (18:2cisDelta9,12) and saturated FA decreased. The developmental and substrate specificity characteristics of microsomal DAG acyltransferase (DGAT, EC 2.3.1.20) and lysophosphatidic acid acyltransferase (LPAAT, EC 2.3.1.51) were examined using cultivar AC Emerson. The maximal acyltransferase specific activities occurred in the range of 8-14 DAF, during rapid lipid accumulation on a per seed basis. Acyl-CoA of EPA (20:5cisDelta5,8,11,14,17) or DHA (22:6 cis4,7,10,13,16,19) were included in the specificity studies. DGAT displayed enhanced specificity for alpha-18:3-CoA, whereas the preferred substrate of [PAAT was 18:2-CoA. Both enzymes could use EPA- or DHA-CoA to varying extents. Developing flax embryos were able to take up and incorporate these nutritional FA into TAG and other intermediates in the TAG-formation pathway. This study suggests that if the appropriate acyl-CoA-dependent desaturation/elongation pathways are introduced and efficiently expressed in flax, this may lead to the conversion of alpha-18:3-CoA into EPA-CoA, thereby providing an activated substrate for TAG formation.

TANMEI/EMB2757 encodes a WD repeat protein required for embryo development in Arabidopsis.

We identified the Arabidopsis (Arabidopsis thaliana) tanmei/emb2757 (tan) mutation that causes defects in both embryo and seedling development. tan mutant embryos share many characteristics with the leafy cotyledon (lec) class of mutants in that they accumulate anthocyanin, are intolerant of desiccation, form trichomes on cotyledons, and have reduced accumulation of storage proteins and lipids. Thus, TAN functions both in the early and late phases of embryo development. Moreover, the TAN and LEC genes interact synergistically, suggesting that they do not act in series in the same genetic pathway but, rather, that they have overlapping roles during embryogenesis. tan mutants die as embryos, but immature mutant seeds can be germinated in culture. However, tan mutant seedlings are defective in shoot and root development, their hypocotyls fail to elongate in the dark, and they die as seedlings. We isolated the TAN gene and showed that the predicted polypeptide has seven WD repeat motifs, suggesting that TAN forms complexes with other proteins. Together, these results suggest that TAN interacts with other proteins to control many aspects of embryo development.

Lipids as trophic markers in Antarctic krill. III. Temporal changes in digestive gland lipid composition of Euphausia superba in controlled conditions

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 296:81-91 (2005) - doi:10.3354/meps296081 Lipids as trophic markers in Antarctic krill. III. Temporal changes in digestive gland lipid composition of Euphausia superba in controlled conditions Frédéric Alonzo1,2,3,5,*, Patti Virtue1, Stephen Nicol2, Peter D. Nichols3,4 1Institute of Antarctic and Southern Ocean Studies, University of Tasmania, Private Bag 77, Hobart, Tasmania 7001, Australia2Australian Antarctic Division, Channel Highway, Kingston, Tasmania 7050, Australia3CSIRO Marine Research, Castray Esplanade, GPO Box 1538, Hobart, Tasmania 7001, Australia4Antarctic Cooperative Research Centre, University of Tasmania, GPO Box 252-77, Hobart, Tasmania 7001, Australia5Present address: Institut de Radioprotection et de Sûreté Nucléaire, Centre de Cadarache, BP 3, 13115 Saint-Paul-Lez-Durance cedex, France *Email: fred.alonzo@wanadoo.fr ABSTRACT: Two phytoplankton diets with markedly different fatty acid (FA) profiles (thraustochytrid and diatom over a range of concentrations) were fed to juvenile Euphausia superba Dana to evaluate the time lapse required for the development of trophic lipid signatures in the digestive gland (DG). Krill were collected after 5, 10 and 20 d of feeding on thraustochytrids. Krill fed on thraustochytrids for 20 d were also collected after a further 5, 10 and 20 d of feeding on diatoms. An accumulation of polar lipid (0.4 mg per DG) was observed after 5 d of feeding on thraustochytrids at every concentration of food. The deposition of triacylglycerol (0.25 mg per DG) required 10 d of feeding and only occurred at the 2 highest food concentrations. Amounts of thraustochytrid-derived FA, 14:0, 16:0 22:6ω3 and 22:5ω6, increased after 10 d of feeding. The final concentration of 22:6ω3 was dependent on food concentration. Though it was absent in the diet, the essential FA 20:5ω3 accumulated from the retroconversion of 22:6ω3 after 20 d. Changing the krill diet to diatoms resulted in a marked decline in thraustochytrid FA, particularly 14:0 and 22:6ω3, after 5 d, whilst diatom FA, 16:1ω7 and 20:5ω3, remained constant. Thraustochytrid signatures were completely erased from the FA profiles of DG after 20 d feeding on diatoms. Slow accumulation of storage lipid and short residence time of dietary FA suggest that lipid turnover rates are high in Antarctic krill DG. KEY WORDS: Euphausia superba · Antarctic krill · Lipid class · Fatty acid · Digestive gland · Trophic signature · Temporal scale Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 296. Online publication date: July 12, 2005 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2005 Inter-Research.

Synthesis of novel lipids in Saccharomyces cerevisiae by heterologous expression of an unspecific bacterial acyltransferase.

The bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT) is the key enzyme in storage lipid accumulation in the gram-negative bacterium Acinetobacter calcoaceticus ADP1, mediating wax ester, and to a lesser extent, triacylglycerol (TAG) biosynthesis. Saccharomyces cerevisiae accumulates TAGs and steryl esters as storage lipids. Four genes encoding a DGAT (Dga1p), a phospholipid:diacylglycerol acyltransferase (Lro1p) and two acyl-coenzyme A:sterol acyltransferases (ASATs) (Are1p and Are2p) are involved in the final esterification steps in TAG and steryl ester biosynthesis in this yeast. In the quadruple mutant strain S. cerevisiae H1246, the disruption of DGA1, LRO1, ARE1, and ARE2 leads to an inability to synthesize storage lipids. Heterologous expression of WS/DGAT from A. calcoaceticus ADP1 in S. cerevisiae H1246 restored TAG but not steryl ester biosynthesis, although high levels of ASAT activity could be demonstrated for WS/DGAT expressed in Escherichia coli XL1-Blue in radiometric in vitro assays with cholesterol and ergosterol as substrates. In addition to TAG synthesis, heterologous expression of WS/DGAT in S. cerevisiae H1246 resulted also in the accumulation of fatty acid ethyl esters as well as fatty acid isoamyl esters. In vitro studies confirmed that WS/DGAT is capable of utilizing a broad range of alcohols as substrates comprising long-chain fatty alcohols like hexadecanol as well as short-chain alcohols like ethanol or isoamyl alcohol. This study demonstrated the highly unspecific acyltransferase activity of WS/DGAT from A. calcoaceticus ADP1, indicating the broad biocatalytic potential of this enzyme for biotechnological production of a large variety of lipids in vivo in prokaryotic as well as eukaryotic expression hosts.

Characterization of Plastidial Thioredoxins from Arabidopsis Belonging to the New y-Type

The plant plastidial thioredoxins (Trx) are involved in the light-dependent regulation of many enzymatic activities, owing to their thiol-disulfide interchange activity. Three different types of plastidial Trx have been identified and characterized so far: the m-, f-, and x-types. Recently, a new putative plastidial type, the y-type, was found. In this work the two isoforms of Trx y encoded by the nuclear genome of Arabidopsis (Arabidopsis thaliana) were characterized. The plastidial targeting of Trx y has been established by the expression of a TrxGFP fusion protein. Then both isoforms were produced as recombinant proteins in their putative mature forms and purified to characterize them by a biochemical approach. Their ability to activate two plastidial light-regulated enzymes, NADP-malate dehydrogenase (NADP-MDH) and fructose-1,6-bisphosphatase, was tested. Both Trx y were poor activators of fructose-1,6-bisphosphatase and NADP-MDH; however, a detailed study of the activation of NADP-MDH using site-directed mutants of its regulatory cysteines suggested that Trx y was able to reduce the less negative regulatory disulfide but not the more negative regulatory disulfide. This property probably results from the fact that Trx y has a less negative redox midpoint potential (-337 mV at pH 7.9) than thioredoxins f and m. The y-type Trxs were also the best substrate for the plastidial peroxiredoxin Q. Gene expression analysis showed that Trx y2 was mainly expressed in leaves and induced by light, whereas Trx y1 was mainly expressed in nonphotosynthetic organs, especially in seeds at a stage of major accumulation of storage lipids.

Diapause in the calanoid freshwater copepod Eudiaptomus graciloides

The seasonal appearance and the intensity of diapausing-egg production in Eudiaptomus graciloides in five lakes of different size and trophic level were studied. In all lakes, diapausing eggs were produced in autumn. In the large mesotrophic lake Selenter See, the population’s shift to the production of diapausing eggswas morecompletethan in other lakes.Weexamined daylength, temperatureand foodasproximate factors for the production of diapausing eggs with laboratory experiments. Eudiaptomus graciloides produced diapausing eggs in all treatments, but a significantly higher percentage of diapausing eggs was found under short day conditions except when algal food was abundant and temperature was high. To investigate the adaptive significance of diapause in E. graciloides, we compared the survival of adult and juvenile at different temperatures for E. graciloides with Eudiaptomus gracilis, a sympatric species that does not exhibit diapause. At 8 � C, adult E. graciloides survived better than adult E. gracilis and exhibited reduced gut contents and accumulation of storage lipids, traits characteristic of adult diapause. Eudiaptomus graciloides nauplii did not reach the copepodid stage at 6 � C, but E. gracilis nauplii exhibited high mortality and developed very slowly. We hypothesize that adult diapause and production of diapausing eggs facilitate the survival of E. graciloides during cold periods and enhance coexistence with its congener, E. gracilis, in temperate zones.

Natural variation for seed oil composition in Arabidopsis thaliana

The biochemical pathways involved in the biosynthesis and accumulation of storage lipids in seeds have been extensively studied. However, the regulatory mechanisms of those pathways, their environmental interactions and the ecological implications of variation are poorly understood. We have initiated a new approach: the analysis of natural variation in Arabidopsis thaliana. Three hundred and sixty accessions were surveyed for content of oil, very long chain fatty acids (VLCFAs) and polyunsaturated fatty acids (PUFAs) in their seeds. The results revealed extensive natural variation. A core set of accessions, the seeds of which reproducibly contain extreme amounts of oil, VLCFAs and PUFAs have been identified. Reproducible oil content ranged from 34.6 to 46.0% of seed dry weight. VLCFA content ranged from 13.0 to 21.2% of total fatty acids. PUFA content, ranged from 53.3 to 66.1% of total fatty acids. Interactions were also identified for PUFA and VLCFA content of seeds with vernalisation of plants. Mapping of the regions of the genome involved in controlling the traits was conducted in an F 2 population and indicated that natural variation at the loci FAE1 and FAD3 might be involved in the regulation of VLCFA and PUFA content, respectively. A set of accessions, which capture a broad range of the natural variation for these traits available in A. thaliana, has been selected to form a core set which can be used to further dissect the genetics of the regulation of seed lipid traits and to identify the genes involved.

Biochemical and physiological studies of Arabidopsis thaliana transgenic lines with repressed expression of the mitochondrial pyruvate dehydrogenase kinase.

Pyruvate dehydrogenase kinase (PDHK), a negative regulator of the mitochondrial pyruvate dehydrogenase complex (mtPDC), plays a pivotal role in controlling mtPDC activity, and hence, the TCA cycle and cell respiration. Previously, the cloning of a PDHK cDNA from Arabidopsis thaliana and the effects of constitutively down-regulating its expression on plant growth and development has been reported. The first detailed analyses of the biochemical and physiological effects of partial silencing of the mtPDHK in A. thaliana using antisense constructs driven by both constitutive and seed-specific promoters are reported here. The studies revealed an increased level of respiration in leaves of the constitutive antisense PDHK transgenics; an increase in respiration was also found in developing seeds of the seed-specific antisense transgenics. Both constitutive and seed-specific partial silencing of the mtPDHK resulted in increased seed oil content and seed weight at maturity. Feeding 3-(14)C pyruvate to bolted stems containing siliques (constitutive transgenics), or to isolated siliques or immature seeds (seed-specific transgenics) confirmed a higher rate of incorporation of radiolabel into all seed lipid species, particularly triacylglycerols. Neither constitutive nor seed-specific partial silencing of PDHK negatively affected overall silique and seed development. Instead, oil and seed yield, and overall plant productivity were improved. These findings suggest that a partial reduction of the repression of the mtPDC by antisense PDHK expression can alter carbon flux and, in particular, the contribution of carbon moieties from pyruvate to fatty acid biosynthesis and storage lipid accumulation in developing seeds, implicating a role for mtPDC in fatty acid biosynthesis in seeds.

Biochemical events leading to the diversion of carbon into storage lipids in the oleaginous fungi Mucor circinelloides and Mortierella alpina.

The biochemical events associated with the onset of lipid accumulation in Mucor circinelloides and Mortierella alpina, under conditions of nitrogen-limited growth, have been elucidated; they differ in key aspects from those described in oleaginous yeasts. The NAD+:isocitrate dehydrogenases of Mc. circinelloides and Mort. alpina were not absolutely dependent on AMP for activity. Furthermore, changes in the cellular adenine nucleotide pools and energy charge were different from those reported for oleaginous yeasts. In Mc. circinelloides ATP, ADP and AMP concentrations all decreased by 50% after nitrogen limitation, leading to a constant energy charge at the expense of the size of the total adenylate pool. Pyruvate carboxylase in Mc. circinelloides was cytosolic, having implications for the organization of lipid synthesis in filamentous fungi. As a result of the data obtained, a revised and more concerted mechanism for the initiation of storage lipid accumulation is put forward for filamentous fungi.

Patterns of gene expression in developing Arabidopsis seeds during storage lipid synthesis

We have used microarrays to explore factors that regulate the overall partitioning of seed reserves into storage compounds, and particularly the accumulation of seed oil. Arabidopsis can be used as a model to study lipid synthesis in oil crops, because triacylglycerides are the major reserve in its seeds. To this end, we have produced microarrays containing about 5000 clones selected from a developing Arabidopsis seed-specific cDNA library, and used them to study gene expression profiles during seed filling. This approach allowed us to simultaneously compare mRNA levels of genes for fatty acid synthesis (FAS), storage proteins, glycolysis, etc. from the same material. Analysis of expression profiles at different stages of development identified about 2000 genes whose expression changed significantly during the accumulation of the storage reserves. Several genes encoding FAS enzymes (acetyl-CoA carboxylase subunits, KAS I, and acyl-carrier protein) showed coordinated expression. Typically, the highest gene expression coincided with the most active storage lipid accumulation, and then decreased rapidly. This pattern was distinctly different from that for many glycolytic enzymes, storage proteins and oleosins, as well as some fatty acid modifying enzymes (FAD3 and FAE1). These observations imply different regulatory controls over gene expression for different storage components in seeds. We also identified several transcription factors, kinases and phosphatases, whose expression changed during the seed development, and which may be involved in the regulation of the storage compound synthesis. Moreover, cluster analysis of the expression profiles established distinct groups of co-regulated genes.

Expression of the FAE1 gene and FAE1 promoter activity in developing seeds of Arabidopsis thaliana.

Plant fatty acid elongase which catalyzes very-long-chain fatty acid (VLCFA) biosynthesis is a membrane-bound multienzyme complex. It is composed of four enzymes, a 3-ketoacyl-CoA synthase (condensing enzyme), a 3-ketoacyl-CoA reductase, a 3-hydroxyacyl-CoA dehydrase, and an enoyl-CoA reductase required for completion of each step of 2-carbon elongation of fatty acids. To improve our understanding of the overall regulation of the fatty acid elongase, we investigated the spatial and temporal expression of its key component, the FAE1-condensing enzyme, and examined the activity of the promoter of the FAE1 gene in Arabidopsis. In situ hybridization results revealed that FAE1 transcripts were found exclusively in the embryo. RNA blot analysis and histochemical analysis of GUS activity in pFAE1::GUS transgenic Arabidopsis lines demonstrated that the FAE1 gene was already transcribed in the early torpedo stage embryos 4-5 days after flowering, with transcription reaching its peak 9-11 days after flowering. VLCFA deposition closely paralleled FAE1 transcript accumulation. FAE1 promoter was highly active and embryo-specific. Because its timing coincides with the period of major storage lipid accumulation, and because its in vivo activity in Arabidopsis is superior to the napin promoter, FAE1 promoter may be ideal for genetic engineering of seed oil composition.

Lipid biosynthesis in cultures of oilseed rape

This review focuses on how microspore-derived (MD) embros and cell suspension cultures of oilseed rape have been used to advance our understanding of the biochemistry and molecular biology of lipid biosynthesis in plants. Both types of cultures are easily maintained and circumvent the difficulties associated with using developing seeds for investigations of lipid biosynthesis. Developing MD embryos exhibit a similar storage lipid accumulation profile and fatty acid composition to developing seed. The use of dihaploids derived from plantlets of MD embryos have accelerated breeding programs and have proven useful in the detection of recessive mutations. MD embryos and MD cell suspension cultures have been particularly useful in investigating the properties of key enzymes involved in triacylglycerol (TG) bioassembly. MD cell suspension cultures, however, offer the advantage of being able to study lipid metabolism in the absence of cellular differentiation. TG accumulation can be induced in MD cell suspension cultures by increasing the sucrose concentration of the growth medium thereby providing a useful system to investigate gene expression and the proteomics of lipid biosynthesis.