ABSTRACT
The expression of the FATTY ACID ELONGATION1 genes was characterised to provide insight into the regulation of very long chain fatty acid (VLCFA) biosynthesis in Brassica napus embryos. Each of the two rapeseed homoeologous genes (Bn-FAE1.1 and Bn-FAE1.2) encoding isozymes of 3-keto-acylCoA synthase, a subunit of the cytoplasmic acyl-CoA elongase complex that controls the production of elongated fatty acids, are expressed predominantly in developing seeds. The proximal regions of the Bn-FAE1.1 and Bn-FAE1.2 promoters possess strong sequence identity suggesting that transcriptional control of expression is mediated by this region which contains putative cis-elements characteristic of those found in the promoters of genes expressed in embryo and endosperm. Histochemical staining of rapeseed lines expressing Bn-FAE1.1 promoter:reporter gene fusions revealed a strong expression in the embryo cotyledon and axis throughout the maturation phase. Quantitative analyses revealed the region, -331 to -149, exerts a major control on cotyledon specific expression and the level of expression. A second region, -640 to -475, acts positively to enhance expression levels and extends expression of Bn-FAE1.1 into the axis and hypocotyl but also acts negatively to repress expression in the root meristem. The expression of the Bn-FAE1.1 gene was not restricted to the seed but was also detected in the vascular tissues of germinating seedlings and mature plants in the fascicular cambium tissue present in roots, stem and leaf petiole. We propose that Bn-FAE1.1 expression in vascular tissue may contribute VLCFA for barrier lipid synthesis and reflects the ancestral function of FAE1 encoded 3-keto-acylCoA synthase.
Subject(s)
Brassica napus/embryology , Brassica napus/genetics , Gene Expression Regulation, Plant , Plant Proteins/genetics , Plant Vascular Bundle/embryology , Plant Vascular Bundle/genetics , Base Sequence , Gene Expression Regulation, Developmental , Genes, Plant , Molecular Sequence Data , Plant Proteins/metabolism , Plants, Genetically Modified , Promoter Regions, Genetic , Seeds/genetics , Sequence AlignmentABSTRACT
In higher plants, lysophosphatidic acid acyltransferase (LPAAT), located in the cytoplasmic endomembrane compartment, plays an essential role in the synthesis of phosphatidic acid, a key intermediate in the biosynthesis of membrane phospholipids in all tissues and storage lipids in developing seeds. In order to assess the contribution of LPAATs to the synthesis of storage lipids, we have characterized two microsomal LPAAT isozymes, the products of homoeologous genes that are expressed in rapeseed (Brassica napus). DNA sequence homologies, complementation of a bacterial LPAAT-deficient mutant, and enzymatic properties confirmed that each of two cDNAs isolated from a Brassica napus immature embryo library encoded a functional LPAAT possessing the properties of a eukaryotic pathway enzyme. Analyses in planta revealed differences in the expression of the two genes, one of which was detected in all rapeseed tissues and during silique and seed development, whereas the expression of the second gene was restricted predominantly to siliques and developing seeds. Expression of each rapeseed LPAAT isozyme in Arabidopsis (Arabidopsis thaliana) resulted in the production of seeds characterized by a greater lipid content and seed mass. These results support the hypothesis that increasing the expression of glycerolipid acyltransferases in seeds leads to a greater flux of intermediates through the Kennedy pathway and results in enhanced triacylglycerol accumulation.
Subject(s)
Acyltransferases/metabolism , Brassica rapa/enzymology , Plant Oils/metabolism , Plant Proteins/metabolism , Seeds/metabolism , Acyltransferases/genetics , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis/metabolism , Brassica rapa/genetics , Cloning, Molecular , DNA, Plant/genetics , Gene Expression Regulation, Plant , Gene Library , Isoenzymes/genetics , Isoenzymes/metabolism , Molecular Sequence Data , Plant Proteins/genetics , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Promoter Regions, Genetic , Seeds/genetics , Sequence Alignment , Triglycerides/biosynthesisABSTRACT
In algae, the biosynthesis of docosahexaenoic acid (22:6omega3; DHA) proceeds via the elongation of eicosapentaenoic acid (20:5omega3; EPA) to 22:5omega3, which is required as a substrate for the final Delta4 desaturation. To isolate the elongase specific for this step, we searched expressed sequence tag and genomic databases from the algae Ostreococcus tauri and Thalassiosira pseudonana, from the fish Oncorhynchus mykiss, from the frog Xenopus laevis, and from the sea squirt Ciona intestinalis using as a query the elongase sequence PpPSE1 from the moss Physcomitrella patens. The open reading frames of the identified elongase candidates were expressed in yeast for functional characterization. By this, we identified two types of elongases from O. tauri and T. pseudonana: one specific for the elongation of (Delta6-)C18-PUFAs and one specific for (Delta5-)C20-PUFAs, showing highest activity with EPA. The clones isolated from O. mykiss, X. laevis, and C. intestinalis accepted both C18- and C20-PUFAs. By coexpression of the Delta6- and Delta5-elongases from T. pseudonana and O. tauri, respectively, with the Delta5- and Delta4-desaturases from two other algae we successfully implemented DHA synthesis in stearidonic acid-fed yeast. This may be considered an encouraging first step in future efforts to implement this biosynthetic sequence into transgenic oilseed crops.
