Thomas Roscoe

A rapeseed FAE1 gene is linked to the E1 locus associated with variation in the content of erucic acid

Abstract The synthesis of very long chain fatty acids occurs in the cytoplasm via an elongase complex. A key component of this complex is the β-ketoacyl-CoA synthase, a condensing enzyme which in Arabidopsis is encoded by the FAE1 gene. Two sequences homologous to the FAE1 gene were isolated from a Brassica napus immature embryo cDNA library. The two clones, CE7 and CE8, contain inserts of 1647 bp and 1654 bp, respectively. The CE7 gene encodes a protein of 506 amino acids and the CE8 clone, a protein of 505 amino acids, each having an approximate molecular mass of 56 kDa. The sequences of the two cDNA clones are highly homologous yet distinct, sharing 97% nucleotide identity and 98% identity at the amino acid level. Southern hybridisation showed the rapeseed β-ketoacyl-CoA synthase to be encoded by a small multigene family. Northern hybridisation showed the expression of the rapeseed FAE1 gene(s) to be restricted to the immature embryo. One of the FAE1 genes is tightly linked to the E1 locus, one of two loci controlling erucic acid content in rapeseed. The identity of the second locus, E2, is discussed.

Expression of Rapeseed Microsomal Lysophosphatidic Acid Acyltransferase Isozymes Enhances Seed Oil Content in Arabidopsis

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.

Mutations in the fatty acid elongation 1 gene are associated with a loss of β‐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 β‐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 β‐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 β‐ketoacyl‐CoA synthase proteins.

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.

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.

Regulation of FATTY ACID ELONGATION1 expression in embryonic and vascular tissues of Brassica napus

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.

Synthesis of Structural and Storage Lipids by the ER

The endoplasmic reticulum (ER) in plants plays a key role in the synthesis of a wide rangeof lipids which are essential structural components of all cellular membranes. Lipids also represent themajor form of storage carbon in the seeds, pollen and fruit of many plant species and in some cases over75% of the dry mass of these tissues has been metabolised by the ER. The world vegetable-oils market isworth over

Life and death among plant lysophosphatidic acid acyltransferases.

30 billion per year and is of great importance to the agricultural economy. There is thereforeparticular interest in aspects of ER function relating to triacylglycerol synthesis. In the epidermis,lipids made by the ER are exported to form the cuticular barrier protecting the plant against water loss,biotic and abiotic stresses. In addition, ER-derived glycerolipids, sphingolipids and sterols have essentialroles as components of signal transduction pathways. This chapter describes the biochemical pathways ofmembrane and storage lipid synthesis in the plant ER and charts progress in the identification and characterisationof the genes involved.

Improving Quality and Content of Oils in Seeds: Strategies, Approaches, and Applications Towards Engineering New Oilseed Crop Plants

The tetraploid Brassica napus possesses several seed-expressed microsomal lysophosphatidic acid acyltransferases (LPAAT ) including BAT1.5, which has been retained after genome duplication as a consequence of a subfunctionalisation of the gene encoding the ubiquitously expressed Kennedy pathway enzyme BAT1.13. Next, cDNA BAT1.3, encoding a LPAAT was subsequently isolated from an embryo library. The rapeseed LPAAT encoded by BAT1.3 is orthologous to the Arabidopsis thaliana At1g51260 gene product possibly associated with tapetum development and male fertility. However, BAT1.3 expression is predominant during the mid stages of embryo development in seeds of Brassica napus. Functional characterisation of BAT1.3 provides further support for a hypothesis of gene dosage sensitivity of LPAATs as does an analysis of the chromosomal localisation of LPAAT genes in Arabidopsis thaliana. The pattern of retention or loss of LPAAT genes after polyploidisation or segmental duplication is consistent with a model of balanced gene drive.

Characterization of Protein Factors Interacting with the rDNA Promoter Region

Plant oils are the major sources for human nutrition. There is increasing interest in the use of plant oils as renewable sources of industrial feedstocks. In order to alleviate the increasing demand for plant oils, omics approaches have been adopted to facilitate modification of the fatty acid composition in order to improve the nutritional properties and to generate specific physiochemical properties for industrial uses. An overview of the application of omics to aid progress in the engineering of oil quality and seed yield is presented in this chapter.