Acyl-CoA elongase expression during seed development in Brassica napus

Abstract

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.

Introduction

In many higher plants, C16:1 and C18:1 fatty acids are the major components of the seed storage triacylglycerols (TAG). High erucic acid rapeseed (HEAR) oil is different since erucic acid (C22:1, Δ13) represents from 45 to 60% of the total fatty acids. Erucic acid has many industrial uses, for instance in lubricants, plastics, etc. [1], and research programs attempting to increase the erucic acid content in rapeseed by genetic manipulations have been undertaken.

In rapeseed, it has been demonstrated that lysophosphatidic acid acyltransferase (LPAAT) is a key enzyme since it does not allow the esterification of the sn-2 position of the glycerol backbone by erucoyl-CoA in rapeseed [2], [3]. Transgenic rapeseeds expressing the LPAAT from Limnanthes alba, which are able to esterify erucic acid at the sn-2 position, synthesize trierucin but the overall content of erucic acid in the seed remains unchanged [4]. This result is generally interpreted as reflecting a low erucoyl-CoA pool available for TAG biosynthesis [4]. Thus, elongases which are responsible for erucoyl-CoA synthesis are also key enzymes in trierucin biosynthesis. In higher plants, the existence of at least two types of elongases, the ATP-dependent elongase and the acyl-CoA elongase, has been demonstrated [5], [6]. ATP-dependent elongase elongates an unknown endogenous substrate, whereas the acyl-CoA elongase, a multienzymatic and membrane-bound complex [6], uses exogenous acyl-CoA and does not require ATP. The situation is further complicated as it has been shown in rapeseed microsomes that conditions that allow maximum oleoyl-CoA elongation partially inhibit ATP-dependent elongation, and at maximum rates ATP-dependent elongation decreases oleoyl-CoA elongation [6]. These results strongly suggest that the two elongation systems cannot operate in vitro at the same time or in the same cell compartment.

The acyl-CoA elongase has been extensively studied and partly purified from many sources [5], [7], [8], [9], including Brassica napus seeds [10]. This elongation complex comprises four different proteins with apparent molecular masses ranging from 54 to 65 kDa [8], [9], [10]. Four successive reactions are involved in the elongation process: condensation of C18:1-CoA to malonyl-CoA to form a 3-ketoacyl-CoA; reduction of the 3-ketoacyl-CoA; dehydration of the resulting 3-hydroxyacyl-CoA and reduction of the trans-(2,3)-enoyl-CoA [11], [12], [13]. The gene encoding the first enzyme, the 3-ketoacyl-CoA synthase, has been characterized in Arabidopsis thaliana (FAE1) [14] and jojoba [15] and two homologous sequences (Bn-FAE1.1 and Bn-FAE1.2) have been isolated from embryos of B. napus. Bn-FAE1.2 encodes a protein of 505 amino acids and Bn-FAE1.1, a protein of 506 amino acids, both proteins sharing 98.2% identity [16].

It has also been demonstrated that very long chain fatty acid biosynthesis is controlled through the expression and specificity of the condensing enzyme [17]. In B. napus, the understanding of the role of Bn-FAE1.1 and Bn-FAE1.2 and their regulation during the development of the seed is necessary to implement strategies to increase the erucic acid content. Despite numerous investigations, fatty acid regulation at the molecular level is far from being understood. The pioneering work by Norton and Harris [18] reported that changes in fatty acid composition in developing rapeseed occurred in three consecutive phases, and that erucic acid accumulation took place in the last phase. To date, most of the studies have been carried out using microspore-derived cell cultures [19]. It has been shown that: (i) the fatty acid synthesis and acyltransferase activities increase from the 2nd to the 4th week of growth, and then decrease; (ii) the diacylglycerol acyltransferase (DAGAT) activity and the oleosin gene expression increase when the sucrose concentration in the culture medium is raised from 2 to 22% (w/v) [20], [21]; (iii) abscisic acid (ABA) and temperature stimulate erucic acid biosynthesis by different mechanisms [22] leading to an accumulation of very long chain monounsaturated fatty acids (VLCMFA) correlated with a stimulation of the elongase activity [23]. The expression of Bn-FAE1 transcripts is induced by 10 μM ABA within 1 h and is further increased up to 6 h while, during the same time, the VLCMFA content doubles [24].

Canola or low erucic acid rapeseed (LEAR) varieties, characterized by a near-absence of VLCMFA, were created by the introduction of recessive alleles at two loci that control the elongation of fatty acids [25]. Elongase activities are present in HEAR embryos but absent from embryos of LEAR varieties [26], as is the case with the FAE1 (fatty acid elongation) mutants of Arabidopsis [27], that exhibit a similar phenotype to LEAR. The Arabidopsis FAE1 gene encodes a protein that shares homologies with condensing enzymes [14]. The jojoba homologue of FAE1 encoding a 3-ketoacyl-CoA synthase restored elongation activity to developing embryos of LEAR, leading to the conclusion that the mutations that gave rise to the LEAR phenotype are associated with either the structural gene encoding 3-ketoacyl-CoA synthase or with genes regulating its expression [15]. In rapeseed, the two elongation steps from oleoyl-CoA to erucic acid are each controlled by alleles at two loci, E1 and E2, which exhibit additive gene action [28]. A gene encoding the rapeseed 3-ketoacyl-CoA synthase, Bn-FAE1.1, was shown by Barret et al. [16] to be tightly linked to the E1 locus and the homologous gene, Bn-FAE1.2, was assigned to the E2 locus.

Preliminary experiments indicate that Bn-FAE1 could also be transcribed in LEAR embryos [29]. Since this observation was obtained using seeds harvested 9 weeks after pollination (WAP), no data concerning a post-transcriptional degradation, modification or regulation of this protein during seed development are available. Therefore, we investigated the enzymatic activities and regulation of Bn-FAE1.1 and Bn-FAE1.2 gene expression during rapeseed development using HEAR and LEAR cultivars.