Evidence that oleoyl‐CoA and ATP‐dependent elongations coexist in rapeseed (Brassica napus L.)

Abstract

The elongation of different substrates was studied using several subcellular fractions from Brassica napus rapeseed. In the presence of malonyl-CoA, NADH and NADPH, very-long-chain fatty acid (VLCFA) synthesis was observed from either oleoyl-CoA (acyl-CoA elongation) or endogenous primers (ATP-dependent elongation). No activity was detected using oleic acid as precursor. Acyl-CoA and ATP-dependent elongation activities were mainly associated with the 15 000 g/25 min membrane fraction. Reverse-phase TLC analysis showed that the proportions of fatty acids synthesized by these activities were different. Acyl-CoA elongation increased up to 60 mm oleoyl-CoA, and ATP-dependent elongation was maximum at 1 mm ATP. Both activities increased with malonyl-CoA concentration (up to 200 mm). Under all conditions tested, acyl-CoA elongation was higher than ATP-dependent elongation, and, in the presence of both ATP and oleoyl-CoA, the elongation activity was always lower. ATP strongly inhibited acyl-CoA elongation, whereas ATP-dependent elongation was slightly stimulated by low oleoyl-CoA concentrations (up to 15 mm) and decreased in the presence of higher concentrations. CoA (up to 150 mm) had no effect on the ATP-dependent elongation, whereas it inhibited the acyl-CoA elongation. These marked differences strongly support the presence in maturing rapeseed of two different elongating activities differently modulated by ATP and oleoyl-CoA.Keywords: acyl-CoA elongase; acyl-CoAs; ATP-dependent elongation; erucic acid; plant lipids.Very-long-chain fatty acids (VLCFAs, fatty acids with at least 20 carbon atoms) are widely distributed in nature. In plants, they are primarily found in epicuticular waxes (mostly saturated VLCFAs) and in seed storage oils [mostly monounsaturated VLCFAs, generally belonging to the cis (n-9) series]. The enzymes responsible for their synthesis, referred to as elongases, are membrane-bound proteins that utilize malonyl-CoA as the C2-unit donor and primers having at least 18 carbon atoms [1,2].Genetic [3,4] and biochemical [5,6] studies have suggested the existence of several elongases. Several studies using leek epidermis microsomes [7±9] have hypothesized the existence of at least two different elongating activities. In the presence of labeled malonyl-CoA, VLCFAs could be synthesized from either acyl-CoAs (acyl-CoA elongation) or endogenous acyl components of the microsomal lipids; in the latter case, there is an absolute requirement for ATP (ATP-dependent elongation). In the case of acyl-CoA elongation, Lessire et al. [9] demonstrated that successive additions of C2 units to stearoyl-CoA (C 18 : 0 -CoA) were responsible for the sequential synthesis of arachidonoyl-CoA (C 20 : 0 -CoA), behenoyl-CoA (C 22 : 0 -CoA) and lignoceroyl-CoA (C 24 : 0 -CoA). On the other hand, ATP-dependent elongation involves a one-step condensation from an endogenous primer (Cn) with malonylCoA to a Cn + 2 [2,7]. Using linear sucrose-gradient centrifugation, Moreau et al.[10] studied the intracellular localization of these el...

Seed-Specific Heterologous Expression of a Nasturtium FAE Gene in Arabidopsis Results in a Dramatic Increase in the Proportion of Erucic Acid

“…Although there has been considerable debate regarding the acyl substrate for elongase activity in developing oilseeds (Hlousek-Radojcic et al, 1995), recent studies of developing seeds of B. napus have revealed the presence of two types of elongation activity in vitro: an acyl-CoA-dependent activity and an ATP-dependent activity that apparently utilizes an endogenous acyl primer (Domergue et al, 1999). A 15,000g particulate fraction was isolated from nasturtium embryos collected at mid-development (at 14-17 d after pollination), the stage which exhibited the highest enrichment in acyl-CoA-dependent elongase activity.…”

Inheritance and variation of erucic acid content in a transgenic rapeseed (Brassica napus L.) doubled haploid population

“…There is some evidence that the cytosolic pool of oleoyl-CoA available for elongation may be limiting erucic acid biosynthesis (Bao et al 1998;Domergue et al 1999). Crossing of conventional HEAR to rapeseed with reduced contents of linoleic acid (18:2) and linolenic acid (18:3) did result in recombinant high erucic low polyunsaturated fatty acid (HELP) F 3 plants which, however, did not show an increased erucic acid content compared to the parental HEAR genotype (50% erucic acid; Sasongko and Möllers 2005).…”

Action of fentrazamide on protein metabolism and cell division in plants

“…No ATP was included in the assay since it reduced the elongation rate. 13) In untreated controls, the incorporation of radioactivity was similar between rice and early watergrass.…”