Long chain fatty acid synthesis in rapeseed cotyledons : a thesis presented for the fulfilment of the requirements for the degree of Master of Science in Botany, Massey University, New Zealand

Abstract

When developing rapeseed cotyledons were incubated with [ 14 C] acetate, approximately 70% of the label was found in triacylglycerol, in which erucate was the most heavily labelled fatty acid. Oxidative degradation studies to determine the distribution of radioactivity in oleate, eicosenoate and erucate of this labelled triacylglycerol showed that (a) the specific radioactivity of oleate, and of the oleoyl portions of eicosenoate and erucate were similar. Since the masses of these three fatty acids in the triacylglycerol of the cotyledons used were different, this suggests that a particular fatty acid is incorporated into triacylglycerol in proportion to the amount of each fatty acid already present in the oil. (b) the specific radioactivities of the oleoyl portions of eicosenoate and erucate were much lower than those of the carboxyl terminal carbons added by chain elongation, indicating that the specific radioactivities of the acetate utilized for de novo synthesis and that used for chain elongation were different; this suggests that there are distinct pools of acetate for these two processes. In in vitro assays, rapeseed oil body preparations incorporated label from [ 14 C] malonyl CoA mainly into eicosenoate and erucate, whereas crude homogenates utilized the [ 14 C] malonyl CoA mainly for de novo synthesis of palmitate and stearate. In assays containing oil bodies, incorporation was dependent on the presence of freshly prepared dithiothreitol; NADPH was the most efficient reductant, and ATP was required for maximum incorporation. The addition of oleoyl or eicosenoyl CoA to assays did not stimulate incorporation but markedly affected the amounts of radioactive eicosenoate and erucate synthesized, providing evidence that long chain CoAs are substrates for the chain elongation reaction. The lack of any dependence on acyl CoAs suggests that they were present in oil body preparations or synthesized during the assays. Generally the level of labelled long chain acyl CoAs was low and most of the radioactive fatty acids synthesized in vitro were found in triacylglycerol and phosphatidic acid. From in vivo and in vitro studies it is suggested that the synthesis of eicosenoate and erucate involves the formation of the corresponding CoAs by elongation of oleoyl (or eicosenoyl) CoA with malonyl CoA and NADPH in a manner analogous to malonate-dependent elongation in mammalian microsomes, and that the synthesis of oleate and its subsequent elongation to eicosenoate and erucate occur at different sites in the cell utilizing different acetate pools. The intracellular location of chain elongation and the mechanism by which the fatty acyl CoA products of chain elongation are incorporated into triacylglycerol are discussed.