The biosynthesis of erucic acid in developing embryos of brassica rapa

Abstract

The prevailing hypothesis on the biosynthesis of erucic acid in developing seeds is that oleic acid, produced in the plastid, is activated to oleoyl-coenzyme A (CoA) for malonyl-CoA-dependent elongation to erucic acid in the cytosol. Several in vivo-labeling experiments designed to probe and extend this hypothesis are reported here. To examine whether newly synthesized oleic acid is directly elongated to erucic acid in developing seeds of Brassica rapa L., embryos were labeled with [14C]acetate, and the ratio of radioactivity of carbon atoms C-5 to C-22 (de novo fatty acid synthesis portion) to carbon atoms C-1 to C-4 (elongated portion) of erucic acid was monitored with time. If newly synthesized 18:1 (oleate) immediately becomes a substrate for elongation to erucic acid, this ratio would be expected to remain constant with incubation time. However, if erucic acid is produced from a pool of preexisting oleic acid, the ratio of 14C in the 4 elongation carbons to 14C in the methyl-terminal 18 carbons would be expected to decrease with time. This labeling ratio decreased with time and, therefore, suggests the existence of an intermediate pool of 18:1, which contributes at least part of the oleoyl precursor for the production of erucic acid. The addition of 2-[3-chloro-5-(trifluromethyl)-2-pyridinyloxyphenoxy] propanoic acid, which inhibits the homodimeric acetyl-CoA carboxylase, severely inhibited the synthesis of [14C]erucic acid, indicating that essentially all malonyl-CoA for elongation of 18:1 to erucate was produced by homodimeric acetyl-CoA carboxylase. Both light and 2-[3-chloro-5-(trifluromethyl)-2-pyridinyloxyphenoxy]-propanoic acid increased the accumulation of [14C]18:1 and the parallel accumulation of [14C]phosphatidylcholine. Taken together, these results show an additional level of complexity in the biosynthesis of erucic acid.

Figure 1

Accumulation of fatty acids during B. rapa development. Lipids were extracted from 20 pooled embryos at the times indicated and fatty acid methyl esters were separated and quantified by GC. The total 18:1 derivatives were obtained by adding 18:1, 18:2, 18:3, 20:1, and 22:1 together. The accumulation rate of 18:1 derivatives (16 nmol h⁻¹ embryo⁻¹) was calculated for the embryos at 28 DAF when labeling experiments were conducted.

Figure 2

Effects of light on the synthesis of 22:1 (A), 18:1 (B), TAG (C), and PC (D). ▪, Dark; □, light. Radioactivity is expressed per embryo.

Figure 3

Labeling of fatty acids by [¹⁴C]acetate in developing B. rapa embryos. A, The percentage of [¹⁴C]acetate incorporated into total 18:1 (18:1 plus the derived 18:1 portion of 20:1 and 22:1) that appears in 22:1 is plotted versus time. The incubation was conducted either in the light (□) or in the dark (▪). B, Time course of ¹⁴C accumulation into total 18:1 derivatives (18:1 plus the derived 18:1 portion of 20:1 and 22:1) in the light. All of the fatty acids were derived from total lipid extracts, and the numbers represent radioactivity calculated per embryo.

Figure 4

Effect of increasing concentrations of haloxyfop on fatty acid and lipid synthesis. B. rapa embryos were pretreated with the indicated concentrations of herbicide for 30 min, then incubated for 1 h in the light with the addition of [¹⁴C]acetate. Radioactivity of lipid and fatty acid are quantified on the basis of dpm per embryo.