Synthesis of medium-chain fatty acids and their incorporation into triacylglycerols by cell-free fractions from Cuphea embryos

Abstract

During their rapid maturation period, seeds of Cuphea wrightii A. Gray mainly accumulate medium-chain fatty acids (C8 to C14) in their storage lipids. The rate of lipid deposition (40-50 mg·d(-1)·(g fresh weight)(-1)) is fourfold higher than in seeds of Cuphea racemosa (L. f.) Spreng, which accumulate long-chain fatty acids (C16 to C18). Measurements of the key enzymes of fatty-acid synthesis in cell-free extracts of seeds of different maturities from Cuphea wrightii show that malonyl-CoA synthesis may be a triggering factor for the observed high capacity for fatty-acid synthesis. Experiments on the incorporation of [1-(14)C]acetate into fatty acids by purified plastid preparations from embryos of Cuphea wrightii have demonstrated that the biosynthesis of medium-chain fatty acids (C8 to C14) is localized in the plastid. Thus, in the presence of cofactors for lipid synthesis (ATP, NADPH, NADH, acyl carrier protein, and sn-glycerol-3-phosphate), purified plastid fractions predominantly synthesized free fatty acids, 30% of which were of medium chain length. Transesterification of the freshly synthesized fatty acids to coenzyme A and recombination with the microsomal fraction of the embryo homogenate induced triacylglycerol synthesis. It also stimulated fatty-acid synthesis by a factor 2-3 and increased the relative amount of medium-chain fatty acids bound to triacylglycerols, which corresponded to about 60-80% in this lipid fraction.

Condensing enzymes from Cuphea wrightii associated with medium chain fatty acid biosynthesis

“…The appearance of β-ketoacyl-ACP reductase, the enzyme that reduces the product of the condensation reaction, was coincident with accumulation of the synthases (Figure 4c). Induction of two C. wrightii FatB thioesterases follows this same time-course , and acetyl-CoA carboxylase activity has been shown to peak 10-14 days after flowering in C. wrightii (Deerberg et al, 1990). Taken together, these results indicate co-ordinate regulation of the enzymes required for synthesis of storage lipids, with massive accumulation in a 1-2-day period.…”

“…Although in vitro experiments indicated the presence of cerulenin-resistant synthases that extend C4-C8 substrates, uninhibited developing embryo extracts readily extended [1-14 C]acetyl-CoA to long chain acyl-ACP. Previous workers have also observed that homogenates of developing Cuphea seeds or preparations of lysed embryo plastids synthesize primarily long chain fatty acids (Deerberg et al, 1990;Fuhrmann and Heise, 1993;Singh et al, 1984). An unresolved question therefore is whether production of large amounts of MCFA in vivo is solely a consequence of thioesterases with appropriate chain length specificity acting on available acyl-ACP pools, or is also dependent on an unknown feature of intraplastidial enzyme organization that is lost upon homogenization.…”

Broad-Range and Binary-Range Acyl-Acyl-Carrier-Protein Thioesterases Suggest an Alternative Mechanism for Medium-Chain Production in Seeds

“…The seed-specific expression of a different enzyme, one involved in fatty acyl elongation, may explain the respective seed-oil compositions. Indirect evidence for modified P-ketoacyl synthases in medium-chain producers has accumulated in the past (Deerberg et al, 1990;Heise and Fuhrmann, 1994), and partially purified condensing enzymes from C. lanceolata hardly elongate 10:O-ACP (Schuch et al, 1993). Very recently, it was demonstrated that a seed-specific /3-ketoacyl synthase from Cuphea wrightii selectively boosts the production of specific medium chains in transgenic Arabidopsis after co-expression with a broad-range FatB enzyme (M. Slabaugh, Oregon State University, personal communication).…”

Structure and Expression of an Arabidopsis Acetyl-Coenzyme A Carboxylase Gene

“…ACCase may also be the site of feedback inhibition of fatty acid synthesis in tobacco suspension cells supplemented with exogenous fatty acids (Shintani and Ohlrogge, 1993). Furthermore, ACCase activity increases in association with lipid deposition in developing seeds of oilseed crops (Simcox et al, 1979;Tumham and Northcote, 1983;Charles et al, 1986;Deerburg et al, 1990). These observations have prompted speculation that ACCase may be a rate-limiting enzyme for oilseed fatty acid synthesis, but conclusive evidence supporting this hypothesis has not been obtained.…”