Laurate can be produced in the seed reserve oil of Brassica napus (rapeseed) by the expression of an heterologous lauroyl acyl‐carrier protein thioesterase under the control of a napin seed‐storage protein promoter. Analysis of a large number of transgenic events, and their progeny after self‐pollination, shows that laurate can accumulate to nearly 60% of the triglyceride acyl groups. Up to 40 mol% laurate the phenotype is correlated positively with the number of thioesterase gene copies. The use of a tandem gene construct elevates the average laurate content. This effect correlates with an increased average number of T‐DNA insertions per event; no cis‐inactivation of tandem genes is apparent. Above 40 mol% laurate other factors apparently limit the phenotype. The expression timing conferred by the napin promoter is unlikely to be limiting, as it covers almost the entire period of oil deposition. A more significant limitation resides in the second acylation reaction of oil biosynthesis, as shown by the very low incorporation of laurate at the sn‐2 acyl group. The novel, high‐laurate oil is consequently rich in sn‐1,3‐dilauroyl triglycerides, but its unusual composition appears to pose no problems for mobilization during seed germination.
Abstract.We have examined production of mediumchain fatty acids by Brassica napus L. plants transformed with a California bay (Umbellularia californica) medium-chain acyl-acyl carrier protein (ACP) thioesterase (UcFatB1) cDNA under the control of the constitutive cauliflower mosaic virus 35S promoter. These plants were found to accumulate medium-chain fatty acids in seeds but not in leaves or roots. Assay of thioesterase activity in extracts of leaves indicated that lauroyl-ACP thioesterase activity is comparable to oleoyl-ACP thioesterase (EC 3.1.2.14) activity in transformant leaves. Furthermore, leaf lauroyl-ACP thioesterase activity was in excess of that which produced a significant increase in the amount of laurate (12:0) in seed. Studies in which isolated chloroplasts were 1*C-labelled were used to evaluate whether medium-chain fatty acids were produced in transformed leaves. Up to 34% of the fatty acids synthesized in vitro by isolated chloroplasts were 12:0. These results demonstrate that the normally seed-localized lauroyl-ACP thioesterase can be expressed in active form in leaves, imported into chloroplasts and can access acyl-ACP intermediates of leaf de-novo fatty acid synthesis. The most likely explanation for the lack of accumulation of 12:0 in transformed leaves is its rapid,degradation by ~-oxidation. In support of this hypothesis, isocitrate lyase (EC 4.1.3.1) activity was found to be significantly increased in plants transformed with 35S-UcFatB1. Key words: [~-Oxidation -Brassica -Fatty acid synthesisLeaf Medium-chain acyl-ACP thioesterase -Umbellularia Abbreviations: ACP = acyl carrier protein; CaMV = cauliflower mosaic virus; control = Brassica napus cultivar 212/86; event 8 = pCGN3831-212/86-8; event 11 = pCGN3831-212/86-11; FAS = fatty acid synthase; IL = isocitrate lyase; KAS = 13-keto-acyl ACP synthase; MS = malate synthase; OTE = oleoyl-ACP thioesterase; TAG = triacylglycerol; UcFatB1 = California bay medium-chain acyl-ACP thioesterase
l h e seed oil of Cuphea palusfris has an unusual fatty-acyl composition, whereby the principal fatty-acyl groups, myristate (64%) and caprylate (20%), differ by more than two methylenes. We have isolated two thioesterase (TE) cDNAs from C. palustris, encoding proteins designated Cp FatBl and Cp FatB2, which, when expressed in Escherichia coli, have TE activities specific for 8:0/10:0-and 14:0/16:0-acyl carrier protein substrates, respectively. The specific activities of the recombinant affinity-purified enzymes indicate that Cp FatB2 is kinetically superior to Cp FatB1. This result is consistent with the predominance of 14:O in the seed oil, despite apparently equal mRNA abundance of the two transcripts in the seed. In C. palustris the expression of both sequences is confined to the seed tissues. Based on these findings we propose that these two enzymes are major factors determining the bimodal chain-length composition of C. palustris oil. Analysis of the immature and mature seed oil by reverse-phase high-performance liquid chromatography confirmed that the principal triglycerides contain both 8:O and 14:o. This result indicates that both fatty acids are synthesized at the same time and in the same cells at all developmental stages during oil deposition, suggesting that the two TEs act together in the same fatty acid synthesis system.The end products of plant fatty acid synthetase activities are usually 16-and 18-carbon fatty acids (Harwood, 1988). There are, however, severa1 plant families that store large amounts of 8-to 14-carbon (medium-chain) fatty acids in their oilseed. Several mechanisms have been proposed for the synthesis of medium-chain fatty acids in plants. A specific acyl-ACP TE might terminate fatty acid synthesis by hydrolyzing the thioester bond of a particular acyl-ACP, resulting in the release of both ACP and free fatty acid (Stumpf, 1987). Alternatively, a specific 3-ketoacyl-ACP synthase (condensing enzyme) or an acyl-ACP acyltransferase might be involved (Harwood, 1988). At the time these mechanisms were proposed, however, there were limited experimental data to support these hypotheses. More recently, studies with Umbellularia californica (California bay), a plant that produces seed oil rich in lauric acid, have demonstrated the existence of a medium-chain-specific isozyme of acyl-ACP TE in the seed plastids (Pollard et al., 1991). These authors were able to separate the activity of a long-chain TE from a mediumchain TE, suggesting that there is medium-chain TE in-
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