Enzymatic studies of high stearic acid sunflower seed mutants Sara Cantisán, Enrique Martínez-Force, Rafael Garcés* Instituto de la Grasa, Consejo Superior de Investigaciones Científicas, Apartado 1078, 41080 Sevilla, Spain * Author to whom correspondence should be addressed (fax +34 954616790; e-mail rgarces@cica.es) (Received 24 November 1999; accepted 28 January 2000) Abstract – Three high stearic acid sunflower (Helianthus annuus L.) mutants, CAS-3, CAS-4 and CAS-8, accumulating 28, 15 and 14 % of stearic acid in the seed lipids have been biochemically characterised. In vivo conversion rate of palmitic acid into stearic acid is not altered in the mutants but the conversion rate of stearic acid into oleic acid shows a reduction that correlated with the total stearic acid content of seed lipid mutants. Two enzymatic activities are found to be involved in the mutant phenotype, the acyl-ACP thioesterase (EC 3.1.2.14) and the stearoyl-ACP desaturase (EC 1.12.99.6). Our data suggest that the high stearic phenotype is due to the combined effect of a reduced stearoyl-ACP desaturase activity and an acyl-ACP thioesterase with higher activity on stearoyl-ACP. The same thioesterase activity increment, found on stearoyl-ACP, was also found on palmitoyl-ACP, suggesting that the affected thioesterase activity could be a FatB type. © 2000 Éditions scientifiques et médicales Elsevier SAS Acyl-ACP thioesterase / fatty acid mutant / Helianthus (seed) / stearic acid / stearoyl-ACP desaturase ACP, acyl carrier protein / DAF, days after flowering / FAS, fatty acid synthetase / KAS II, -keto-acyl-ACP synthetase II / SAD, stearoyl-ACP desaturase / TE, acyl-ACP thioesterase 1. INTRODUCTION The primary product of fatty acid intraplastidial plant biosynthesis is palmitoyl-acyl carrier protein (palmitoyl-ACP), which is synthesised by a multien- zyme complex called fatty acid synthetase I (FAS I). Palmitoyl-ACP is elongated by two carbon atoms to produce stearoyl-ACP by the fatty acid synthetase II (FAS II), which differs from the FAS I only in the first enzyme of the complex, the -keto-acyl-ACP syn- thetase II (KAS II; EC 2.3.1.41). Later, the stearoyl- ACP can follow two pathways: (a) in most cases stearoyl-ACP is desaturated to oleoyl-ACP in a reac- tion catalysed by the stearoyl-ACP desaturase (SAD; EC 1.12.99.6), which introduces a double bond between carbon atoms 9 and 10; (b) occasionally, stearoyl-ACP is hydrolysed from the ACP by a second enzyme, an acyl-ACP thioesterase (TE; EC 3.1.2.14). The fatty acid is then exported outside the plastid and used for the biosynthesis of storage oils. Thus, the stearic acid content in cultivated oilseeds is mainly determined by the action of these two intraplastidial enzymes, stearoyl- ACP desaturase and acyl-ACP thioesterase. In culti- vated oilseeds, the stearic acid content ranges from 2 to 6 %. Because the SAD catalyses the first reaction of desaturation, it plays a key role in determining the ratio of total saturated to unsaturated fatty acids. TEs have been classified into two types according to their specificity: FatA and FatB [11]. Thioesterases of the FatA group hydrolyse oleoyl-ACP preferentially, while those of the FatB group show more specificity towards saturated acyl-ACPs. Previous works have shown that TEs are the key determinant of the chain length of fatty acid from de novo synthesis. When Arabidopsis thaliana was transformed with the lauroyl-ACP thioesterase from Umbellularia californica, a plant that accumulates 60 % of lauric acid, transgenic Ara- bidopsis accumulated high amounts of lauric acid in the oil [24]. However, more recent studies have demonstrated that TEs are not the only enzymes involved, but that KAS and SAD, which compete with TE for substrates, are also implicated in determining chain length and degree of desaturation of fatty acids [3, 23]. In order to avoid the use of animal fats and/or hydrogenation of vegetable oils, a healthier vegetable oil with increased saturated fatty acid content, prefer- ably stearic acid that has a neutral effect on serum Plant Physiol. Biochem., 2000, 38 (5), 377-382 / © 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S0981942800007580/FLA Plant Physiol. Biochem., 0981-9428/00/5/© 2000 E ´ ditions scientifiques et médicales Elsevier SAS. All rights reserved