The first committed step in the conversion of cycloartenol into ⌬ 5 C24-alkyl sterols in plants is catalyzed by an S-adenosylmethionine-dependent sterol-C24-methyltransferase type 1 (SMT1). We report the consequences of overexpressing SMT1 in tobacco (Nicotiana tabacum), under control of either the constitutive carnation etched ring virus promoter or the seed-specific Brassica napus acyl-carrier protein promoter, on sterol biosynthesis in seed tissue. Overexpression of SMT1 with either promoter increased the amount of total sterols in seed tissue by up to 44%. The sterol composition was also perturbed with levels of sitosterol increased by up to 50% and levels of isofucosterol and campesterol increased by up to 80%, whereas levels of cycloartenol and cholesterol were decreased by up to 53% and 34%, respectively. Concomitant with the enhanced SMT1 activity was an increase in endogenous 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, from which one can speculate that reduced levels of cycloartenol feed back to up-regulate 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and thereby control the carbon flux into sterol biosynthesis. This potential regulatory role of SMT1 in seed sterol biosynthesis is discussed.

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Acyl-carrier protein (ACP) is a key component involved in the regulation of fatty acid biosynthesis in plants. cDNA clones encoding ACP from Brassica napus (oil seed rape) embryos have been isolated using oligonucleotide probes derived from heterologous ACPs. Analysis of the DNA sequence data, in conjunction with N-terminal amino acid sequence data, revealed ACP to be synthesized from nuclear DNA as a precursor containing a 5 1 -amino-acid N-terminal extension.Immunocytochemical studies showed ACP to be localised solely within the plastids of B. nupus seed tissue and it would therefore appear that the N-terminal extension functions as a transit peptide to direct ACP into these organelles. Analysis of several cDNA clones revealed sequence heterogeneity and thus evidence for an ACP multigene family. From ten cDNA clones, six unique genes, encoding five different mature ACP polypeptides, were identified. Northern blot hybridisation studies provide evidence that the seed and leaf forms of rape ACP are encoded by structurally distinct gene sets.De novo synthesis of fatty acids is catalysed by fatty acid synthetase which consists of seven or eight catalytic domains. In animals [l] and yeast [2] the domains are present on one or two multifunctional polypeptide chains (type I fatty acid synthetase), which are localised within the cytoplasm. In contrast, in plants [3] the fatty acid synthetase domains exist as discreet, monofunctional activities (type 11) which are organellar in location.Some insight into the genetic regulation of type I1 fatty acid synthetase systems has recently been obtained through cloning of genes from both yeast [4] and mammals [5]. Our interest lies in understanding the genetic control of fatty acid biosynthesis in plants, in particular within developing oil seeds. As a first step towards that objective we report here the molecular cloning of cDNA encoding seed-expressed acylcarrier protein (ACP) from Brassica napus (oil seed rape).ACP is a key component of the plant Fatty acid biosynthetic machinery, serving both as a component of fatty acid synthetase and also as an acyl donor in desaturation and acyltransfer reactions [6]. Recent studies have shown two major ACP isoforms to be expressed in leaf tissue [7, 81, but apparently only one major isoform in seeds [8]. To date, characterisation of plant ACP has been largely confined to the leafexpressed forms. Thus, spinach [S] and barley [7] isoforms have been purified and N-terminal analysis suggests that, in both species, the isoforms are products of distinct genes. Using ACP as a representative marker protein, the site of fatty acid biosynthesis in leaves has been identified as the chloroplast [9]. In developing soybean seeds, ACP levels increase in close correlation with storage lipid synthesis [lo] suggestive of a regulatory role for ACP in this process. Despite this important role, ACP has not previously been localised within, or purified from, a seed source.This paper provides the first insight into the origin, structure and expression of gene...

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