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.

No abstract

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...

No abstract

Xyloglucan endotransglycosylase (XET) catalyses the breaking and re-joining of xyloglucan molecules. It may be involved in plant growth, functioning either to anchor newly synthesized xyloglucan polymers into the cell wall or to reversibly loosen the cellulose-xyloglucan network permitting turgor-driven cell expansion. Consistent with a role in growth, levels of XET activity have been shown to correlate with elongation in, for example, maize roots (Pritchard et al., 1993). Our group has recently isolated a cDNA clone from nasturtium (de Silva et al., 1993) encoding a seed enzyme with XET activity (Edwards et al., 1986; Fanutti et al., 1993). The biological function of XET is being investigated via in vivo manipulation. We have used the nasturtium sequence to isolate several cDNA clones from tomato whose encoded polypeptide sequences exhibit approximately 40% homology with nasturtium XET. Heterologous expression in E. coli has been used to confirm XET activity. An isoform of XET has been purified from tomato fruit and a polyclonal antibody raised. The antibody and gene-specific nucleic acid probes are being used to monitor developmental changes in XET protein and mRNA levels, respectively. Preliminary results suggest that tomato XET is encoded by a family of genes which show different developmental patterns of expression. Agrobacterium transformation of tomato has been carried out using the nasturtium cDNA (sense orientation) and a fruit-expressed tomato cDNA (antisense orientation) under the control of a constitutive promoter. The phenotypic consequences of altering levels of XET are being investigated.

The level of two thioesterases, acyl-CoA thioesterase and acyl-ACP thioesterase was determined during seed maturation in oil seed rape. Both thioesterase activities rose markedly prior to the onset of lipid accumulation, but the induction kinetics suggest that the activities reside on distinct polypeptides. Acyl-ACP thioesterase (EC 3.1.2.14) was purified 2000-fold using a combination of ion exchange, ACP-affinity chromatography, chromatofocusing and gel filtration. Using native gel electrophoresis, and assays for enzymic activity, two polypeptides were identified on SDS-PAGE as associated with the activity. Cleveland mapping of these polypeptides, of 38 kDa component and 33 kDa respectively, demonstrated that they are related. An antibody was prepared against the 38 kDa component, and this also recognises the 33 kDa polypeptide in highly purified preparations. Western blotting of a crude extract identifies one band at 38 kDa consistent with the 33 kDa component being a degradation product generated during purification. The native molecule has a M(r) of 70 kDa indicating a dimeric structure. The enzyme has a pH optimum of 9.5 and shows strong preference for oleoyl-ACP as substrate. The intact enzyme has an N-terminus blocked to protein sequencing. We also found that two other polypeptides co-purify with acyl-ACP thioesterase under native conditions. The N-terminal amino-acid sequence of these polypeptides is shown and their possible identity is discussed.

No abstract

No abstract