Knockout of the regulatory site of 3-ketoacyl-ACP synthase III enhances short- and medium-chain acyl-ACP synthesis

3-ketoacyl-acyl carrier protein synthase (KAS) III catalyses the first condensing step of the fatty acid synthase (FAS) type II reaction in plants and bacteria, using acetyl CoA and malonyl-acyl carrier protein (ACP) as substrates. Enzymatic characterization of recombinant KAS III from Cuphea wrightii embryo shows that this enzyme is strongly inhibited by medium-chain acyl-ACP end products of the FAS reaction, i.e. inhibition by lauroyl-ACP was uncompetitive towards acetyl CoA and non-competitive with regard to malonyl-ACP. This indicated a distinct attachment site for regulatory acyl-ACPs. Based on alignment of primary structures of various KAS IIIs and 3-ketoacyl CoA synthases, we suspected the motif G290NTSAAS296 to be responsible for binding of regulatory acyl-ACPs. Deletion of the tetrapeptide G290NTS293 led to a change of secondary structure and complete loss of KAS III condensing activity. Exchange of asparagine291 to aspartate, alanine294 to serine and alanine295 to proline, however, produced mutant enzymes with slightly reduced condensing activity, yet with insensitivity towards acyl-ACPs. To assess the potential of unregulated KAS III as tool in oil production, we designed in vitro experiments employing FAS preparations from medium-chain fatty acid-producing Cuphea lanceolata seeds and long-chain fatty acid-producing rape seeds, each supplemented with a fivefold excess of the N291D KAS III mutant. High amounts of short-chain acyl-ACPs in the case of C. lanceolata, and of medium-chain acyl-ACPs in the case of rape seed preparations, were obtained. This approach targets regulation and offers new possibilities to derive transgenic or non-transgenic plants for production of seed oils with new qualities.     

The role of β-ketoacyl-acyl carrier protein synthase III in the condensation steps of fatty acid biosynthesis in sunflower

The β-ketoacyl-acyl carrier protein synthase III (KAS III; EC 2.3.1.180) is a condensing enzyme catalyzing the initial step of fatty acid biosynthesis using acetyl-CoA as primer. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus L.) developing seeds, a cDNA coding for HaKAS III (EF514400) was isolated, cloned and sequenced. Its protein sequence is as much as 72% identical to other KAS III-like ones such as those from Perilla frutescens, Jatropha curcas, Ricinus communis or Cuphea hookeriana. Phylogenetic study of the HaKAS III homologous proteins infers its origin from cyanobacterial ancestors. A genomic DNA gel blot analysis revealed that HaKAS III is a single copy gene. Expression levels of this gene, examined by Q-PCR, revealed higher levels in developing seeds storing oil than in leaves, stems, roots or seedling cotyledons. Heterologous expression of HaKAS III in Escherichia coli altered their fatty acid content and composition implying an interaction of HaKAS III with the bacterial FAS complex. Testing purified HaKAS III recombinant protein by adding to a reconstituted E. coli FAS system lacking condensation activity revealed a novel substrate specificity. In contrast to all hitherto characterized plant KAS IIIs, the activities of which are limited to the first cycles of intraplastidial fatty acid biosynthesis yielding C6 chains, HaKAS III participates in at least four cycles resulting in C10 chains.     

KAS IV: a 3-ketoacyl-ACP synthase from Cuphea sp . is a medium chain specific condensing enzyme

cDNA clones encoding a novel 3-ketoacyl-ACP synthase (KAS) have been isolated from Cuphea . The amino acid sequence of this enzyme is different from the previously characterized classes of KASs, designated KAS I and III, and similar to those designated as KAS II. To define the acyl chain specificity of this enzyme, we generated transgenic Brassica plants over-expressing the cDNA encoded protein in a seed specific manner. Expression of this enzyme in transgenic Brassica seeds which normally do not produce medium chain fatty acids does not result in any detectable modification of the fatty acid profile. However, co-expression of the Cuphea KAS with medium chain specific thioesterases, capable of production of either 12:0 or 8:0/10:0 fatty acids in seed oil, strongly enhances the levels of these medium chain fatty acids as compared with seed oil of plants expressing the thioesterases alone. By contrast, co-expression of the Cuphea KAS along with an 18:0/18:1-ACP thioesterase does not result in any detectable modification of the fatty acids. These data indicate that the Cuphea KAS reported here has a different acyl-chain specificity to the previously characterized KAS I, II and III. Therefore, we designate this enzyme KAS IV, a medium chain specific condensing enzyme.     

β-Ketoacyl-acyl carrier protein synthase III from pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.): properties,inhibition by a novel thiolactomycin analogue and isolation of a cDNA clone encoding the enzyme

A beta-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III; short-chain condensing enzyme) has been partly purified from pea leaves. The enzyme, which had acetyl-CoA:ACP acyltransferase (ACAT) activity, was resolved from a second, specific, ACAT protein. The KAS III enzyme had a derived molecular mass of 42 kDa (from its cDNA sequence) and operated as a dimer. Its enzymological characteristics were similar to those of two other plant KAS III enzymes except for its inhibition by thiolactomycin. A derivative of thiolactomycin containing a longer (C8 saturated) hydrophobic side-chain (compound 332) was a more effective inhibitor of pea KAS III and showed competitive inhibition towards malonyl-ACP whereas thiolactomycin showed uncompetitive characteristics at high concentrations. This difference may be due to the better fit of compound 332 into a hydrophobic pocket at the active site. A full-length cDNA for the pea KAS III was isolated. This was expressed in Escherichia coli as a fusion protein with glutathione S-transferase in order to facilitate subsequent purification. Demonstrated activity in preparations from E. coli confirmed that the cDNA encoded a KAS III enzyme. Furthermore, the expressed KAS III had ACAT activity, showing that the latter was inherent. The derived amino acid sequence of the pea cDNA showed 81-87% similarity to that for other plant dicotyledon KAS IIIs, somewhat less for Allium porrum (leek, 71%) and for Porphyra spp. (62%), Synechocystis spp. (65%) and various bacteria (42-65%). The pea KAS III exhibited four areas of homology, three of which were around the active-site Cys(123), His(323) and Asn(353). In addition, a stretch of 23 amino acids (residues 207-229 in the pea KAS III) was almost completely conserved in the plant KAS IIIs. Modelling this stretch showed they belonged to a peptide fragment that fitted over the active site and contained segments suggested to be involved in substrate binding and in conformational changes during catalysis, as well as an arginine suggested to participate in the acid-base catalytic mechanism.     

Modification of Brassica napus seed oil by expression of the Escherichia coli fabH gene,encoding 3-ketoacyl-acyl carrier protein synthase III

The Escherichia coli fabH gene encoding 3-ketoacyl-acyl carrier protein synthase III (KAS III) was isolated and the effect of overproduction of bacterial KAS III was compared in both E. coli and Brassica napus. The change in fatty acid profile of E. coli was essentially the same as that reported by Tsay et al. (J Biol Chem 267 (1992) 6807–6814), namely higher C14:0 and lower C18:1 levels. In our study, however, an arrest of cell growth was also observed. This and other evidence suggests that in E. coli the accumulation of C14:0 may not be a direct effect of the KAS III overexpression, but a general metabolic consequence of the arrest of cell division. Bacterial KAS III was expressed in a seed- and developmentally specific manner in B. napus in either cytoplasm or plastid. Significant increases in KAS III activities were observed in both these transformation groups, up to 3.7 times the endogenous KAS III activity in mature seeds. Only the expression of the plastid-targeted KAS III gene, however, affected the fatty acid profile of the storage lipids, such that decreased amounts of C18:1 and increased amounts of C18:2 and C18:3 were observed as compared to control plants. Such changes in fatty acid composition reflect changes in the regulation and control of fatty acid biosynthesis. We propose that fatty acid biosynthesis is not controlled by one rate-limiting enzyme, such as acetyl-CoA carboxylase, but rather is shared by a number of component enzymes of the fatty acid biosynthetic machinery.     

Production of high levels of 8:0 and 10:0 fatty acids in transgenic canola by overexpression of Ch FatB2, a thioesterase cDNA from Cuphea hookeriana

The Mexican shrub Cuphea hookeriana accumulates up to 75% caprylate (8:0) and caprate (10:0) in its seed oil. An acyl-ACP thioesterase cDNA from C. hookeriana , designated Ch FatB2 , has been identified, which, when expressed in Escherichia coli , provides thioesterase activity specific for 8:0- and 10:0-ACP substrates. Expression of this clone in seeds of transgenic canola, an oilseed crop that normally does not accumulate any 8:0 and 10:0, resulted in a dramatic increase in the levels of these two fatty acids accompanied by a preferential decrease in the levels of linoleate (18:2) and linolenate (18:3). The Ch FatB2 differs from Ch FatB1 , another Cuphea hookeriana thioesterase reported recently, in both substrate specificity and expression pattern. The Ch FatB1 has a broad substrate specificity with strong preference for 16:0-ACP and is expressed throughout the plant; whereas Ch FatB2 is specific for 8:0/10:0-ACP and its expression is confined to the seed. It is proposed that the amplified expression of Ch FatB2 in the embryo provides the hydrolytic enzyme specificity determining the fatty acyl composition of Cuphea hookeriana seed oil.     

Purification of a jojoba embryo wax synthase, cloning of its cDNA, and production of high levels of wax in seeds of transgenic arabidopsis

Wax synthase (WS, fatty acyl-coenzyme A [coA]: fatty alcohol acyltransferase) catalyzes the final step in the synthesis of linear esters (waxes) that accumulate in seeds of jojoba (Simmondsia chinensis). We have characterized and partially purified this enzyme from developing jojoba embryos. A protein whose presence correlated with WS activity during chromatographic fractionation was identified and a cDNA encoding that protein was cloned. Seed-specific expression of the cDNA in transgenic Arabidopsis conferred high levels of WS activity on developing embryos from those plants. The WS sequence has significant homology with several Arabidopsis open reading frames of unknown function. Wax production in jojoba requires, in addition to WS, a fatty acyl-CoA reductase (FAR) and an efficient fatty acid elongase system that forms the substrates preferred by the FAR. We have expressed the jojoba WS cDNA in Arabidopsis in combination with cDNAs encoding the jojoba FAR and a beta-ketoacyl-CoA synthase (a component of fatty acid elongase) from Lunaria annua. (13)C-Nuclear magnetic resonance analysis of pooled whole seeds from transgenic plants indicated that as many as 49% of the oil molecules in the seeds were waxes. Gas chromatography analysis of transmethylated oil from individual seeds suggested that wax levels may represent up to 70% (by weight) of the oil present in those seeds.     

Beta-ketoacyl-acyl carrier protein synthase III (FabH) is essential for bacterial fatty acid synthesis

beta-Ketoacyl-acyl carrier protein (ACP) synthase III (KAS III, also called acetoacetyl-ACP synthase) encoded by the fabH gene is thought to catalyze the first elongation reaction (Claisen condensation) of type II fatty acid synthesis in bacteria and plant plastids. However, direct in vivo evidence that KAS III catalyzes an essential reaction is lacking, because no mutant organism deficient in this activity has been isolated. We report the first bacterial strain lacking KAS III, a fabH mutant constructed in the Gram-positive bacterium Lactococcus lactis subspecies lactis IL1403. The mutant strain carries an in-frame deletion of the KAS III active site region and was isolated by gene replacement using a medium supplemented with a source of saturated and unsaturated long-chain fatty acids. The mutant strain is devoid of KAS III activity and fails to grow in the absence of supplementation with exogenous long-chain fatty acids demonstrating that KAS III plays an essential role in cellular metabolism. However, the L. lactis fabH deletion mutant requires only long-chain unsaturated fatty acids for growth, a source of long-chain saturated fatty acids is not required. Because both saturated and unsaturated fatty acids are required for growth when fatty acid synthesis is blocked by biotin starvation (which prevents the synthesis of malonyl-CoA), another pathway for saturated fatty acid synthesis must remain in the fabH deletion strain. Indeed, incorporation of [1-14C]acetate into fatty acids in vivo showed that the fabH mutant retained about 10% of the fatty acid synthetic ability of the wild-type strain and that this residual synthetic capacity was preferentially diverted to the saturated branch of the pathway. Moreover, mass spectrometry showed that the fabH mutant retained low levels of palmitic acid upon fatty acid starvation. Derivatives of the fabH deletion mutant strain were isolated that were octanoic acid auxotrophs consistent with biochemical studies indicating that the major role of FabH is production of short-chain fatty acid primers. We also confirmed the essentiality of FabH in Escherichia coli by use of a plasmid-based gene insertion/deletion system. Together these results provide the first genetic evidence demonstrating that FabH conducts the major condensation reaction in the initiation of type II fatty acid biosynthesis in both Gram-positive and Gram-negative bacteria.     

Development of ultra-high erucic acid oil in the industrial oil crop Crambe abyssinica

Erucic acid (22 : 1) is a major feedstock for the oleochemical industry. In this study, a gene stacking strategy was employed to develop transgenic Crambe abyssinica lines with increased 22 : 1 levels. Through integration of the LdLPAAT, BnFAE1 and CaFAD2-RNAi genes into the crambe genome, confirmed by Southern blot and qRT-PCR, the average levels of 18 : 1, 18 : 2 and 18 : 3 were markedly decreased and that of 22 : 1 was increased from 60% in the wild type to 73% in the best transgenic line of T4 generation. In single seeds of the same line, the 22 : 1 level could reach 76.9%, an increase of 28.0% over the wild type. The trierucin amount was positively correlated to 22 : 1 in the transgenic lines. Unlike high erucic rapeseed, the wild-type crambe contains 22 : 1 in the seed phosphatidylcholine and in the sn-2 position of triacylglycerols (5% and 8%, respectively). The transgenic line with high 22 : 1 had decreased 22 : 1 level in phosphatidylcholine, and this was negatively correlated with the 22 : 1 level at the sn-2 position of TAG. The significances of this study include (i) achieving an unprecedented level of 22 : 1 in an oil crop; (ii) disclosing mechanisms in the channelling of a triacylglycerol-specific unusual fatty acid in oil seeds; (iii) indicating potential limiting factors involved in the erucic acid biosynthesis and paving the way for further increase of this acid and (iv) development of an added value genetically modified oil crop having no risk of gene flow into feed and food crops.     

Effects of genomic position and copy number of Acyl-ACP thioesterase transgenes on the level of the target fatty acids in Brassica napus L.

The effects of genomic position and copy number of acyl-acyl carrier protein (ACP) thioesterase (TE) transgenes on the major target fatty acid, either lauric acid (C12:0) or palmitic acid (C16:0) depending on the TE, in transgenic Brassica napus seed oil were investigated. Four transgenic parental lines, transformed individually with the bay-TE (Uc FatB1), elm-TE (Ua FatB1), nutmeg-TE (Mf FatB1) and Cuphea-TE (Ch FatB1) transgenes, were crossed with the non-transgenic recipient genotypes '212/86' or 'QO4'. Bay-TE and Cuphea-TE F₁ seeds, which carry half the number of the construct copies compared to the self-pollinated seeds of the transgenic parents, showed significantly lower levels of the target fatty acid. Doubled haploid (DH) lines were developed through microspore culture from F₁ hybrids with the elm-TE or the Cuphea-TE transgenes. DH lines carrying one to five copies of the Cuphea-TE transgene displayed a positive correlation between transgene copy number and the target fatty acid C16:0 level (r = 0.77**). DH lines with elm-TE transgene copies at four different loci showed different C16:0 levels, with one of the loci (E-II) leading to significantly higher C16:0 levels. This study supports the importance of the selection of high transgene copy number and/or the optimum genomic integration site in order to achieve maximum expression levels of the target fatty acid in transgenic oil quality modification.     

Novel E. coli β-ketoacyl-acyl carrier protein synthase III inhibitors as targeted antibiotics

β-Ketoacyl-acyl carrier protein synthase (KAS) III is a condensing enzyme that initiates fatty acid biosynthesis in most bacteria. We determined three pharmacophore maps from receptor-oriented pharmacophore-based in silico screening of the X-ray structure of Escherichia coli KAS III (ecKAS III) and choose 16 compounds as candidate ecKAS III inhibitors. Binding inhibitors were characterized using saturation-transfer difference NMR spectroscopy (STD-NMR), and binding constants were determined with fluorescence quenching experiments. Based on the results, we propose that the antimicrobial compound, 4-cyclohexyliminomethyl-benzene-1,3-diol (YKAs3003), is a potent inhibitor of pathogenic KAS III, displaying minimal inhibitory concentration (MIC) values in the range 128–256 μg/mL against various bacteria.     

Molecular aspects of beta-ketoacyl synthase (KAS) catalysis

Crystal structure data for Escherichia coli beta-ketoacyl synthase (KAS) I with C(10) and C(12) fatty acid substrates bound in conjunction with results from mutagenizing residues in the active site leads to a model for catalysis. Differences from and similarities to the other Claisen enzymes carrying out decarboxylations reveal two catalytic mechanisms, one for KAS I and KAS II, the other for KAS III and chalcone synthase. A comparison of the structures of KAS I and KAS II does not reveal the basis of chain-length specificity. The structures of the Arabidopsis thaliana KAS family are compared.     

Seed-specific overexpression of an endogenous Arabidopsis phytoene synthase gene results in delayed germination and increased levels of carotenoids,chlorophyll, and abscisic acid

Phytoene synthase catalyzes the dimerization of two molecules of geranylgeranyl pyrophosphate to phytoene and has been shown to be rate limiting for the synthesis of carotenoids. To elucidate if the capacity to produce phytoene is limiting also in the seed of Arabidopsis (Wassilewskija), a gene coding for an endogenous phytoene synthase was cloned and coupled to a seed-specific promoter, and the effects of the overexpression were examined. The resulting transgenic plants produced darker seeds, and extracts from the seed of five overexpressing plants had a 43-fold average increase of beta-carotene and a total average amount of beta-carotene of approximately 260 microg g-1 fresh weight. Lutein, violaxanthin, and chlorophyll were significantly increased, whereas the levels of zeaxanthin only increased by a factor 1.1. In addition, substantial levels of lycopene and alpha-carotene were produced in the seeds, whereas only trace amounts were found in the control plants. Seeds from the transgenic plants exhibited delayed germination, and the degree of delay was positively correlated with the increased levels of carotenoids. The abscisic acid levels followed the increase of the carotenoids, and plants having the highest carotenoid levels also had the highest abscisic acid content. Addition of gibberellic acid to the growth medium only partly restored germination of the transgenic seeds.     

3-Ketoacyl-acyl carrier protein synthase III from spinach (Spinacia oleracea) is not similar to other condensing enzymes of fatty acid synthase.

A cDNA clone encoding spinach (Spinacia oleracea) 3-ketoacyl-acyl carrier protein synthase III (KAS III), which catalyzes the initial condensing reaction in fatty acid biosynthesis, was isolated. Based on the amino acid sequence of tryptic digests of purified spinach KAS III, degenerate polymerase chain reaction (PCR) primers were designed and used to amplify a 612-bp fragment from first-strand cDNA of spinach leaf RNA. A root cDNA library was probed with the PCR fragment, and a 1920-bp clone was isolated. Its deduced amino acid sequence matched the sequences of the tryptic digests obtained from the purified KAS III. Northern analysis confirmed that it was expressed in both leaf and root. The clone contained a 1218-bp open reading frame coding for 405 amino acids. The identity of the clone was confirmed by expression in Escherichia coli BL 21 as a glutathione S-transferase fusion protein. The deduced amino acid sequence was 48 and 45% identical with the putative KAS III of Porphyra umbilicalis and KAS III of E. coli, respectively. It also had a strong local homology to the plant chalcone synthases but had little homology with other KAS isoforms from plants, bacteria, or animals.     

In-vitro evidence for feed-back regulation of β-ketoacyl-acyl carrier protein synthase III in medium-chain fatty acid biosynthesis

The cerulenin-insensitive -ketoacyl-acyl carrier protein (ACP) synthase III (KAS III, EC 2.3.1.41) catalyzes the first condensing step of the fatty-acid synthase (FAS) reaction in plants and bacteria, using directly acetyl-CoA as substrate for condensation with malonyl-ACP. In order to identify a possible site for regulation of the biosynthesis of medium-chain fatty acids, the influence of acyl-ACPs of different chain-lengths (C₄,C₆,C₈ and C₁₀) on the activity of KAS III was investigated in vitro using an FAS preparation from seeds of Cuphea lanceolata Ait. (a crop accumulating up to 90% decanoic acid into triacylglycerols) that had been treated with 100 M cerulenin. All acyl-ACPs investigated led to a decrease in the activity of KAS III towards acetyl-CoA, an effect apparently related to the length of the acyl chain. Analysis of the reaction products of the assay revealed that short-chain acyl-ACPs elongated to a very small extent simultaneously with acetyl-CoA. This extent of elongation did not correlate with the decrease in KAS III-activity levels. These data excluded the possibility of competition between acetyl-CoA and acyl-ACPs, but indicated that acyl-ACPs inhibited the enzyme. Decanoyl-ACP caused the highest decrease in enzyme activity (IC₅₀ = 0.45 M), thus being a potent inhibitor of KAS III. Michaelis-Menten kinetics revealed that the inhibition of KAS III by decanoyl-ACP was non-competitive in relation to malonyl-ACP and uncompetitive in relation to acetyl-CoA. Moreover, our data indicate that KAS III has a strict specificity for the elongation of acetyl-CoA. An inhibition of KAS III by acyl-ACPs was observed in experiments using FAS preparations from rape seeds and spinach leaves, but the inhibition of KAS III from C. lanceolata seeds by decanoyl-ACP was approximately 1.5-fold higher. The data provide evidence that acyl-ACPs are involved in the modulation of plant fatty-acid biosynthesis by a feed-back mechanism.Abbreviations ACP acyl carrier protein - DTT dithiothreitol - TCA trichloroacetic acid - ecACP acyl carrier protein from Escherichia coli - FAS fatty-acid synthase - IC₅₀ concentration causing 50% inhibition - KAS -ketoacyl-ACP synthase - NEM N-ethylmaleimide In honour of Professor Hartmut K. Lichtenthaler's sixtieth birthdayThis work was supported by a grant from the German Ministry of Research and Technology (BMFT) and in part by the Fonds der Chemischen Industrie and the Ministry of Science and Research of the State Northrhine-Westfalia. The authors wish to thank Prof. G. Röbbelen (University of Göttingen, Göttingen, Germany) for kindly providing the plant material. This paper is part of the doctoral thesis of Fritzi Maike Brück.     

Enhanced seed oil production in canola by conditional expression of Brassica napus LEAFY COTYLEDON1 and LEC1-LIKE in developing seeds

The seed oil content in oilseed crops is a major selection trait to breeders. In Arabidopsis (Arabidopsis thaliana), LEAFY COTYLEDON1 (LEC1) and LEC1-LIKE (L1L) are key regulators of fatty acid biosynthesis. Overexpression of AtLEC1 and its orthologs in canola (Brassica napus), BnLEC1 and BnL1L, causes an increased fatty acid level in transgenic Arabidopsis plants, which, however, also show severe developmental abnormalities. Here, we use truncated napin A promoters, which retain the seed-specific expression pattern but with a reduced expression level, to drive the expression of BnLEC1 and BnL1L in transgenic canola. Conditional expression of BnLEC1 and BnL1L increases the seed oil content by 2% to 20% and has no detrimental effects on major agronomic traits. In the transgenic canola, expression of a subset of genes involved in fatty acid biosynthesis and glycolysis is up-regulated in developing seeds. Moreover, the BnLEC1 transgene enhances the expression of several genes involved in Suc synthesis and transport in developing seeds and the silique wall. Consistently, the accumulation of Suc and Fru is increased in developing seeds of the transgenic rapeseed, suggesting the increased carbon flux to fatty acid biosynthesis. These results demonstrate that BnLEC1 and BnL1L are reliable targets for genetic improvement of rapeseed in seed oil production.     

A jojoba beta-Ketoacyl-CoA synthase cDNA complements the canola fatty acid elongation mutation in transgenic plants.

beta-Ketoacyl-coenzyme A (CoA) synthase (KCS) catalyzes the condensation of malonyl-CoA with long-chain acyl-CoA. This reaction is the initial step of the microsomal fatty acyl-CoA elongation pathway responsible for formation of very long chain fatty acids (VLCFAs, or fatty acids with chain lengths > 18 carbons). Manipulation of this pathway is significant for agriculture, because it is the basis of conversion of high erucic acid rapeseed into canola. High erucic acid rapeseed oil, used as an industrial feedstock, is rich in VLCFAs, whereas the edible oil extracted from canola is essentially devoid of VLCFAs. Here, we report the cloning of a cDNA from developing jojoba embryos involved in microsomal fatty acid elongation. The jojoba cDNA is homologous to the recently cloned Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene that has been suggested to encode KCS. We characterize the jojoba enzyme and present biochemical data indicating that the jojoba cDNA does indeed encode KCS. Transformation of low erucic acid rapeseed with the jojoba cDNA restored KCS activity to developing embryos and altered the transgenic seed oil composition to contain high levels of VLCFAs. The data reveal the key role KCS plays in determining the chain lengths of fatty acids found in seed oils.     

Overexpression of Arabidopsis thaliana farnesyl diphosphate synthase (FPS1S) in transgenic Arabidopsis induces a cell death/senescence-like response and reduced cytokinin levels

To investigate the contribution of farnesyl diphosphate synthase (FPS) to the overall control of the mevalonic acid pathway in plants, we have generated transgenic Arabidopsis thaliana overexpressing the Arabidopsis FPS1S isoform. Despite high levels of FPS activity in transgenic plants (8- to 12-fold as compared to wild-type plants), the content of sterols and the levels of 3-hydroxy-3-methylglutaryl-CoA reductase activity in leaves were similar to those in control plants. Plants overexpressing FPS1S showed a cell death/senescence-like phenotype and grew less vigorously than wild-type plants. The onset and the severity of these phenotypes directly correlated with the levels of FPS activity. In leaves of plants with increased FPS activity, the expression of the senescence activated gene SAG12 was prematurely induced. Transgenic plants grown in the presence of either mevalonic acid (MVA) or the cytokinin 2-isopentenyladenine (2-iP) recovered the wild-type phenotype. Quantification of endogenous cytokinins demonstrated that FPS1S overexpression specifically reduces the levels of endogenous zeatin-type cytokinins in leaves. Altogether these results support the notion that increasing FPS activity without a concomitant increase of MVA production leads to a reduction of IPP and DMAPP available for cytokinin biosynthesis. The reduced cytokinin levels would be, at least in part, responsible for the phenotypic alterations observed in the transgenic plants. The finding that wild-type and transgenic plants accumulated similar increased amounts of sterols when grown in the presence of exogenous MVA suggests that FPS1S is not limiting for sterol biosynthesis.