Expression of a Brassica Isopropylmalate Synthase Gene in Arabidopsis Perturbs Both Glucosinolate and Amino Acid Metabolism

Abstract

Isopropylmalate synthase (IPMS) is a key enzyme in the biosynthesis of the essential amino acid leucine, and thus primary metabolism. In Arabidopsis, the functionally similar enzyme, methythiolalkylmalate synthase (MAM), is an important enzyme in the elongation of methionine prior to glucosinolate (GSL) biosynthesis, as part of secondary metabolism. We describe the cloning of an IPMS gene from Brassica, BatIMS, and its functional characterisation by heterologous expression in E. coli and Arabidopsis. Over expression of BatIMS in Arabidopsis resulted in plants with an aberrant phenotype, reminiscent of mutants in GSL biosynthesis. Metabolite analyses showed that these plants had both perturbed amino acid metabolism and enhanced levels of GSLs. Microarray profiling showed that BatIMS over expression caused up regulation of the genes for methionine-derived GSL biosynthesis, and down regulation of genes involved in leucine catabolism, in addition to perturbed expression of genes involved in auxin and ethylene metabolism. The results illustrate the cross talk that can occur between primary and secondary metabolism within transgenic plants.

From Amino Acid to Glucosinolate Biosynthesis: Protein Sequence Changes in the Evolution of Methylthioalkylmalate Synthase in Arabidopsis

“…After the loss of the regulatory domain, a strong selection against any remaining IPMS activity could be expected because of the negative effects of unregulated Leu formation. Overexpression of a Brassica IPMS gene in Arabidopsis resulted in aberrant morphology and perturbed amino acid metabolism (Field et al, 2006).…”

Transcriptomic and Reverse Genetic Analysesof Branched-Chain Fatty Acid and Acyl Sugar Production inSolanum pennelliiandNicotiana benthamiana

“…Direct evidence for the existence of novel extended BCAAs in Solanaceae trichomes has not yet been provided. Nevertheless, it is noteworthy that extended Leu-based amino acids have been detected in leaves of Arabidopsis overexpressing MAM1, one of the IPMS genes for glucosinolate production, without any apparent detrimental effect on the plant (Field et al, 2006). How and where the acyl-CoAs produced by the BCKD complex become substrates for acyl sugar formation in N. benthamiana also remain to be established.…”

“…Analogous to glucosinolate biosynthesis in Arabidopsis (Arabidopsis thaliana), this entails extended cycling through isopropyl malate synthase (IPMS), isopropyl malate dehydrogenase (IPMD), and isopropylmalate dehydratase (IPDS) to yield medium branched-chain keto acids. In primary metabolism, these steps are associated with Leu synthesis, but here choice of substrate is evidently wider (Kroumova and Wagner, 2003;Field et al, 2006). Interestingly, iso-and anteiso-branched chains are also significant components of wax alkanes, epicuticular esterified alcohols, and fatty acids in tobacco species, but according to isotope-labeling studies, the latter are generated by FAS-mediated elongation of branched-chain precursors rather than a-KAE (Kaneda, 1967;Kolattukudy, 1968;Kroumova and Wagner, 1999).…”

Two Arabidopsis Genes (IPMS1 and IPMS2) Encode Isopropylmalate Synthase, the Branchpoint Step in the Biosynthesis of Leucine

“…Conversely, purified MAM3 can catalyze the reaction with 2-oxoisovalerate (S. Textor, unpublished data), even though both of these reactions occur at such low rates that they probably do not play a major role in planta. In this respect, it is remarkable that ectopic overexpression of a presumptive IPMS from B. atlantica (BatIMS) in Arabidopsis caused a doubling of aliphatic glucosinolates in the leaves of the T 1 generation in addition to perturbed amino acid levels (Field et al, 2006). However, these changes seem unlikely to be the result of an overlap in substrate specificity between IPMS and MAM, since this transformant displayed extensive morphological, metabolic, and transcriptional changes, including the up-regulation of at least four of the 10 known genes of the aliphatic glucosinolate biosynthetic pathway.…”

“…Just recently, a MAM sequence (GenBank accession no. DQ143886) was reported from Brassica atlantica that shares a high amino acid identity (90% and 86%) with the predicted IPMS homologs of Arabidopsis and is able to restore the growth of an IPMS-null Escherichia coli mutant in the absence of Leu (Field et al, 2006), and thus was called an IPMS (BatIMS). Such a rescue of an IPMS-null E. coli mutant was also described for At1g74040 and furthermore for both MAM-encoding genes, MAM1 and MAM3 (Junk and Mourad, 2002;S.…”