Glucosinolate and Amino Acid Biosynthesis in Arabidopsis

Abstract

Enzymes that catalyze the condensation of acetyl coenzyme A and 2-oxo acids are likely to be important in two distinct metabolic pathways in Arabidopsis. These are the synthesis of isopropylmalate, an intermediate of Leu biosynthesis in primary metabolism, and the synthesis of methylthioalkylmalates, intermediates of Met elongation in the synthesis of aliphatic glucosinolates (GSLs), in secondary metabolism. Four Arabidopsis genes in the ecotype Columbia potentially encode proteins that could catalyze these reactions. MAM1 and MAML are adjacent genes on chromosome 5 at the Gsl-elong locus, while MAML-3 and MAML-4 are at opposite ends of chr 1. The isopropylmalate synthase activity of each member of the MAM-like gene family was investigated by heterologous expression in an isopropylmalate synthase-null Escherichia coli mutant. Only the expression of MAML-3 restored the ability of the mutant to grow in the absence of Leu. A MAML knockout line (KO) lacked long-chain aliphatic GSLs, which were restored when the KO was transformed with a functional MAML gene. Variation in expression of MAML did not alter the total levels of Met-derived GSLs, but just the ratio of chain lengths. MAML overexpression in Columbia led to an increase in long-chain GSLs, and an increase in 3C GSLs. Moreover, plants overexpressing MAML contained at least two novel amino acids. One of these was positively identified via MS/MS as homo-Leu, while the other, with identical mass and fragmentation patterns, was likely to be homo-Ile. A MAML-4 KO did not exhibit any changes in GSL profile, but had perturbed soluble amino acid content.

A robust omics-based approach for the identification of glucosinolate biosynthetic genes

“…The reactions involved in Met side-chain elongation are similar to those involved in Leu biosynthesis; moreover, the enzymes involved in Met side-chain elongation and Leu biosynthesis are presumably encoded by homologous genes belonging to the same gene families. In fact, MAM genes and IPMS (isopropylmalate synthase) genes, which are responsible for Met side-chain elongation and Leu synthesis, respectively, share sequence similarity with each other and with bacterial IPMS (de Kraker et al 2007;Field et al 2004;Kroymann et al 2001). All of the above-mentioned candidate genes are under the transcriptional regulation involving Myb28 (Hirai et al 2007).…”

“…ATTED-II analysis using a whole dataset showed weak correlation between ESM1 and ESP (Epithiospecifier protein, At1g54040) (data not shown). MAM genes that encode one of the Met side-chain elongation enzymes were also identified and characterized on the basis of the QTL analysis (Field et al 2004;Textor et al 2004;Kroymann et al 2001Kroymann et al , 2003. To my knowledge, however, some other genes that are involved in Met side-chain elongation process, namely, MAM-I (coding for methylthioalkylmalate isomerase) and MAM-D (coding for methylthioalkylmalate dehydrogenase) have not been identified by QTL analysis, presumably because natural variation of these genes does not result in metabolic natural variation.…”

Mathematical modelling of aliphatic glucosinolate chain length distribution in Arabidopsis thaliana leaves

“…Moreover, it should be noted that scaling all reaction rates in a metabolic network by the same factor leaves all steadystate metabolite concentrations unchanged (cf. Heinrich and Schuster 1996;Fell 1997). Instead of V max values for MAM3, we started from the values that are usually measured in experiment-the turnover numbers normalized by the molar mass of the enzyme, that is, the maximal velocity normalized by mass concentration of the enzyme (Table III in Textor et al 2007).…”

“…Despite knowledge of the substrate kinetics of the chain elongation cycle enzymes MAM1 and MAM3, mutations in the corresponding genes lead to glucosinolate profiles differing from those of wild-type plants that are not fully explainable: MAM3 missense or knockout mutants show almost no change in C 3 glucosinolates and only a slight increase in C 4 , but have a complete lack or much reduced level of longchain glucosinolates C 6 -C 8 , which can be restored by transgenic expression of MAM3 (Field et al 2004;Textor et al 2007). MAM1 mutants also show an interesting glucosinolate profile: whereas C 4 glucosinolates are dominant in wild-type plants, in MAM1 missense or knockout mutants C 3 glucosinolates are more abundant (Haughn et al 1991;Kroymann et al 2001;Textor et al 2007).…”

“…Heinrich and Schuster 1996;Fell 1997;Fong and Palsson 2004). It is a trademark of modern systems biology (cf.…”