Like many other plant defense compounds, glucosinolates are present constitutively in plant tissues, but are also induced to higher levels by herbivore attack. Of the major glucosinolate types, indolic glucosinolates are most frequently induced regardless of the type of herbivore involved. Over 90% of previous studies found that herbivore damage to glucosinolate-containing plants led to an increased accumulation of indolic glucosinolates at levels ranging up to 20-fold. Aliphatic and aromatic glucosinolates are also commonly induced by herbivores, though usually at much lower magnitudes than indolic glucosinolates, and aliphatic and aromatic glucosinolates may even undergo declines following herbivory. The glucosinolate defense system also requires another partner, the enzyme myrosinase, to hydrolyze the parent glucosinolates into biologically active derivatives. Much less is known about myrosinase induction after herbivory compared to glucosinolate induction, and no general trends are evident. However, it is clear that insect feeding stimulates the formation of various myrosinase associated proteins whose function is not yet understood. The biochemical mechanism of glucosinolate induction involves a jasmonate signaling cascade that leads eventually to increases in the transcript levels of glucosinolate biosynthetic genes. Several recently described transcription factors controlling glucosinolate biosynthesis are activated by herbivory or wounding. Herbivore induction of glucosinolates has sometimes been demonstrated to increase protection against subsequent herbivore attack, but more research is needed to evaluate the costs and benefits of this phenomenon.

In Arabidopsis thaliana and related plants, glucosinolates are a major component in the blend of secondary metabolites and contribute to resistance against herbivorous insects. Methylthioalkylmalate synthases (MAM) encoded at the MAM gene cluster control an early step in the biosynthesis of glucosinolates and, therefore, are central to the diversification of glucosinolate metabolism. We sequenced bacterial artificial chromosomes containing the MAM cluster from several Arabidopsis relatives, conducted enzyme assays with heterologously expressed MAM genes, and analyzed MAM nucleotide variation patterns. Our results show that gene duplication, neofunctionalization, and positive selection provide the mechanism for biochemical adaptation in plant defense. These processes occur repeatedly in the history of the MAM gene family, indicating their fundamental importance for the evolution of plant metabolic diversity both within and among species.biochemical neofunctionalization ͉ glucosinolate metabolism ͉ methylthioalkylmalate synthase ͉ plant-enemy interactions P lants synthesize an immense number of secondary compounds, so called because their significance for processes of basic growth and development is not immediately evident. More than 200,000 known secondary metabolites provide an increasingly exploited reservoir for the generation of pharmaceutically active agents (1), and many more await discovery. Classic hypotheses that seek to explain this vast metabolic diversity propose a stepwise and reciprocal process of adaptation and counteradaptation between plants and their natural enemies, molded by mutual selection (2). In Arabidopsis thaliana and other crucifers, glucosinolates are a major component in the mélange of secondary metabolites. More than 120 glucosinolates are known, which share a chemical core structure but differ in their amino acid-derived side chain. Glucosinolate composition and quantity varies among and within species (3-5). Upon tissue disruption, myrosinase-catalyzed hydrolysis of glucosinolates generates biologically active compounds, which play an important ecological role in plant defense against herbivorous insects (4, 6-9). However, insects can adapt to glucosinolate profiles or evade deleterious effects from glucosinolate hydrolysis by counteradaptation (9-12). In A. thaliana, a modular genetic system regulates diversity in glucosinolate profiles among accessions and may permit the rapid generation of new glucosinolate combinations in response to challenges imposed by the biotic environment (3). Several genetic loci (3, 9), each present in several alleles, interact epistatically and, together with modifying proteins (7), determine the blend of bioactive products that emerges during glucosinolate hydrolysis (9). One genetic locus central for glucosinolate diversity, the methylthioalkylmalate synthase gene (MAM) cluster, controls an early step in the biosynthesis of aliphatic glucosinolates, the predominant glucosinolate class in A. thaliana. This locus comprises a small family of MAM genes, ...

Chain elongated, methionine (Met)-derived glucosinolates are a major class of secondary metabolites in Arabidopsis (Arabidopsis thaliana). The key enzymatic step in determining the length of the chain is the condensation of acetyl-coenzyme A with a series of v-methylthio-2-oxoalkanoic acids, catalyzed by methylthioalkylmalate (MAM) synthases. The existence of two MAM synthases has been previously reported in the Arabidopsis ecotype Columbia: MAM1 and MAM3 (formerly known as MAM-L). Here, we describe the biochemical properties of the MAM3 enzyme, which is able to catalyze all six condensation reactions of Met chain elongation that occur in Arabidopsis. Underlining its broad substrate specificity, MAM3 also accepts a range of non-Met-derived 2-oxoacids, e.g. converting pyruvate to citramalate and 2-oxoisovalerate to isopropylmalate, a step in leucine biosynthesis. To investigate its role in vivo, we identified plant lines with mutations in MAM3 that resulted in a complete lack or greatly reduced levels of long-chain glucosinolates. This phenotype could be complemented by reintroduction of a MAM3 expression construct. Analysis of MAM3 mutants demonstrated that MAM3 catalyzes the formation of all glucosinolate chain lengths in vivo as well as in vitro, making this enzyme the major generator of glucosinolate chain length diversity in the plant. The localization of MAM3 in the chloroplast suggests that this organelle is the site of Met chain elongation.

Arabidopsis and other Brassicaceae produce an enormous diversity of aliphatic glucosinolates, a group of methionine (Met)-derived plant secondary compounds containing a-thio-glucose moiety, a sulfonated oxime, and a variable side chain. We fine-scale mapped GSL-ELONG, a locus controlling variation in the side-chain length of aliphatic glucosinolates. Within this locus, a polymorphic gene was identified that determines whether Met is extended predominantly by either one or by two methylene groups to produce aliphatic glucosinolates with either three-or four-carbon side chains. Two allelic mutants deficient in four-carbon side-chain glucosinolates were shown to contain independent missense mutations within this gene. In cell-free enzyme assays, a heterologously expressed cDNA from this locus was capable of condensing 2-oxo-4-methylthiobutanoic acid with acetyl-coenzyme A, the initial reaction in Met chain elongation. The gene methylthioalkylmalate synthase1 (MAM1) is a member of a gene family sharing approximately 60% amino acid sequence similarity with 2-isopropylmalate synthase, an enzyme of leucine biosynthesis that condenses 2-oxo-3-methylbutanoate with acetyl-coenzyme A.