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, ...