Glucosinolates are sulfur-rich plant secondary metabolites whose breakdown products have a wide range of biological activities in plant-herbivore and plant-pathogen interactions and anticarcinogenic properties. In Arabidopsis thaliana, hydrolysis by the enzyme, myrosinase, produces bioactive nitriles, epithionitriles, or isothiocyanates depending upon the plant's genotype and the glucosinolate's structure. A major determinant of this structural specificity is the epithiospecifier locus (ESP), whose protein causes the formation of epithionitriles and nitriles. A quantitative trait locus (QTL) on chromosome 3 epistatically affects nitrile formation in combination with ESP; this QTL has been termed EPITHIOSPECIFIER MODIFIER1 (ESM1). We identified a myrosinase-associated protein as the ESM1 QTL in Arabidopsis using map-based cloning with recombinant inbred lines, natural variation transcriptomic analysis, and metabolic profiling. In planta and in vitro analyses with natural ESM1 alleles, ESM1 knockouts, and overexpression lines show that ESM1 represses nitrile formation and favors isothiocyanate production. The glucosinolate hydrolysis profile change influenced by ESM1 is associated with the ability to deter herbivory by Trichoplusia ni. This gene could provide unique approaches toward improving human nutrition.
Glucosinolates (GSLs) are amino acid-derived secondary metabolites with diverse biological activities dependent on chemical modifications of the side chain. We previously identified the flavin-monooxygenase FMO GS-OX1 as an enzyme in the biosynthesis of aliphatic GSLs in Arabidopsis (Arabidopsis thaliana) that catalyzes the S-oxygenation of methylthioalkyl to methylsulfinylalkyl GSLs. Here, we report the fine mapping of a quantitative trait locus for the S-oxygenating activity in Arabidopsis. In this region, there are three FMOs that, together with FMO GS-OX1 and a fifth FMO, form what appears to be a crucifer-specific subclade. We report the identification of these four uncharacterized FMOs, designated FMO GS-OX2 to FMO GS-OX5 . Biochemical characterization of the recombinant protein combined with the analysis of GSL content in knockout mutants and overexpression lines show that FMO GS-OX2 , FMO GS-OX3 , and FMO GS-OX4 have broad substrate specificity and catalyze the conversion from methylthioalkyl GSL to the corresponding methylsulfinylalkyl GSL independent of chain length. In contrast, FMO GS-OX5 shows substrate specificity toward the long-chain 8-methylthiooctyl GSL. Identification of the FMO GS-OX subclade will generate better understanding of the evolution of biosynthetic activities and specificities in secondary metabolism and provides an important tool for breeding plants with improved cancer prevention characteristics as provided by the methylsulfinylalkyl GSL.Glucosinolates (GSLs) are amino acid-derived secondary metabolites present in the order Brassicales.
Glucosinolates are secondary metabolites found almost exclusively in the order Brassicales. They are synthesized from a variety of amino acids and can have numerous side chain modifications that control biological function. We investigated the biosynthesis of 2-hydroxybut-3-enyl glucosinolate, which has biological activities including toxicity to Caenorhabditis elegans, inhibition of seed germination, induction of goiter disease in mammals, and production of bitter flavors in Brassica vegetable crops. Arabidopsis (Arabidopsis thaliana) accessions contain three different patterns of 2-hydroxybut-3-enyl glucosinolate accumulation (present in leaves and seeds, seeds only, or absent) corresponding to three different alleles at a single locus, GSL-OH. Fine-scale mapping of the GSL-OH locus identified a 2-oxoacid-dependent dioxygenase encoded by At2g25450 required for the formation of both 2R-and 2S-2-hydroxybut-3-enyl glucosinolate from the precursor 3-butenyl glucosinolate precursor. Naturally occurring null mutations and T-DNA insertional mutations in At2g25450 exhibit a complete absence of 2-hydroxybut-3-enyl glucosinolate accumulation. Analysis of herbivory by the generalist lepidopteran Trichoplusia ni showed that production of 2-hydroxybut-3-enyl glucosinolate provides increased resistance. These results show that At2g25450 is necessary for the hydroxylation of but-3-enyl glucosinolate to 2-hydroxybut-3-enyl glucosinolate in planta and that this metabolite increases resistance to generalist herbivory.
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