A Novel 2-Oxoacid-Dependent Dioxygenase Involved in the Formation of the Goiterogenic 2-Hydroxybut-3-enyl Glucosinolate and Generalist Insect Resistance in Arabidopsis    

Abstract

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.

Understanding the Evolution of Defense Metabolites in Arabidopsis thaliana Using Genome-wide Association Mapping

“…To visualize the relationships of these gene-to-trait associations and to test the breadth of phenotypic effects (i.e., whether detected associations control specific traits or suites of traits), we divided GSL traits into four biosynthetic groups on the basis of their biochemical and genetic regulation (Figure 1) (Kliebenstein et al 2002a;Gigolashvili et al 2007a,b;Hirai et al 2007;Sønderby et al 2007;Hansen et al 2008;Kliebenstein 2009). These groups were INDOLE, all indolic GSL traits; OHBUT, all 2-hydroxy-but-3-enyl traits; SC, all 3-and 4-carbon-long aliphatic GSL-related traits except the OHBUTs; and LC, all 5 carbon and longer aliphatic GSL-related traits.…”

“…with epistatic interactions that control insect resistance in A. thaliana (Lambrix et al 2001;Kliebenstein et al 2005a;Bidart-Bouzat and Kliebenstein 2008;Hansen et al 2008;Kliebenstein 2009). This suggests that discounting phenotype-genotype association of any SNP because it shows linkage with a causal polymorphism may not be biologically warranted, especially if the queried trait is subject to selection.…”

Seed-Specific Expression of a Feedback-Insensitive Form of CYSTATHIONINE-γ-SYNTHASE in Arabidopsis Stimulates Metabolic and Transcriptomic Responses Associated with Desiccation Stress

“…The first is a 2-oxoacid-dependent dioxygenase involved in the hydroxylation of glucosinolates as part of their secondary modifications, and the second is a b-THIOGLUCOSIDE GLUCOHYDROLASE2 (TGG; At5g25980), which degrades glucosinolates to produce toxins, such as isothiocyanate, that deter herbivores ( Fig. 7; Hansen et al, 2008). The expression levels of SAMDC (At3g02470) and ARG DECARBOXYLASE2 (At4g34710), two rate-limiting enzymes that catalyze the first steps of polyamine biosynthesis, were also increased (Table III; Fig.…”

Coselected genes determine adaptive variation in herbivore resistance throughout the native range of Arabidopsis thaliana

“…Our second strongest association, located on chromosome 5, was situated near the GS-ELONG locus, containing the genes MAM1 (AT5G23010, methylthioalkylmalate synthase 1) and MAM3 (AT5G23020, methylthioalkylmalate synthase-like) (hereafter "MAM region"). Next, a region of chromosome 2 contained four significantly associated SNPs located within 6 kbp (kb) of the GS-OH locus (AT2G25450), which is known to regulate hydroxylation of alkenyl GSLs (hereafter the "GS-OH" region) (19). Associations near GS-OH were detected when mapping the leaf concentrations of progoitrin (2-hydroxy-3-butenyl), consistent with previous studies (1,19), as well as napoleiferin (2-hydroxy-4-pentenyl).…”

“…Next, a region of chromosome 2 contained four significantly associated SNPs located within 6 kbp (kb) of the GS-OH locus (AT2G25450), which is known to regulate hydroxylation of alkenyl GSLs (hereafter the "GS-OH" region) (19). Associations near GS-OH were detected when mapping the leaf concentrations of progoitrin (2-hydroxy-3-butenyl), consistent with previous studies (1,19), as well as napoleiferin (2-hydroxy-4-pentenyl). Although other SNPs distributed throughout the genome were significantly associated with GSL variation, these were primarily individual markers with no obvious candidates located within 20 kbp.…”