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SummaryGlucosinolates are a class of secondary metabolites with important roles in plant defense and human nutrition. Here, we characterize a putative UDP-glucose:thiohydroximate S-glucosyltransferase, UGT74B1, to determine its role in the Arabidopsis glucosinolate pathway. Biochemical analyses demonstrate that recombinant UGT74B1 specifically glucosylates the thiohydroximate functional group. Low K m values for phenylacetothiohydroximic acid (approximately 6 lM) and UDP-glucose (approximately 50 lM) strongly suggest that thiohydroximates are in vivo substrates of UGT74B1. Insertional loss-of-function ugt74b1 mutants exhibit significantly decreased, but not abolished, glucosinolate accumulation. In addition, ugt74b1 mutants display phenotypes reminiscent of auxin overproduction, such as epinastic cotyledons, elongated hypocotyls in lightgrown plants, excess adventitious rooting and incomplete leaf vascularization. Indeed, during early plant development, mutant ugt74b1 seedlings accumulate nearly threefold more indole-3-acetic acid than the wild type. Other phenotypes, however, such as chlorosis along the leaf veins, are likely caused by thiohydroximate toxicity. Analysis of UGT74B1 promoter activity during plant development reveals expression patterns consistent with glucosinolate metabolism and induction by auxin treatment. The results are discussed in the context of known mutations in glucosinolate pathway genes and their effects on auxin homeostasis. Taken together, our work provides complementary in vitro and in vivo evidence for a primary role of UGT74B1 in glucosinolate biosynthesis.

SUMMARYThe study of glucosinolates and their regulation has provided a powerful framework for the exploration of fundamental questions about the function, evolution, and ecological significance of plant natural products, but uncertainties about their metabolism remain. Previous work has identified one thiohydroximate S-glucosyltransferase, UGT74B1, with an important role in the core pathway, but also made clear that this enzyme functions redundantly and cannot be the sole UDP-glucose dependent glucosyltransferase (UGT) in glucosinolate synthesis. Here, we present the results of a nearly comprehensive in vitro activity screen of recombinant Arabidopsis Family 1 UGTs, which implicate other members of the UGT74 clade as candidate glucosinolate biosynthetic enzymes. Systematic genetic analysis of this clade indicates that UGT74C1 plays a special role in the synthesis of aliphatic glucosinolates, a conclusion strongly supported by phylogenetic and gene expression analyses. Finally, the ability of UGT74C1 to complement phenotypes and chemotypes of the ugt74b1-2 knockout mutant and to express thiohydroximate UGT activity in planta provides conclusive evidence for UGT74C1 being an accessory enzyme in glucosinolate biosynthesis with a potential function during plant adaptation to environmental challenge.

Increasing global atmospheric CO2 has been shown to affect important plant traits, including constitutive levels of defensive compounds. However, little is known about the effects of elevated CO2 on the inducibility of chemical defenses or on plant mechanical defenses. We grew Brassica rapa (oilseed rape) under ambient and elevated CO2 to determine the effects of elevated CO2 on constitutive levels and inducibility of carbon-based phenolic compounds, and on constitutive trichome densities. Trichome density increased by 57% under elevated CO2. Constitutive levels of simple, complex, and total phenolics also increased under elevated CO2, but inducibility of each decreased. Induction of simple phenolics occurred only under ambient CO2. Although induction of complex and total phenolics occurred under both ambient and elevated CO2, the damage-induced increases were 64% and 75% smaller, respectively, under elevated CO2. Constitutive phenolic levels were positively correlated with leaf C:N ratio, and inducibility was positively correlated with leaf N and negatively correlated with leaf C:N ratio, as would be expected if inducibility were constrained by nitrogen availability under elevated CO2. We conclude that B. rapa is likely to exhibit higher constitutive levels of both chemical and mechanical defenses in the future, but is also likely to be less able to respond to herbivore damage by inducing phenolic defenses. To our knowledge, this is only the second study to report a negative effect of elevated CO2 on the inducibility of any plant defense.

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