Arabidopsis glucosyltransferase UGT74B1 functions in glucosinolate biosynthesis and auxin homeostasis

Abstract

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.

Cyanogenic glycosides: a case study for evolution and application of cytochromes P450

“…In Arabidopsis, tryptophan-derived oximes are key intermediates in formation of the phytohormone indole acetic acid (Woodward and Bartel 2005) as well as in biosynthesis of glucosinolates ) and the indole alkaloid camalexin Phytochem Rev (2006) (Glawischnig et al 2004). CYP83A1 and CYP83B1 convert oximes into the corresponding thiohydroximates (Bak and Feyereisen 2001;Bak et al 2001;Hansen et al 2001;Naur et al 2003) which are further metabolized by SUR1 (Mikkelsen et al 2004) and UGT74B1 to desulphoglucosinolates (Douglas et al 2004) which are finally sulphurylated (Piotrowski et al 2004;Klein et al 2006 EIC 323 TIC C lism. A disturbance of oxime metabolism affects phenylpropanoid metabolism and the monomer composition of lignin, but the nature of this interaction is not yet understood (Hemm et al 2003).…”

Indolic glucosinolates at the crossroads of tryptophan metabolism

“…First, CYP79B2 overexpression leads to increased IAA (Zhao et al 2002). Second, as discussed in more detail below, mutants that block IG production at steps downstream of IAOx formation have excess levels of IAA (Barlier et al 2000;Boerjan et al 1995;Grubb et al 2004). How IAOx would be converted to IAA is still not clear.…”

Cytochromes P450 in the biosynthesis of glucosinolates and indole alkaloids

“…The knockout mutant ugt74b1-1, of the S-glucosyltransferase in the glucosinolate pathway, exhibited a phenotype reminiscent of auxin overproduction such as elongated hypocotyls, epinastic cotyledons and excessive adventitious root formation in addition to being dwarfed and partially sterile. The severe phenotype of ugt74b1-1 is more likely to reflect pleiotropic effects due to the toxicity of accumulating thiohydroximic acid than a possible auxin activity of thiohydroximic acids (Douglas et al 2004). …”

“…Piotrowski et al (2004) proposed another C-S lyase, CORI-3, as a potential player in the biosynthesis of indole glucosinolates based on specific upregulation of indole-3-ylmethyl and N-methoxyindole-3-methyl-glucosinolate combined with the concurrent upregulation of CORI-3 upon treatment with coronatine, a phytotoxin produced by many pathovars of Pseudomonas syringae. Based on sequence similarity to an in vivo confirmed thiohydroximic acid S-glucosyltransferase (S-GT) in Brassica napus, the glycosyltransferase UGT74B1 was identified and afterwards shown to glucosylate phenylacetothiohydroximic acid in vitro (Douglas et al 2004). The knockout mutant, ugt74b1, displays a phenotype similar to auxin overproduction and is severely reduced in total amount of glucosinolates.…”

“…The knockout mutant, ugt74b1, displays a phenotype similar to auxin overproduction and is severely reduced in total amount of glucosinolates. The fact that ugt74b1 contains glucosinolates makes it evident that other glucosyltransferases are involved in the glucosylation process (Douglas et al 2004). Recently, on the basis of a hierarchical clustering of publicly available gene expression data, wherein UGT74B1 was also pulled out, UGT74C1 was proposed to be a candidate S-glucosyltransferase as it co-regulated with the genes in the synthesis of aliphatic glucosinolates (Gachon et al 2005) The final sulfation step was shown to be catalysed by three desulfoglucosinolate:PAPS sulfotransferases (STs) in Arabidopsis, ST5a, ST5b and ST5c (Klein et al 2006;Piotrowski et al 2004).…”