Abstract

We describe the characterization of four genes designated BNST1 to - 4 encoding steroid sulfotransferases from Brassica napus . Recombinant BNST3 catalyzes the sulfonation of brassinosteroids and of mammalian estrogenic steroids. The enzyme is stereospecific for 24-epibrassinosteroids, with a substrate preference for 24-epicathasterone, a metabolic precursor of 24-epibrassinolide. The bean second internode bioassay was used to demonstrate that enzymatic sulfonation of 24-epibrassinolide abolishes its biological activity. This mechanism of hormone inactivation is similar to the modulation of estrogen biological activity observed in mammals. The sulfonation of an intermediate in the biosynthesis of 24-epibrassinolide may prevent the formation of biologically active end products. BNST genes are inducible by salicylic acid, a signal molecule in the defense response, suggesting that plants respond to pathogen infection by modulating steroid-dependent growth and developmental processes. BNST genes along with alcohol dehydrogenase ( ADH ) and xyloglucan endotransglycosylases ( XET ) genes are also inducible by ethanol. BNST4 displays the fastest response among BNST genes, with peak mRNA levels within one hour after treatment. Plants respond to low oxygen stress by a switch to ethanolic fermentation and the induction of anaerobic proteins, including ADH and XET. BNST proteins are induced under low oxygen stress, suggesting that endogenous ethanol may act as a chemical signal regulating gene expression. The localization of BNST2 and - 3 expression was studied in transgenic A. thaliana expressing promoter fusions with the Ý-glucoronidase reporter gene. Expression was observed at the apex of leaf organs, including cotyledons, rosette and cauline leaves. Expression was also observed in the transition zone of seedlings. The tissue distribution of expression is compatible with a function of the sulfotransferases in brassinosteroid inactivation. We tested this hypothesis in transgenic A. thaliana expressing BNST3 under the control of the constitutive CaMV35S promoter. No effect of the transgene was observed on plant growth and development, and the response of transgenic lines to exogenous 24-epiteasterone or 24-epibrassinolide was similar to that of the wild-type. Alternative experimental approaches will be required in order to determine the function of the sulfotransferases. The results of preliminary experiments performed in order to purify endogenous substrates of the BNST enzymes are discussed.