PURIFICATION OF MULTIPLE GLUTATHIONE TRANSFERASES INVOLVED IN HERBICIDE DETOXIFICATION FROM WHEAT (TRITICUM-AESTIVUM L.) TREATED WITH THE SAFENER FENCHLORAZOLE-ETHYL

Multiple isoenzymes of glutathione transferase (GST), purified from th e shoots of wheat seedlings treated with the herbicide safener fenchlo razole-ethyl, could be resolved into polar and hydrophobic types using hydrophobic interaction chromatography. Both types of GSTs could also be resolved based on their affinities for S-hexyl-glutathione-agarose . A minor proportion of the GSTs could be eluted from the column with a salt wash and this loosely bound fraction contained polypeptides whi ch were recognized by an antiserum raised against the theta-type maize GST ZmGST I-II. The major proportion could only be recovered using S- hexyl-glutathione and these GSTs were characterized in detail. These i soenzymes catalyzed the glutathione conjugation of xenobiotics, includ ing herbicides, and showed additional activities as glutathione peroxi dases. Each GST was composed of two subunits, with four distinct class es of subunit being determined. A 25-kDa polypeptide, termed Triticum aestivum GST 1 (TaGST 1), was the most abundant subunit and could be r esolved into two variants, TaGST la and TaGST Ib by reversed-phase HPL C. This GST subunit was recognised by an antiserum raised against the maize GST ZmGST V-VI, which is a tau-type GST. In addition to TaGST 1, two 26-kDa polypeptides, TaGST 2 and TaGST 3, and a 24-kDa polypeptid e, TaGST 4, could be resolved by a combination of hydrophobic interact ion chromatography, SDS-PAGE, and reversed-phase HPLC. In the shoots o f untreated wheat seedlings the major isoenzyme was TaGST la-lb, while in the shoots of fenchlorazole-ethyl-treated plants the heterodimers TaGST 1-2, TaGST 1-3, and TaGST 1-4 also accumulated. Significantly, o nly the safener-inducible TaGST 1-2, TaGST 1-3, and TaGST 1-4 isoenzym es catalyzed the detoxification of fenoxaprop-ethyl and this may help to explain why fenchlorazole-ethyl enhances the glutathione-mediated m etabolism and also the tolerance of wheat toward this herbicide. All i soenzymes were active in detoxifying the herbicides metolachlor and fl uorodifen, bur only TaGST 1-2 and TaGST 1-3 showed any activity toward atrazine. (C) 1997 Academic Press.