CHARACTERIZATION OF MULTIPLE GLUTATHIONE TRANSFERASES CONTAINING THE GST-I SUBUNIT WITH ACTIVITIES TOWARD HERBICIDE SUBSTRATES IN MAIZE (ZEA-MAYS)

The glutathione transferases (GSTs) of maize with activities toward ch loroacetanilide herbicides are relatively well characterised, but thei r range of substrate specificities has not been determined in detail. GST activities toward an extensive range of chemically diverse xenobio tic substrates, including the herbicides atrazine, alachlor, metolachl or and fluorodifen, have been determined in crude and purified prepara tions from the roots and shoots of dark-grown maize seedlings treated with and without the herbicide-safener dichlormid. With the exception of the activity toward atrazine, specific activities were higher in th e roots than in the shoots in all cases. In untreated shoots activitie s were in the order atrazine = alachlor = metolachlor > fluorodifen wi th safener-treatment selectively increasing the activity toward the ch loroacetanilides and fluorodifen. In the roots the highest GST activit ies toward herbicides were toward the chloroacetanilides. Dichlormid t reatment resulted in an increase in activities toward all four herbici des in the roots of one maize cultivar (Pioneer 3394) but only enhance d the activities toward the chloroacetanilides and fluorodifen in cult ivar Artus. Using the non-herbicide 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, anion-exchange chromatography showed that the roots and shoots contained a similar range of GST isoenzymes. All of these isoe nzymes were enhanced in response to safeners, though the extent of thi s induction was organ-dependent. GST isoenzymes containing the GST I s ubunit were purified from safener-treated roots by a combination of hy drophobic interaction chromatography and affinity chromatography using Orange A agarose, Three isoenzymes could then be purified following r esolution by anion-exchange chromatography. The three GSTs were termed GST I/I, GST I/II and GST I/III with GST I, II and III referring to t he presence of 29 kDa, 27 kDa and 26 kDa subunits respectively. This r evised nomenclature for the maize GSTs was considered necessary in vie w of the continued discovery of new isoenzymes, such as GST I/III, com posed of subunits which have been previously described. GST I/I had me asurable activity toward atrazine, low activities toward the other her bicides and appreciable activities toward a range of other xenobiotic substrates. GST I/II and the novel GST I/III isoenzymes both showed hi gh activities toward the chloroacetanilides and fluorodifen but lower activities toward the other substrates and negligible activities towar d atrazine. The GST II subunit of GST I/II also had activity as a glut athione peroxidase. Our results show that the GST I subunit can form d imers with the GST III subunit in addition to the GST I and GST II sub units and that the degree of specificity toward herbicide substrates o f the respective isoenzymes is greater than previously reported. Our r esults also suggest that the safener-inducible GST II subunit has addi tional activities as a glutathione peroxidase.