Two glutathione S-transferase (GST) activities have been identified and purified from etiolated corn tissue. The first, designated GST I enzyme, is constitutively present in corn tissue, and the second, designated GST II enzyme, is present only in tissue which has been treated with chemical antidotes which protect corn against chloroacetanilide herbicides. The total activity constitutes approximately 2% of the soluble protein in these tissues. The native forms of these enzymes have molecular weights of approximately 50 000 as determined by Sephadex G-100 chromatography. On sodium dodecyl sulfate-polyacrylamide gels, GST I enzyme migrates primarily as a single band of molecular weight 29 000, and GST II enzyme migrates as primarily two bands of molecular weight 29 000 and 27 000. Both enzymes catalyze the formation of a glutathione-herbicide conjugate in vitro when the herbicide alachlor is used as a substrate. This conjugation results in elimination of the biological activity of the herbicide.

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Glutathione-S-transferases (GST's) in maize represent a family of enzymes which conjugate glutathione to several major classes of pre-emergent, selective herbicides. Chemicals termed safeners have been demonstrated to increase the tolerance of maize toward such herbicides when the maize seed has been previously treated with safeners. It has subsequently been shown that corresponding increases in glutathione-S-transferase species occur. To determine whether these compounds act at a transcriptional level we have used synthetic oligonucleotide probes to isolate cDNA clones encoding the major GST polypeptide subunit, designated GST A. The identity of the clones has been confirmed by hybrid-selected mRNA translation and immunoprecipitation using antibodies made against this GST species as well as by production of active GST in yeast cells transformed with an expression vector containing the cloned DNA. GST A has been found to be encoded in a mRNA of 1.1 kb. Sequencing of cDNA products obtained by primer extension of maize mRNA using our oligonucleotide probes is consistent with this mRNA corresponding to the isolated cDNA clone. Using the clone as a probe for Northern analysis we have found a three to four-fold increase in the steady state level of this mRNA in maize tissue grown from safener-treated seeds. The level of safener which gives this induction is comparable to that required to obtain herbicide tolerance in the field.

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The poly(A)-contasning mRNA from barley aleurone layers pretreated with gibbereUic acid has been purified by phenol-chloroform extraction and repeated oligold(pT)I-cellulose chromatography. This RNA has been translated in both the wheat germ and reticulocyte lysate in vitr translation systems with greater than 50% of the synthesized protein being a-amylase. The mRNA for a-amylase has been further purified by dimethylsulfoxideformamide-sucrose density gradient centrifugation and by gel electrophoresis. By these methods, its molecular weight has been determined to be 580,000.In barley aleurone layers, the synthesis and secretion of several hydrolases begins as early as 6 h after the addition of GA3 (6,17,18). One of these hydrolases, a-amylase, accounts for more than 60o of all in vivo protein synthesis in these tissues after 10-12 h of hormone treatment. The induction of this enzyme is inhibited by the addition of cordycepin, an RNA synthesis inhibitor in these tissues (9). Higgins et al. (8) have shown that during the first 10 h after addition of GA3, the level of translatable mRNA for aamylase increases to a maximum level and remains constant thereafter. However, they showed that only 12-25% of in vitro synthesized peptides were a-amylase (8). There is also evidence that the addition of GA3 increases synthesis of poly(A) RNA in these tissues and the incorporation of added radiolabeled nucleosides into RNA in aleurone layers (12).Poly (