Benoxacor Treatment Increases Glutathione S-Transferase Activity in Suspension Cultures of Zea mays

Abstract

Increases of glutathione S-transferase (GST) activity were evaluated in cell suspension cultures of Zea mays (cv. Black Mexican Sweet) treated with the herbicide safener benoxacor. A time course analysis of [14C]benoxacor uptake indicated that increases of extractable radioactivity were detected as early as 15 min after benoxacor treatment and continued increasing through 48 h after treatment. A time course analysis of benoxacor-induced increases of GST activity determined that GST activity, assayed with the herbicide metolachlor as a substrate, was detectably increased 4 h after 10 μM benoxacor treatment and continued increasing until it reached maximum levels 24 h after treatment. Anion exchange chromatography (on Mono-Q resins) of total GST activity extracted from 7-day-old cultures resolved several constitutively expressed GST activities that were detected when metolachlor or 1-chloro-2,4-dinitrobenzene were used as substrates. Three of these activities were increased by 10 μM benoxacor treatment. Proteins in anion exchange chromatography fractions containing GST activity were immunoblotted and probed with polyclonal antisera raised against maize GST I or a mixture of maize GST I and GST III. No more than two polypeptides were detected in three fractions, indicating that GST I, GST III, or polypeptides with sequence homology to GST I or GST III are expressed in BMS cultures. The results obtained from anion exchange chromatography and immunoblotting analyses of protein extracts from benoxacor-treated BMS cells were similar to previously reported results obtained from the same analyses using benoxacor-treated maize seedlings. Both basal levels and benoxacor-induced increases of GST activity, as well as the incorporation of [3H]leucine into total soluble protein, were reduced by 10 and 100 μM cycloheximide treatment. This suggests that de novo protein synthesis is required for both basal levels and benoxacor-induced increases of GST activity. Similarly, 10 and 100 μM cordycepin treatment reduced both benoxacor-induced increases of GST activity and the incorporation of [3H]uracil into total RNA. This suggests that benoxacor induces the de novo synthesis of mRNA transcripts required for increases of GST activity.