Partial Characterization of Glutathione S-Transferase Isozymes Induced by the Herbicide Safener Benoxacor in Maize

Abstract

The effects of the dichloroacetamide safener benoxacor on maize (Zea mays 1. var Pioneer 3906) growth and glutathione Stransferase (CST) activity were evaluated, and CST isozymes induced by benoxacor were partially separated, characterized, and identified. Protedion from metolachlor injury was closely correlated with CST activity, which was assayed with metolachlor as a substrate, as benoxacor concentration increased from 0.01 to 1 p~. CST adivity continued to increase at higher benoxacor concentrations (10 and 100 PM), but no further protection was observed.Total CST activity with metolachlor as a substrate increased 2.6-to 3.8-fold in response to 1 p~ benoxacor treatment. Total CST activity from maize treated with or without 1 p~ benoxacor was resolved by fast protein liquid chromatography anion-exchange chromatography into four major activities, designated adivity peaks A, B, C, and D in their order of elution. These CST activity peaks were enhanced to varying degrees by benoxacor. Activity peak B showed the least induction, whereas activity peak A was absent constitutively and thus highly induced by benoxacor. In contrast to earlier reports, there appear to be not one, but at least two, major constitutive isozymes (adivity peaks A and D) having activity with metolachlor as substrate; there were at least three such isozymes in benoxacor-treated maize (activity peaks A, C, and D). l h e elution volumes of activity peaks A, B, C, and D were compared with those of partially purified maize CST I and CST 11; also, the reactivity of polypeptides in these activity peaks with antisera to CST I or CST 1/111 (mixture) was evaluated. Evidence from these experiments indicated that adivity peak B contained CST I, and adivity peak C contained CST II and CST 111. Adivity peaks A and D contained unique CSTs that may play a major role in metolachlor metabolism and in the safening activity of benoxacor in maize. lsozymes present in adivity peaks A and D were not detected in earlier reports because of the very low activity with the artificial substrate l-chloro-2,4-dinitrobenzene. Immunoblotting experiments also indicated the presence of numerous unidentified CST subunits, including multiple subunits in chromatography fractions containing single peaks of CST adivity; this is indicative of the likely complexity and diversity of the maize CST enzyme family.GST enzymes catalyze the conjugation of GSH to electrophilic, lipophilic molecules. The GSH conjugates formed are

Purification and Characterization of a Glutathione S-Transferase from Benoxacor-Treated Maize (Zea mays)

“…Each purification step yielded increases in the specific activity of the enzyme. Severa1 GST isozymes in com seedlings are capable of utilizing metolachlor as a substrate (Dean et al, 1991;Fuerst et al, 1993); therefore, enzyme recovery and purification, calculated from the total GST-M activity in crude extracts, is most likely underestimated. Greater than 90% of the total GST-M activity was precipitated between 40 and 70% (NH4)2S04 saturation.…”

“…Treatment of com with benoxacor causes elevated levels of total GST activity, which subsequently results in the enhanced metabolism of metolachlor to its GSH conjugate (Viger et al, 1991). The total GST activity extracted from benoxacor-treated corn seedlings can be separated by anion-exchange FPLC into several distinct GST activities that differ in both inducibility and substrate specificity (Dean et al, 1991;Fuerst et al, 1993). We have previously described (Fuerst et al, 1993) a GST isozyme that is highly induced by benoxacor treatment and could facilitate the conjugation of metolachlor, but not CDNB (a model GST substrate), with GSH.…”

A New 1-Aminocyclopropane-1-Carboxylic Acid-Conjugating Activity in Tomato Fruit

“…GST-catalyzed conjugation reactions are ubiquitous in bacteria, fungi, animals, and plants (Pickett and Lu, 1989), and plant GSTs are receiving increased attention in relation to their induction by herbicides, herbicide safeners, and pathogen attack and their role in conjugation and detoxification of herbicides (Meyer and Goldsbrough, 1991;Fuerst et al, 1993;Irzyk and Fuerst, 1993;Mauch and Dudler, 1993). GSTs catalyze the nucleophilic addition of GSH through a thioether linkage to hydrophobic acceptors containing electrophilic centers, such as aryl and alkyl halides, olefins, organic hydroperoxides, quinones, alkenes, and www.plantphysiol.org on May 11, 2018 -Published by Downloaded from Copyright © 1995 American Society of Plant Biologists.…”

Herbicide Safener-Binding Protein of Maize1

“…Examples of these classes of compound are shown in Figure 1. Although it is well established that in maize dichloroacetamide safeners elevate GSH levels and induce novel GSH S-transferases, the mechanism by which they do so is not known (Lay et al, 1975;Fuerst et al, 1993).Etiolated maize seedlings contain a soluble, highaffinity-binding activity (K d , 0.12 m) for a tritiated form of the dichloroacetamide safener Saf (Fig. 1) (Walton and Casida, 1995).…”

“…Examples of these classes of compound are shown in Figure 1. Although it is well established that in maize dichloroacetamide safeners elevate GSH levels and induce novel GSH S-transferases, the mechanism by which they do so is not known (Lay et al, 1975;Fuerst et al, 1993).…”