The Activities Of Herbicide Safeners In Wheat (Triticum aestivum L)

Abstract

Commercialised herbicide safeners (also known as protectants or antidotes) are synthetic chemicals used to enhance herbicide tolerance in cereal crops. They do this by causing an up-regulation in xenobiotic detoxifying enzymes such as glutathione transferases (GSTs). Seedlings of wheat (Triticum aestivum cv ‘Einstein’) were sprayed with the safeners cloquintocet mexyl, fenchlorazole ethyl and mefenpyr diethyl. All three compounds caused an identical up regulation of GSTs from the phi, tau and lambda classes, despite their differences in chemistry. Using cloquintocet mexyl as a classic wheat safener treatment, it was found that GST induction was both dose and time dependent. Safening was found to be associated with the rapid hydrolysis of the parent ester to cloquintocet acid. When the free acid was tested, the GST-induction response obtained was identical to that determined with the parent ester, suggesting that cloquintocet itself is the active safener. GST induction was found to be tissue specific within the wheat shoots, with the lambda GSTs being preferentially expressed in the meristematic tissue. Proteomic 2 D gel analysis revealed that the tau TaGSTU3 was a major up-regulated GST. In addition, six GSTs that were previously shown in literature to be up-regulated by herbicide safeners in wheat were cloned, expressed and

characterized as the respective recombinant enzymes and renamed to bring them in line with existing nomenclature. The GSTs cloned included TaGSTU3, TaGSTU6, TaGSTF4, TaGSTF10 and TaGSTL1. Metabolism studies showed that following the hydrolysis of cloquintocet mexyl, no further down- stream metabolites could be identified and none of the up-regulated GSTs showed any activity toward the safener. However TaGSTU3 was found to bind and be inhibited by cloquintocet free acid as determined by isothermal titration calorimetry. Safener treatment also led to a transient inhibition of GST activity in crude wheat extracts after spraying the seedlings. In addition to the induction of GSTs, safener treatments also resulted in an enhanced growth of wheat seedlings. The work presented in this thesis confirms that very different compounds can induce apparently identical downstream events at the level of GST enhancement and that these induction events underpin wider changes in plant physiology.