Characterisation of the unique lambda class of plant glutathione transferases

The glutathione transferases (GSTs) are an adaptable group of proteins found in all aerobic organisms with multiple roles in metabolism and counteracting oxidative stress. In man, their protective activity is well recognised, with GSTs being important in detoxifying ingested natural and synthetic toxins. On occasions, this activity is problematic, as high levels of GSTs in tumours can prevent chemotherapeutic agents from killing cancer cells. As such medicinal chemists have developed a range of selective inhibitors to inactivate GSTs present in carcinomas, but not in healthy tissue, thereby over-coming drug resistance. Our own interests in GSTs relate to the multiple functions of these proteins in plants. In the course of evolution, plant GSTs have been recruited to fulfil multiple roles in foreign compound detoxification, amino acid and antioxidant metabolism and the transport of reactive natural products around the cell. However, these functions are poorly understood, and the complex regulation of GSTs during plant development and exposure to stress suggests that many other important functions for these enzymes are yet to be determined. An excellent example is seen with the lambda class GSTs (=GSTLs). These plant specific proteins are up-regulated in wheat by a group of agrochemicals called herbicide safeners. Their increased expression is then associated with an enhanced tolerance of herbicides. More disturbingly, when wild grasses start to over-express GSTLs they also become resistant to herbicides and this can result in the weeds out-competing the cereal crops due to the loss of selective chemical control. It would therefore be very interesting to determine the role of these GSTLs in both cereals and weeds and use this information to develop new crop protection strategies. However, both wheat and grass weeds contain multiple genes encoding GSTLs and establishing their functions is problematic, especially in weeds where we do not have access to the necessary genetic information and tools to test their activities. Instead, taking a lead from medicinal chemistry, we propose to test for GSTL function using chemical probes which selectively inhibit these enzymes, thereby disrupting their function. By testing a panel of chemistries against a library of different GSTs, we have identified a class of inhibitor which selectively inactivates GSTLs. Using this as a starting point, we propose to synthesise a series of GSTL inhibitors and test that they give us accurate information about GSTL function by using them in the model plant Arabidopsis thaliana, where we also have the ability to disrupt the expression of these proteins using conventional genetic methods. In each case we will treat the plants with the inhibitors and look for changes in metabolites and proteins. By showing that the inhibitors give the same biochemical responses to those seen when the respective GSTL gene is 'knocked out' we have a means of robustly validating their use. Once confident of their selectivity, we can then use the inhibitors in wheat and grass weeds, determining the roles of GSTLs in herbicide safening and resistance respectively. This project therefore represents a useful example of how we can use information and tools derived from investigating the functions of genes in model plants to studying important agronomic traits in crops and weeds. The long-term objectives of this work are to use this information to counteract herbicide resistance in grass weeds and improve stress tolerance and crop yields in cereals.

The lambda class glutathione transferases (GSTLs) are a group of plant specific proteins integrally linked to responses to chemically imposed stress and herbicide tolerance in crops and weeds. The functions of the GSTL family are not known, though their active site chemistry is different from the herbicide-detoxifying GSTs and catalyzes glutathione-dependent redox reactions. It is proposed to functionally characterize the GSTLs linked to multiple herbicide resistance in wheat and the associated problem weed black-grass using chemical probes based on haloenol lactone chemistries which selectively inactivate the unique active sites of this protein family. Importantly, we will first validate the use of these 'chemotyping' inhibitors using the GSTLs of Arabidopsis as a model system in a multi-tiered approach by; 1. Defining the biochemical phenotype associated with selectively over-expressing and knocking out members of the GSTL family in Arabidopsis using standard molecular genetic approaches. 2. Identifying ligands and substrates of family members in planta using selective protein pull-down experiments with enzymically active and inactive GSTLs. 3. Synthesizing and testing a series of specific GSTL-chemotype inhibitors based on the selective structural elaboration of haloenol lactones, including the generation of probes which can be used to selectively label targeted proteins using 'click' chemistry. 4. Validating the GSTL chemotyping agents in Arabidopsis by comparing the biochemical phenotypes obtained with the inhibitors with those observed with the respective gene knock-outs (see objective 1). 5. Using the inhibitors to probe the roles of GSTLs in herbicide tolerance in wheat treated with safeners and in black-grass, displaying multiple herbicide resistance. 6. Further examining the roles of GSTLs in wheat through the generation and analysis of stably transformed over-expressing and KO plants.