The plant glutathione transferases (GSTs) are a super-family of proteins with diverse roles in metabolism and redox homeostasis which underpin important agronomic traits such as tolerance to biotic and abiotic stress. Recently, we have identified the plant-specific lambda (L) class GSTL proteins as having key roles in determining the resistance of cereal crops and weeds to chemical treatments including herbicides. We hypothesize that GSTLs are glutathione-dependent redox enzymes which provide broad-ranging antioxidant activities either a) at the level of generating reduced metabolites/cofactors which are essential in supporting cytoprotective metabolism, or b) that they limit and reverse damage to important proteins incurred by exposure to chemically-imposed stress. Based on our knowledge of the GSTLs in Arabidopsis we know that these proteins are present in a small gene family which is differentially regulated, with members targeted to both the cytosol and chloroplast. In wheat and weeds such as black-grass where elevated levels of specific related GSTLs are integrally associated with herbicide resistance, the size and organization of the respective gene families are unknown, making their functional characterization by conventional genetic approaches problematic. Instead, in this project we propose to test for GSTL function in wheat and the associated problem weed black-grass by producing inhibitors based on haloenol lactone chemistries which specifically target the unusual active site chemistry of the GSTLs. We will then look for perturbations in the metabolome and proteome of treated plants to define the functions of the GSTL class of proteins. 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 fused to strepactin.
3. Synthesizing and testing a series of specific GSTL-chemotype inhibitors based on the selective structural elaboration of enol 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 (cf: 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.
At the conclusion of the study we will have derived novel insights into the functions of GSTLs in stress tolerance in Arabidopsis, wheat and an associated problem weed species and derived a new set of validated chemical genetic tools to probe GST function in other plants of agronomic interest.
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 overcoming 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.
This 3 year project has focussed on identifying the functional roles of a group of stress responsive proteins, the lambda glutathione transferases (GSTLs), in natural product metabolism and herbicide tolerance in wheat and competing grass weeds. The approach has been to use a combination of chemical inhibitors and molecular genetic approaches in model plant species (Arabidopsis) to probe for their activities. Applying genetic engineering methods, we have altered the expression of members of the GSTL family in Arabidopsis and wheat. The final analysis associated with these long term studies is currently being completed, but our studies in wheat have shown that perturbing the expression of GSTLs alters the content of specific flavonoids, such that on up-regulation specific flavonoids disappear, while on down regulation the same compounds accumulate. We were able to duplicate the same relative effects on flavonoid content by using chemicals (safeners) to either up regulate GSTL expression, or inhibit their enzyme activity using custom made enol lactones.
Using ligand fishing technology, whereby we have captured the compounds which bind to the active sites of GSTLs in plant extracts, we have linked these changes in flavonoid content to the biochemical function of these enzymes. In the course of plant metabolism and stress, flavonoids serve as powerful antioxidant protectants which undergo oxidation to quinone derivatives while preserving cellular components from incurring chemical damage. As these quinones are highly reactive, they react with another cytoprotective component glutathione, to form conjugates which require rapid recycling to re-release the protective antioxidant flavonols. Using ligand fishing technology, whereby we can identify the compounds which bind to the active sites enzynes in plant extracts, we were able to show that GSTLs captured intermediates derived from this quinone-glutathione recycling pathway. These compounds were identified by mass spectrometry and the results suggested that GSTLs were involved in maintaining the reduced pool of flavonoids in the cell during stress events, as mediated by herbicides and safeners. Consistent with such a function, we then demonstrated that GSTLs can regenerate flavonols from the respective quinone conjugates in a glutathione dependent reductive reaction. Importantly, for the first time this enzyme activity links the antioxidant functions of flavonoids to the ubiquitous glutathione redox system which operates to protect all eukaryotic cells. Intriguingly, animals contain a GSTL like protein (omega GST) which is linked to several disease states and may serve comparable functions in recycling protective dietary flavonoid antioxidants in man. Our studies also suggested that based on differences in substrate selectivity, that the GSTLs may be responsible for recycling other oxidised polyphenolics formed during metabolism, suggestive of broader ranging redox functions.
|Effective start/end date||1/10/10 → 30/09/11|