Bryant, David (2004) Glutathione conjugation of herbicides and fungicides in plants and fungi:: functional characterization of glutathione transferases from phytopathogens. Doctoral thesis, Durham University.
The aim of this research was to determine the relative importance of glutathione S-transferase (GST) mediated herbicide and fungicide detoxification in wheat plants and to functionally characterise GSTs cloned from phytopathogenic fungi. Wheat seedlings treated with and without safeners were used for in vivo and in vitro pesticide metabolism studies with the herbicide fenoxaprop-P-ethyl and the fungicide fluquinconazole, which are both known to undergo glutathione conjugation. Fenoxaprop-P-ethyl underwent rapid detoxification by this route and the rates of metabolism were enhanced in safener treated wheat. In contrast, fluquinconazole was poorly metabolised with only a small proportion detoxified by glutathlonylation irrespective of whether or not the plants were safener treated. Subsequent studies confirmed that whereas fenoxaprop was a substrate for wheat GSTs, fluquinconazole was not. Similarly, when a diverse range of systemic and contact fungicides was tested, none were found to undergo conjugation mediated by wheat GSTs, even though several compounds underwent conjugation with glutathione in alkaline conditions. Metabolite profiling by HPLC-MS established that the alternative thiol present in wheat, hydroxymethyl glutathione, was used in addition to glutathione in fenoxaprop detoxification. The S-conjugates were then further metabolised by peptidases and glucosyl- and malonyl transferases to yield polar derivatives such as the cysteinyl-malonyl-glucosyl derivative. This rapid processing was in contrast to that determined in herbicide-susceptible and herbicide-resistant black-grass, where the glutathione derivative was the major metabolite of fenoxaprop. Using an informatics approach, GSTs in IVIagnaporthe griseae and Phytoptithora infestans were identified cloned and then expressed in E. coli, with the pure enzymes assayed for glutathione-dependent activities. MgGSTX1 of Magnaporthe griseae was found to be a member of a novel class of GSTs (termed xi class) and was shown to function as a thioltransferase, with a specific activity of 50.4 ± 3.4 nKat mg (^-1)protein. Unusually, the catalytic mechanism of thiol transfer was insensitive to the alkylating agent iodoacetamide, indicating that free cysteines were not involved in catalysis. MgGSTX1 did not conjugate any of the experimental xenobiotics tested with GSH. The theta class GST P/GSTT1 cloned from P. infestans exhibited glutathione peroxidase activity and readily detoxified fungitoxic oxylipins produced by potato as antimicrobial defence by potato through the action of lipoxygenases. Using anti-sera raised against the purified recombinant protein, immunoblotting experiments revealed expression of P/GSTT1 in the in vitro cultured fungus as well as during colonisation of potato. P/GSTT1 did not demonstrate GSH conjugating toward any of the xenobiotic substrates tested including herbicides and fungicides. Based on the sequences of GSTs identified in this study an extension of the existing classification system is suggested to include the GSTs of fungal phytopathogens
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||09 Sep 2011 10:01|