Loutre, Caroline (2004) Glucosylation of xenobiotics in maize, soybean and arabidopsis thaliana. Doctoral thesis, Durham University.
Many natural products and xenobiotics become glucosylated in the course of their metabolism in plants. This reaction is catalysed by type 1 UDP-glucose dependent glucosyltransferases (GTs), a super-family of enzymes which differ in their substrate specificity and which are able to glucosylate hydroxy, amino and carboxylic acid groups to form conjugates with altered bioactivities as compared with the parent aglycones. This study has focused both on NGT and OGT enzymes active towards amino and hydroxy groups, respectively, present in natural products and pesticide metabolites in two major crops (Zea mays and Glycine max) and the model plant Arabidopsis thaliana. A sensitive radioactive enzyme assay was developed to monitor conjugating activity in vitro and the substrate specificity of N-GTs and O-GTs determined in the three plant species with respect to xenobiotic detoxification. 3,4-Dichloroaniline was found to be the optimal N-GT substrate and 2,4,5-trichlorophenol the preferred 0-GT substrate in all the species tested. In addition, O-GT activities were also determined with other phenols of both natural and synthetic origin. To confirm the importance of N-GTs and O-GTs in xenobiotic detoxification, plant metabolism studies were carried out with [(^14)C]-p-nitrophenol and [(^14)C]-3,4-dichloroaniline. In each case, O- I N-glucosylation was found to be a major route of detoxification respectively. To determine whether or not herbicide-safeners could enhance the glucosylation of xenobiotics as had been demonstrated for the S-glutathionylation of herbicides in cereals; soybean, maize and Arabidopsis were treated with a range of safeners. The plants were then either fed with radiolabelled [(^14)C]-3,4-dichloroaniline, or extracted and assayed for O-GT and N-GT activities. In all species, safener treatment had no significant effect on the rate of uptake of radioactivity following feeding with [(^14)C]-3,4-dichloroaniline. However, specific safeners were found to enliance N-GT and O-GT activities in etiolated shoots, roots or suspension cultures in all species where tested. Several attempts were made to clone GT enzymes from soybean and maize based on a combination of bioinformatic and PCR approaches, the latter using conserved blocks of sequence in type 1 plant GTs to amplify up partial cDNAs. Using PCR and analysis of soybean expressed tagged accessions, it was possible to assemble a full-length cDNA from soybean which encoded a GT resembling an arbutin synthase GT from Rauvolfia serpentina. Although the resulting GT (GmGT32_l) could be expressed as a recombinant polypeptide in E.coli, the resulting protein was inactive and accumulated in the insoluble inclusion bodies. In the case of maize, a GT termed Z/wRP was identified as a random sequenced clone from a proprietary maize cDNA library. However, ZmRP could not be translated into protein using bacterial expression systems. Instead, an alternative proteomics approach at isolating plant GTs involved in xenobiotic detoxification was undertaken in Arabidopsis, using suspension cultured cells as the starting material. The major N-GT conjugating activity towards 3,4-dichloroaniline was purified 9552-fold using a combination of hydrophobic interaction, ion exchange, and affinity chromatographies. The resulting 50 kDa polypeptide was digested with trypsin and the peptide fragments analysed by MALDI TOF MS. Database analysis unambiguously identified the Arabidopsis protein as UGT72B1 (NM 116337). Following the completion of the Ph.D. programme the activity of GT72B1 towards 3,4-dichloroaniline, 2,4,5-trichlorophenol and other xenobiotics was confirmed and an account of the studies earned out on Arabidopsis GTs published (Lao, et al., 2003), (Loutre, et al., 2003).
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||09 Sep 2011 09:59|