A key role for a glutathione transferase in multiple herbicide resistance in grass weeds
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ABSTRACT: The gene encoding a protein (AmGSTF1) associated with multiple herbicide resistance (MHR) in black-grass was transgenically expressed in Arabidopsis thaliana.The goal of this study was to determine if AmGSTF1 could elicit an MHR phenotype in the transgenic host. Affymetix microarrays were used to detect changes in transcriptional regulation between amgstf1-expressors and wild-type Arabidopsis plants (ecotype Col0). Total RNA was isolated from amgstf1-expressors and wild-type Arabidopsis plants (ecotype Col0) respectively, grown under identical growth room conditions. Performed in biological triplicate.
Project description:The gene encoding a protein (AmGSTF1) associated with multiple herbicide resistance (MHR) in black-grass was transgenically expressed in Arabidopsis thaliana.The goal of this study was to determine if AmGSTF1 could elicit an MHR phenotype in the transgenic host. Affymetix microarrays were used to detect changes in transcriptional regulation between amgstf1-expressors and wild-type Arabidopsis plants (ecotype Col0).
Project description:The aim of this experiment was to identify the genes involved in the detoxification of the toxic pollutant and explosive compound 2,4,6-trinitrotoluene (TNT). Fourteen-day-old, liquid culture grown, Arabidopsis seedlings, ecotype Col0 (NASC stock code N1093), were dosed with 60 uM TNT dissolved in 60 ul dimethyl formamide (DMF) solvent, or 60 ul DMF only. After six hours, RNA was extracted and used for the microarray analysis. Further details and characterisation of glucosyltransferases identified using this method are presented in citation below. 6 samples were used in this experiment
Project description:Multiple-herbicide resistance (MHR) in black-grass (Alopecurus myosuroides) and annual rye-grass (Lolium rigidum) is a global problem leading to a loss of chemical weed control in cereal crops. Although poorly understood, in common with multiple-drug resistance (MDR) in tumors, MHR is associated with an enhanced ability to detoxify xenobiotics. In humans, MDR is linked to the overexpression of a pi class glutathione transferase (GSTP1), which has both detoxification and signaling functions in promoting drug resistance. In both annual rye-grass and black-grass, MHR was also associated with the increased expression of an evolutionarily distinct plant phi (F) GSTF1 that had a restricted ability to detoxify herbicides. When the black-grass A. myosuroides (Am) AmGSTF1 was expressed in Arabidopsis thaliana, the transgenic plants acquired resistance to multiple herbicides and showed similar changes in their secondary, xenobiotic, and antioxidant metabolism to those determined in MHR weeds. Transcriptome array experiments showed that these changes in biochemistry were not due to changes in gene expression. Rather, AmGSTF1 exerted a direct regulatory control on metabolism that led to an accumulation of protective flavonoids. Further evidence for a key role for this protein in MHR was obtained by showing that the GSTP1- and MDR-inhibiting pharmacophore 4-chloro-7-nitro-benzoxadiazole was also active toward AmGSTF1 and helped restore herbicide control in MHR black-grass. These studies demonstrate a central role for specific GSTFs in MHR in weeds that has parallels with similar roles for unrelated GSTs in MDR in humans and shows their potential as targets for chemical intervention in resistant weed management.
Project description:To comprehensively investigate the effects of glutathione on the gene expression, the microarray analysis was performed in the glutathione-fed wild-type Arabidopsis thaliana. Wild-type Arabidopsis (ecotype Columbia-0) were fed with 1 mM oxidized glutathione (GSSG) and 2 mM reduced glutathione (GSH) for comparison at equal nitrogen equivalents. To examine the effects of glutathione other than nitrogen at equal nitrogen equivalents, plants were fed with 3 mM NH4NO3. Plants grown by water were used as a control.
Project description:rs08-03_glutathion - glutathion - How does glutathione content or reduction state affect H2O2-induced changes in the transcriptome? - Three single Arabidopsis mutants were used: cat2, knockout for catalase2 and so enriched in H2O2; cad2, defective in glutathione content; cytGR, knockout for cytosolic glutathione reductase. Cat2 was crossed with cad2 and cyt GR, and col0, 3 single mutants, and 2 double mutants were sampled in controlled growth conditions either in 8h or 16h photoperiod. Keywords: gene knock out
Project description:rs08-03_glutathion - glutathion - How does glutathione content or reduction state affect H2O2-induced changes in the transcriptome? - Three single Arabidopsis mutants were used: cat2, knockout for catalase2 and so enriched in H2O2; cad2, defective in glutathione content; cytGR, knockout for cytosolic glutathione reductase. Cat2 was crossed with cad2 and cyt GR, and col0, 3 single mutants, and 2 double mutants were sampled in controlled growth conditions either in 8h or 16h photoperiod. Keywords: gene knock out 20 dye-swap pairs - CATMA arrays: 40 arrays
Project description:The evolution and growth of multiple-herbicide resistance (MHR) in grass weeds continues to threaten global cereal production. While various processes can contribute to resistance, earlier work has identified the phi class glutathione-S-transferase (AmGSTF1) as a functional biomarker of MHR in black-grass (Alopecurus myosuroides). This study provides further insights into the role of AmGSTF1 in MHR using a combination of chemical and structural biology. Crystal structures of wild-type AmGSTF1, together with two specifically designed variants that allowed the co-crystal structure determination with glutathione and a glutathione adduct of the AmGSTF1 inhibitor 4-chloro-7-nitro-benzofurazan (NBD-Cl) were obtained. These studies demonstrated that the inhibitory activity of NBD-Cl was associated with the occlusion of the active site and the impediment of substrate binding. A search for other selective inhibitors of AmGSTF1, using ligand-fishing experiments, identified a number of flavonoids as potential ligands. Subsequent experiments using black-grass extracts discovered a specific flavonoid as a natural ligand of the recombinant enzyme. A series of related synthetic flavonoids was prepared and their binding to AmGSTF1 was investigated showing a high affinity for derivatives bearing a O-5-decyl-α-carboxylate. Molecular modelling based on high-resolution crystal structures allowed a binding pose to be defined which explained flavonoid binding specificity. Crucially, high binding affinity was linked to a reversal of the herbicide resistance phenotype in MHR black-grass. Collectively, these results present a nature-inspired new lead for the development of herbicide synergists to counteract MHR in weeds.
Project description:The aim of this experiment was to identify the genes involved in the detoxification of the toxic pollutant and explosive compound 2,4,6-trinitrotolune (TNT). Fourteen-day-old, liquid culture grown, Arabidopsis seedlings, ecotype Col0 (NASC stock code N1093), were dosed with 60 uM TNT dissolved in 60 ul dimethyl formamide (DMF) solvent, or 60 ul DMF only. After six hours, RNA was extracted and used for the microarray analysis. Further details and characterisation of glucosyltransferases identified using this method are presented in citation below.
Project description:Freshly harvested Arabidopsis seeds (ecotype Col0 obtained under 10 mM nitrate nutrition, referred to as C10 seeds) are dormant and do not germinate when sown on an agarose-based medium. However, when C10 seeds are sown on 10mM nitrate or when Col0 mother plants are grown on 50 mM nitrate (referred to as C50 seeds), the produced seeds are non dormant and germinate readily on agarose. The G4-3 arabidopsis mutant which is impaired in nitrate assimilation and accumulates nitrate produces likewise non dormant seeds when grown on 10mM nitrate (referred to as G4-3 seeds). The goals of these experiments are to better understand the effects of nitrate feeding of mother plants on the produced seed dormancy.