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Glutathione transferases, also known as Glutathione S-transferases (GSTs), are a diverse group of enzymes that catalyse the conjugation of the tri-peptide glutathione to a wide range of electrophilic substrates. Their biological function in endogenous metabolism in plants is not well characterised, although their role in herbicide metabolism and herbicide selectivity is well documented. Many herbicides used in soybean. Glycine max (L.) Merr., are selective against weeds due to their rapid detoxification in the crop through conjugation with homoglutathione (γ-glu-cys-β-ala), the predominant free thiol in many legumes. However, an in depth characterisation of the GSTs which can potentially catalyse these reactions in soybean has never been performed. This work describes the biochemical and molecular characterisation of GSTs in soybean with emphasis on the identification of specific isoenzymes involved in herbicide metabolism. GST activity toward the chloroacetanilide herbicides acetochlor and metolachlor, the diphenyl ethers acifluorfen and fomesafen and the sulphonyl urea chlorimuron-ethyl were all detected in crude protein extracts from five-day-old suspension cultured soybean cells. GST activity was also determined in five-day-old soybean seedlings, though this activity was significantly lower than that observed with the cell suspension cultures. Treatment of soybean plants with herbicides and herbicide safeners resulted in increased GST activity toward the model substrate l-chloro-2,4-dinitrobenzene (CDNB), but no change in activity toward herbicide substrates. In both plant and cell cultures GST-catalysed conjugation of the diphenyl ethers acifluorfen and fomesafen was over five-fold greater in the presence of homoglutathione as compared with glutathione. The preferential detoxification of these herbicides in the presence of homoglutathione appeared to be an important determinant of their rapid detoxification in soybean and an important factor in herbicide selectivity. GSTs were purified from five-day-old soybean cell cultures using S-hexylglutathione affinity chromatography and anion-exchange chromatography. A combination of reversed-phase HPLC, SDS-PAGE and MALDI-TOF mass spectrometry of the purified fractions indicated the presence of nine putative GST subunits, each with a molecular mass between 25 and 29 kDa. Soybean GST cDNA clones were obtained using a combination of RT-PCR, utilising degenerate oligonucleotides designed to conserved regions within plant GSTs, and screening of cDNA libraries prepared from soybean plants and cell cultures. This process failed to identify any theta-type GSTs, the class associated with herbicide detoxification in maize. In contrast, seven distinct tau-type GSTs were isolated together with a number of clones showing minor variations in individual sequences. Expression of these cDNAs in Escherichia coli showed the purified recombinant GSTs were active toward a diverse range of substrates, and possessed additional glutathione peroxidase activity. GST activities for each recombinant enzyme varied with substrate and thiol type, with a marked preference for homoglutathione with selected substrates. From the work reported in this study it would appear that the tau-type GSTs of soybean are at least as complex as those previously reported in cereals and have an important role in determining herbicide metabolism and selectivity in this major crop.

Our research investigated whether priming switchgrass seeds with water or ethephon would increase stand establishment in the field. ‘Alamo’ seed germinated faster and grew taller than ‘Cave-in-Rock.Seeds primed for six days in water or for one day in ethephon 10 mM had the greatest seedling densities. In growth chamber environments seed priming were tested to hasten germination velocity. Seeds primed for two, four or six days in water germinated faster than unprimed seeds. Ethephon treatments reduced overall germination and germination velocity. Accent and Accent Q herbicides containing nicosulfuron are used to control weeds. To test ‘Alamo’ sensitivity to these herbicides, greenhouse evaluations were conducted. Seedlings treated with Accent Q had lower shoot fresh and dry weights than Accent treated seedlings. Seedling atrazine tolerance was examined in a greenhouse study at various growth stages (1, 2 and 4 true leaves). One and two true leaf were more sensitive to herbicide damage than the 4 leaf seedlings. To investigate difference in atrazine tolerance due to differential atrazine metabolism, 14C atrazine metabolism was examined in 1, 2 and 4 leaf ‘Alamo’ seedlings. 24-48 hours after exposure, 4 leaf seedlings metabolized atrazine at a greater rate than 1 and 2 leaf stage seedlings.

Sustainable weed control in managed agricultural systems requires the judicious use of multiple weed control tactics and prevents over-reliance on any one tactic. In this context, sustainable weed management plays a critical role in the mitigation of one of agriculture's most pressing problems- herbicide resistance. Research conducted in Virginia sought to explore the effects of integrating multiple weed management tactics in corn and cool-season turfgrass. Additionally, research was conducted to confirm nicosulfuron and glyphosate herbicide resistance in Virginia johnsongrass and elucidate the molecular mechanisms conferring those resistances. Rye and hairy vetch cover crop residues, combined with reduced rates of preemergence herbicide and postemergence glyphosate applications, were shown to provide sufficient weed control and corn yield. Cover crop type or residue level did not augment weed control in corn production systems, but the use of glyphosate was essential for late-season weed control. Rye and vetch biculture as a cover crop increased corn yield compared to rye cover crop alone. In cool-season turfgrass, the addition of reduced preemergence herbicide rates to corn gluten meal, an organic herbicide product, reduced crabgrass 25%. Moreover, control was dependent on herbicide choice. Herbicides applied at half of recommended labeled rates or less did not control crabgrass at a commercially-acceptable level, regardless of corn gluten meal addition. In field experiments, Virginia johnsongrass expressed resistance to nicosulfuron and glyphosate. Glyphosate at 0.88 kg ae ha-1 controlled johnsongrass 65%. Nicosulfuron at 0.14 kg ai ha-1 controlled the same population 10%. Greenhouse experiments confirmed differential sensitivity of putative herbicide-resistant johnsongrass seedlings to nicosulfuron and glyphosate when compared to a susceptible population. Herbicide resistance was not conferred via target-site mutation. Five ALS-gene site mutations were confirmed absent in Virginia johnsongrass, while three others were located in coding regions that could not be elucidated in johnsongrass. Further investigations showed glyphosate resistance was not conferred via reduction in herbicide absorption or translocation. The susceptible johnsongrass caused an increase in a polar metabolite at Rf = 0.17 with concomitant reduction in glyphosate over time. Although the mechanism is not clear, these data suggests that glyphosate resistance in johnsongrass may be associated with differential metabolism. / Ph. D.