Characterisation, and inhibition of graminicide-resistant and -susceptible maize acetyl-coenzyme A carboxylase isozyme

Acetyl-coenzyme A carboxylase (ACCase) isozymes were purified from etiolated sethoxydim-resistant and -susceptible maize coleoptiles. Sethoxydim-susceptible maize yielded two ACCase preparations, ACCase220 (extrachloroplastic) and ACCase240 (chloroplastic). ACCase220 showed no affinity for Orange A dye, (acetyl-CoA mimic), was purified 79 fold and contained biotinylated proteins with molecular masses of 220 and 85 kDa. In contrast, ACCase240 bound to Orange A dye was purified approximately 68 fold, and contained 240 and 85 kDa biotinylated proteins. Purification of ACCase from sethoxydim-resistant maize yielded similar results. However, resistant maize contained 3.0 fold more ACCase240 with a 14.5 fold lower specific activity than sethoxydim-susceptible hybrids. Kinetic parameters and cyclohexanedione inhibition of ACCase isozymes were compared for resistant and susceptible hybrids. These results indicated that tolerant ACCase240 was 3.7, $>$77 and 12.8 fold more tolerant to clethodim, sethoxydim and tralkoxydim inhibition, respectively than ACCase from susceptible maize hybrids. ACCase220 from tolerant maize was $>$25 and 7.2 fold more tolerant to sethoxydim and tralkoxydim inhibition, respectively, than susceptible ACCase220. However, sethoxydim-resistant and -susceptible ACCase220 had similar clethodim IC$sb{50}$ values. Comparison of ACCase240 kinetic parameters indicated that the resistant hybrid contained an enzyme which had similar acetyl-CoA K$sb{rm m}$ to the wild type enzyme. Resistant ACCase240 had a k$sb{rm cat}$ 5.5 fold lower than susceptible ACCase240. Using an in situ ACCase activity assay it was determined that ACCase and $beta$-methylcrotonyl-coenzyme A carboxylase (MCCase) co-localised to prolamellar bodies and thylakoid membranes within the chloroplast and to the external endoplasmic reticulum surface. Furthermore, both ACCase and MCCase activity were susceptible to sethoxydim inhibition in susceptible but not in resistant hybrids. Biochemical properties of ACCase isozymes were examined and it was determined that maize ACCase functions as a multimeric enzyme. Dithio-bis(succinimidylpropionate) cross-linking, two-dimensional electrophoresis, photoaffinity labelling, matrix assisted laser desorption/ionisation time-of-flight mass spectrometry, and gel permeation chromatography revealed that multiple, low molecular mass proteins were specifically associated with the ACCase complex. The results presented in the following dissertation prove that maize ACCase does not function solely as a homodimer. Sethoxydim resistance is a result of overexpression of a less efficient chloroplastic isozyme and expression of an altered herbicide-resistant ER localised isozyme.