Date of Award
Doctor of Philosophy
Cyanide is produced throughout a plant's life cycle alongside the hormone ethylene by oxidation of 1-aminocyclopropane-1-carboxylic acid. Production increases during certain developmental stages such as seed germination, seedling elongation, fruit ripening and senescence. Abiotic stresses increase ethylene production giving rise to `stress cyanide'. Cyanide also comes from metabolism of cyanogenic compounds. Cyanide is however, a toxic chemical which readily binds to metallo-enzymes inhibiting primary metabolic processes. Plants have mechanisms to maintain cyanide homeostasis such as the β-cyanoalanine pathway whereby cysteine reacts with cyanide forming β--cyanoalanine, mediated by β-cyanoalanine synthase and cysteine synthase. A dual nitrilase 4 enzyme then converts the β-cyanoalanine into asparagine or aspartate and ammonium. Studies have suggested that the physiological function of the pathway is not restricted to detoxification and assimilation of excess cyanide. The overall research goal was to investigate the role of the pathway in plant tolerance to water deficit and exogenous cyanide exposure in Arabidopsis thaliana. The first objective was to investigate responsiveness of the pathway to duration and intensity of water deficit and cyanide exposure. The second was to investigate the contribution of enzymes associated with the pathway to cyanide metabolism. The questions addressed were whether there is enzymatic redundancy in enzymes associated with the first step of cyanide detoxification and whether there is pathway redundancy between the β-cyanoalanine and an alternative sulfurtransferase pathway. A. thaliana Col-0 and three SALK-line mutants with a T-DNA insertion for the genesAtCysA1, AtCysC1 and AtNIT4 were grown and exposed to water stress. Physiological and biochemical measurements were taken. The results showed a transient increase in cyanide concentration and β-cyanoalanine synthase activity on exposure to stress. The response pattern was similar regardless of intensity or duration of stress. Knocking out AtCysA1 or AtCysC1 did not impair the ability of plants to metabolize cyanide and tolerate stress i.e the enzymes were functionally redundant. The AtNIT4 mutant however, was impaired in cyanide metabolism and exhibited a sensitive phenotype under both stresses, suggesting that the cyanoalanine pathway is the sole pathway in cyanide detoxification. The results show that the pathway may be an important tool in improvement of plant tolerance to abiotic stress.
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