Regulation of ethylene biosynthesis in Festuca novae-zelandiae (Hack.) Cockayne and in Festuca aruninaceae (Schreb.) in response to a water deficit : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Plant Biology at Massey University, Palmerston North, New Zealand

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Changes in ethylene evolution and the associated biosynthetic enzyme ACC oxidase to a water deficit, were examined in intact leaves of Fostuca novae-zelandiae and F. arundinacea cultivar 'Roa' (syn. Schedonorus phoenix). The aim was to establish a role, or otherwise, for ACC oxidase as a regulator of ethylene biosynthesis in response to a water deficit. While ACC synthase has long been recognised as the major rate-limiting enzyme in ethylene biosynthesis, there is mounting evidence to suggest that ACC oxidase may also regulate the ethylene biosynthesic pathway in higher plants. Leaf tissues from the two species were harvested at regular intervals during the experimental dry-down, and dissected into two leaf zones, regions enclosed by the ligule. comprising the meristematic and elongating leaf zone (the enclosed tissue), and exposed regions composing the mature green leaf zones. Leaf proline content and the rate of leaf elongation (LER) were used as internal and external indicators of physiological changes in response to the water-deficit. Ethylene evolution in response to a water-deficit was found to be tissue-specific in F.arundinacea. In the rapidly expanding leaf zones, i.e. enclosed tissue, ethylene was maintained at levels similar to control tissue. In the mature green regions of leaves, ethylene followed changes in the leaf elongation rate (LER) with observed peaks in ethylene evolution occurnng approximately 48 hours after a rapid decline in the LER. This burst of ethylene was found to precede any accumulation of proline. Increases in the proline content in both leaf zones, only became significant after the ethylene evolution had subsided to below base levels. This stage-specific ethylene evolution in leaves suggests preferential protection of the rapidly expanding leaf cells, an observation that has been documented by other authors. ACO specific enzyme activity was greatest at soil water contents of ca. 9% in the enclosed and 10% in the exposed leaf tissues of F.arundinacea. On further purification of the enzyme, two novel proteins were recognised by polyclonal antibodies in water-stressed leaves of F.arundinacea. A 32 kDa protein was identified in the enclosed leaf tissue and a 37 kDa protein was identified in the exposed leaf tissue, by SDS-PAGE. These proteins eluted from a Mono Q column at different points in the separation process, i.e at salt concentrations of 320-340 and 300-320 mM NaCI respectively, indicating that they may represent two distinct isoforms of the ACO enzyme. Both proteins are active at pH 7.5 with saturating substrate (ACC) and co-substrate (Na ascorbate) concentrations of 1 mM and 30 mM respectively, and co-factor concentrations of 0.02 mM Fe² + and 30 mM NaHCO₃. When compared with results from western analyses, maximum specific enzyme activity correlated well with the water-deficit induced protein from partially purified enclosed leaf tissue, but only loosely with the protein identified in the exposed leaf tissue. The presence of high molecular weight proteins in both the crude and the purrfied (Mono Q) leaf extracts of F.arundinacea together with the novel proteins, suggests that the ACO enzyme in this species may exist as a dimer In F.novae-zelandiae, the presence of high molecular weight molecules m the crude and partially purified (Sephadex G-25) extracts also suggests dimensation of the enzyme in this species. From this study however, it is not possible to establish a clear regulatory role for the ACO enzyme in ethylene biosynthesis in either F.arvndinacea or F.novae-zelandiae While two novel water-deficit-induced proteins were associated with increased ACO activity in purified leaf extracts of F. amndinacea, there was no obvious correlation between ethylene evolution and enzyme activity.
Plant enzymes, Ethylene -- Synthesis, Effect of drought on fescue