Characterisation of ACC oxidase isoforms during leaf maturation and senescence in white clover (Trifolium repens L.) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand

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One-aminocyclopropane-1-carboxylic acid (ACC oxidase, the enzyme which catalyses the final step in the ACC-dependent pathway of ethylene biosynthesis in plants, has been studied during leaf maturation and senescence in white clover (Trifolium repens L.). The coding regions from two white clover ACC oxidase genes, designated TR-ACO2 (expressed in mature green leaves) and TR-ACO3 (expressed in senescent leaves), have been expressed in E. coli as fusion proteins. The expression of the two proteins has been optimised in terms of induction time with isopropyl-β-D-thiogalactopyranoside (IPTG) and IPTG concentration. The solubility of the fusion proteins was low but lysis buffer containing 0.5 % (w/v) SDS or 0.5 % (v/v) Triton X-100 produced a higher protein yield. The recombinant TR-ACO2 and TR-ACO3 proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography and had an apparent molecular mass of 38 kDa. Enzyme activities of the purified TR-ACO2 and TR-ACO3 fusion proteins were 0.34 and 0.23 nmol ethylene/h/mg protein, respectively. Activity in vitro of ACC oxidase, extracted from both mature green and senescent leaf tissues, was observed to be very labile at 20°C with lower temperature, ascorbate and 1,10-phenanthroline (PA) required to help stabilise the enzyme activity in vitro during enzyme extraction and purification. Three isoforms of ACC oxidase, one from mature green leaves, designated MGI and two from senescent leaves, designated SEI and SEII, have been identified. Two of the three isoforms (MGI and SEII) were purified to homogeneity as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis with Coomassie Brilliant Blue staining and western analysis. The purified isoforms MGI and SEII had specific enzyme activities of 25.2 and 29.8 nmol ethylene/h/ mg protein at pH 7.5 with approximately 100- and 144-fold purification, respectively. During purification, both isoforms were recognised by an antibody raised against the protein product of TR-ACO2 expressed in E. coli. The native molecular mass of the purified isoforms MGI and SEII was determined to be 37.5 kDa by size exclusion chromatography and molecular masses of MGI and SEII were observed to be 37 kDa and 35 kDa, respectively by SDS-PAGE analysis. The data indicate that both isoforms are active as monomers. Both isoforms were found to be neutral or near neutral proteins with apparent isoelectric points (pIs) of 7.36 for isoform MGI and 7.0 for SEII determined by chromatofocusing. The Optimal pHs for MGI and SEII were 7.5 and 8.5, respectively. The two isoforms also displayed differences in apparent K m and V max values for the substrate ACC. The K m values for MGI and SEII were determined to be 39.7 μM and 110.0 μM, respectively. SEII had a higher V max value for ACC than MGI. The data indicate that MGI displays a higher affinity for ACC, SEII requires a higher ACC concentration to achieve the higher enzyme activity and can operate in an enviroment with higher levels of ACC. In addition, both isoforms exhibited absolute requirements for the co-substrate ascorbate and the cofactors bicarbonate and ferrous iron for maximal enzyme activity in vitro with different optimal concentrations for ascorbate and ferrous iron. The data suggest that the two ACC oxidase isoforms are differentially regulated by pH and ACC concentration and are activated by different levels of cofactors. The significant differences between the two isoforms (pH optimum and K m for ACC) may reflect the distinct physiological status of the leaf tissue in which each isoform is active. These results show that now widely observed transcriptional regulation of the ACC oxidase gene family is also expressed in terms of differential regulation of isoforms of this enzyme in higher plants.
Clover genetics