Characterisation of ACC oxidase 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 at Massey University

ACC oxidase, the enzyme which converts 1-aminocyclopropane-l-carboxylic acid (ACC) to ethylene, has been studied in leaves of a single genotype of white clover (Trifolium repens L., Genotype 10F) during leaf maturation and senescence. Leaf senescence in genotype 10F is associated with an increase in both ACC content and ethylene evolution of the leaves. The increase in levels of ACC slightly precedes that of ethylene production, but occurs concomitantly with the onset of senescence (as judged by chlorophyll loss). The coding regions of two distinct ACC oxidases were generated from leaf tissue of genotype 10F using RT-PCR with degenerate oligonucleotide primers. The coding regions were designated TR-ACO1 and TR-ACO2. TR-ACO1 and 2 are 84 % and 87 % similar in nucleotide and amino acid sequence respectively. Genomic Southern analysis using these regions as probes confirmed that both sequences are encoded by distinct genes, but also suggested that there may be additional genes closely related to both TR-ACO1 and TR-ACO2. Gene expression studies during leaf maturation and senescence were undertaken using these regions as probes. TR-ACO1 hybridised to a single RNA transcript of ca. 1.35 Kb on the northern blot. Expression of this transcript was high in mature green leaves but declined as the leaves senesced. By contract, TR-ACO2 hybridised equally to two RNA transcripts of ca. 1.17 Kb and 1.35 Kb on the northern blot, and unlike expression of TR-ACO1, the levels of these transcripts were low in mature green leaves and increased as the leaves senesced. The 3'-UTRs of TR-ACO1 and TR-ACO2 were generated using 3'-RACE (Randomly Amplified cDNA Ends). Repeating the genomic Southern analysis with these regions as probes indicated that the gene for TR-ACO1 may be polymorphic, and that there may be an additional ACC oxidase gene in the genome that encodes a transcript with a similar coding region, but divergent 3'-UTR to TR-ACO2. The 3'-UTRs of TR-ACO1 and 2 confirmed the expression patterns of their cognate coding regions in northern analysis, except that the 3'-UTR of TR-ACO2 hybridised only to the 1.35 Kb and not the 1.17 Kb transcript. ACC oxidase activity assayed in vitro correlated with the levels of gene expression of TR-ACO1 but not the senescence-associated TR-ACO2, with the activity being highest in mature green leaves and declining as the leaves senesced. The decrease in activity was greatest when expressed on a per unit fresh weight basis (ca. 8-fold) than per unit protein basis (ca. 3-fold). The extraction and assay conditions altered in this study were not able to prevent the decline in ACC oxidase activity in vitro that occurred during leaf senescence. Polyclonal antibodies raised against the translation products of TR-ACO1 and 2 expressed in E. coli recognised a protein of the expected size (ca. 36.4 kDa) for ACC oxidase using western analysis. The pattern of protein accumulation recognised by the antibodies raised against TR-ACO1 (in rabbits) broadly matched gene expression of TR-ACO1 and activity of ACC oxidase. That is, antibody recognition was highest in mature green leaves and declined as the leaves senesced. By comparison, the antibodies raised against TR-ACO2 (in rats) recognised a protein of the same size with weak avidity. The pattern of accumulation was similar to that observed with the TR-ACO1-raised-antibodies, and therefore not consistent with the gene expression pattern of TR-ACO2. It was concluded that these antibodies were cross-reacting with the same protein as that recognised by the TR-ACO1-raised-antibodies and that the lower enzyme activity was due to lower TR-ACO1 ACC oxidase protein. Wounding mature green leaves increased TR-ACO2 gene expression, but as observed for the senescing leaves, increased gene expression of TR-ACO2 was not correlated with increased protein accumulation or ACC oxidase enzyme activity. Potential explanations as to why the increased gene expression of TR-ACO2 during leaf senescence (or wounding) is not accompanied by increased protein accumulation and ACC oxidase activity are discussed.