Regulation of 1-aminocyclopropane-1-carboxylate oxidase gene expression during leaf ontogeny in white clover

The expression of two 1-aminocyclopropane-1-carboxylate (ACC) oxidase (E. C. 1.4.3)(ACO) genes, TR-ACO2 and TR-ACO3 from white clover has been examined in leaf tissue in response to physiological stimuli. In detached mature-green leaves, the expression of TR-ACO3 (a leaf-senescence-associated ACC oxidase) is induced over a 6 h time-course, while expression of TR-ACO2 (expressed constitutively in mature-green leaf tissue) decreased over the same time-course. An almost identical pattern of expression was observed with detached mature-green leaves treated with ethylene, while wounding also did not influence the timing of TR-ACO3 induction, significantly. No increase in overall ACO enzyme activity was detectable in crude extracts from wounded and detached leaf tissue, but after separation of these extracts using hydrophobic interaction column chromatography, two ACO isoforms were identified in non-wounded mature-green leaves (designated MGI and MGII), and the specific activity of one of these (MGII) increased in wounded tissues. In attached leaf tissue, TR-ACO2, but not TR-ACO3 expression was induced by treatment with ethylene; an induction that was not observed with prior treatment of plants with 1-MCP. This induction in response to ethylene was also observed at the translational level using antibodies to TR-ACO2. Analysis, in silico, of the 5′ flanking sequences to identify putative transcriptional binding domains reveals that both the TR-ACO2 and TR-ACO3 5′ flanking sequence contain domains that are associated with an ethylene-response. The significance of these observations in terms of how the expression of these ACO genes is regulated during leaf ontogeny in vivo is discussed.