Topic 22.4

Cloning of the Gene That Encodes ACC Synthase

A major hurdle in the understanding of ethylene biosynthesis was the difficulty in purifying the low abundance, unstable protein and the cloning of the corresponding gene. Researchers overcame this obstacle by raising antisera against partly purified ACC synthase from zucchini fruit slices that were induced to produce large amounts of ethylene. The resulting antisera contained a mixture of antibodies, including antibodies to ACC. The investigators then removed the contaminating antibodies by allowing them to bind to proteins extracted from zucchini tissue that lacked the enzyme. The key to the success of this procedure was the availability of tissue largely lacking the enzyme with which to subtract the non-ACS antibodies from the polyclonal antibody mixture. This circumvented the need for highly purified ACC synthase protein. The purified ACC synthase antibodies were then used to isolate the gene encoding the enzyme (Sato and Theologis 1989).

Once the gene encoding ACS was isolated, it could be used to express the ACC synthase protein in the bacterium E. coli at high levels and isolate purified ACC synthase in large amounts (Sato et al. 1991). The cloned zucchini gene also allowed genes encoding ACC synthase to be isolated from numerous other plant species, using low stringency hybridization and degenerate PCR. The deduced amino acid sequences of ACC synthase genes were found to show a high degree of similarity to the aminotransferase superfamily of enzymes. These enzymes catalyze the transfer of amino groups among amino acids and require pyridoxal phosphate as a cofactor.