Characterization of an ethylene-induced esterase gene isolated from Citrussinensis by competitive hybridization

A simple new method, competitive hybridization, for identification of diffe rentially regulated genes was used to isolate novel genes induced by ethyle ne in citrus (Citrus sinensis [L.] Osbeck cv. Shamouti) leaves. One of the isolated genes, an ethylene-induced esterase gene (EIE), was further charac terized. The deduced protein sequence of this gene shows a similarity to th ose of several plant alpha/beta hydrolase gene family members, which are kn own to be involved in secondary metabolism. Northern blot analysis demonstr ated that EIE mRNA was induced by ethylene within 4 h and accumulated to a very high level 24 h after the initiation of ethylene treatment. Induction of EIE by ethylene could be counteracted by 1-methylcyclopropene, a potent ethylene perception inhibitor, indicating that the expression of EIE is eth ylene-dependent. The bacterially expressed protein of EIE was recognized by antiserum against Pir7b, a naphthol AS esterase induced in rice by the non -host pathogen, Pseudomonas syringae pv. syringae. The EIE protein was iden tified in ethylene-treated leaves using anti-Pir7b antibodies. An alpha -na phthyl acetate esterase accumulated concomitantly with the increase in EIE protein in ethylene-treated citrus leaves. An enzyme activity assay followe d by western analysis confirmed that the esterase was EIE.