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Characterization of an ethylene-induced esterase gene isolated from Citrus sinensis by competitive hybridization
Guang Yan Zhong1, Raphael Goren, Joseph Riov
1The Kennedy-Leigh Centre for Horticultural Research, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel Department of Biochemistry, North Carolina State University, Raleigh, NC 17695-7622, USA Agricultural Research Organization, Newe Ya'ar Research Center, P.O. Box 1021, Ramat Yishay 30095, Israel.
Abstract:
A simple new method, competitive hybridization, for identification of differentially regulated genes was used to isolate novel genes induced by ethylene in citrus (Citrus sinensis [L.] Osbeck cv. Shamouti) leaves. One of the isolated genes, an ethylene-induced esterase gene (EIE), was further characterized. The deduced protein sequence of this gene shows a similarity to those of several plant alpha/beta hydrolase gene family members, which are known to be involved in secondary metabolism. Northern blot analysis demonstrated 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 ethylene-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 identified in ethylene-treated leaves using anti-Pir7b antibodies. An alpha-naphthyl acetate esterase accumulated concomitantly with the increase in EIE protein in ethylene-treated citrus leaves. An enzyme activity assay followed by western analysis confirmed that the esterase was EIE.