LE-ACS4, A FRUIT RIPENING AND WOUND-INDUCED 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE GENE OF TOMATO (LYCOPERSICON-ESCULENTUM) - EXPRESSION IN ESCHERICHIA-COLI, STRUCTURAL CHARACTERIZATION, EXPRESSION CHARACTERISTICS, AND PHYLOGENETIC ANALYSIS

ACC (1-aminocyclopropane-1-carboxylic acid) synthase is the key regula tory enzyme in the biosynthetic pathway of the plant hormone ethylene and is encoded by a highly divergent multigene family in tomato (Rottm ann, W. H., Peter, G. F., Oeller, P. W., Keller, J. A., Shen, N. F., N agy, B. P., Taylor, L. P., Campbell, A. D., and Theologis, A. (1991) J . Mol. Biol. 222, 937-961). Two members of the family, LE-ACS2 and LE- ACS4, are induced during fruit ripening and upon treatment of mature g reen fruits with exogenous ethylene (C2H4) in a dose-dependent manner. Both genes are superinduced by wounding of pericarp tissue during var ious stages of ripening. The wound-induced accumulation of LE-ACS2 mRN A is more rapid and greater than that of LE-ACS4. Both mRNAs accumulat e in the absence of protein synthesis, suggesting that their induction is a primary response to the inducer. The LE-ACS4 gene was isolated a nd structurally characterized. The function of the LE-ACS4 protein (53 ,509 Da, pI 5.4) was verified by expression experiments in Escherichia coli. The promoters of LE-ACS2 and LE-ACS4 contain potential cis-acti ng regulatory elements responsible for induction by ethylene, wounding , and anaerobiosis. In addition, elements for binding the transcriptio nal factors EmBP1, GBF-1, and OCSBF-1 are also present. Phylogenetic a nalysis of 20 ACC synthases from dicots and monocots indicate that the LE-ACS2 and LE-ACS4 proteins belong to an unique sublineage that incl udes an additional member of the tobacco family, NT-ACS1. The divergen ce of this sublineage is a relatively recent event in the evolution of ACC synthase protein.