Apple Ripening - Related cDNA Clone pAP 4 Confers Ethylene - Forming Ability in Transformed Saccharomyces cerevisiae ’ lan

l h e apple ripening-related cDNA insert of clone pAP4 (C.S. ROSS, M.L. Knighton, M. Lay-Yee [1992] Plant MOI Biol 19: 231238) has previously been shown to have considerable nucleic acid and predicted amino acid sequence similarity to the insert of a tomato ripening-related cDNA clone (pTOM13) that i s known to encode the enzyme I-aminocyclopropane-I-carboxylate (ACC) oxidase (A.J. Hamilton, C.W. Lycett, D. Crierson [1990] Nature 346: 284-287; A.J. Hamilton, M. Bouzayen, D. Crierson 119911 Proc Natl Acad Sci USA 88: 7434-7437). l h e cDNA insert from the clone pAP4 was fused between the galactose-inducible promoter and the terminator of the yeast expression vector pYES2. lransformation of Saccharomyces cerevisiae strain F808with this DNA construct and incubation of the yeast in the presence of D[+]galactose allowed these cells to convert ACC to ethylene. l h e transformed yeast converted 1-amino-2-ethylcyclopropane-1-carboxylate isomers to I-butene with the same lR,2S-stereoseledivity as achieved by the native ACC oxidase from apples. Both ascorbate and Fez+ ions stimulated the rate of the production of ethylene from ACC by the transformed yeast, whereas Cu2+ and Co2+ were strongly inhibitory; these are features of ACC oxidase. Northern analysis of the total RNA from nontransformed and transformed yeast showed that the ability to convert the ACC to ethylene was correlated with the synthesis and accumulation of a nove1 1.2-kb mRNA that hybridized to the cDNA clone pAP4. We conclude that the cDNA sequence of the clone pAP4 encodes ACC oxidase.