Apple ripening-related cDNA clone pAP4 confers ethylene-forming ability in transformed Saccharomyces cerevisiae

• Published in: Plant physiology (Bethesda) 1993-07, Vol.102 (3), p.783-788
• Main Authors: Wilson, I.D, Zhu, Y, Burmeister, D.M, Dilley, D.R
• Format: Article
• Language: English
• Subjects: ACIDE ASCORBIQUE; ACIDO ASCORBICO; ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Agronomy. Soil science and plant productions; ANION; ANIONES; ARN MENSAJERO; ARN MESSAGER; Biological and medical sciences; CATION; CATIONES; Cloning, Molecular; Complementary DNA; Cultured cells; DNA, Complementary; Enzymes; ETHYLENE; Ethylene production; Ethylenes - metabolism; ETILENO; EXPRESION GENICA; EXPRESSION DES GENES; FER; Fruit - genetics; Fruit - physiology; Fundamental and applied biological sciences. Psychology; Growth regulators; HIERRO; Kinetics; MALUS PUMILA; Messenger RNA; Metabolism; Molecular Biology and Gene Regulation; Oxidases; OXIDORREDUCTASAS; OXYDOREDUCTASE; Plant physiology and development; Plants; Plasmids; Ripening; RNA; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - growth & development; Saccharomyces cerevisiae - metabolism; SUBSTANCE DE CROISSANCE VEGETALE; SUSTANCIAS DE CRECIMIENTO VEGETAL; TRANSFERENCIA DE GENES; TRANSFERT DE GENE; TRANSFORMACION GENETICA; TRANSFORMATION GENETIQUE; Transformation, Genetic; Yeasts
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.102.3.783

The apple ripening-related cDNA insert of clone pAP4 (G.S. Ross, M.L. Knighton, M. Lay-Yee 19921 Plant Mol Biol 19: 231-238) 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 is known to encode the enzyme 1-aminocyclopropane-1-carboxylate(ACC)oxidase (A.J. Hamilton, G.W. Lycett, D. Grierson [1990] Nature 346: 284-287; A.J. Hamilton, M. Bouzayen, D. Grierson [1991] Proc Natl Acad Sci USA 88: 7434-7437). The cDNA insert from the clone pAP4 was fused between the galactose-inducible promoter and the terminator of the yeast expression vector pYES2. Transformation of Saccharomyces cerevisiae strain F808- with this DNA construct and incubation of the yeast in the presence of D[+]-galactose allowed these cells to convert ACC to ethylene. The transformed yeast converted 1-amino-2-ethylcyclopropane-1-carboxylate isomers to 1-butene with the same 1R,2S-stereoselectivity as achieved by the native ACC oxidase from apples. Both ascorbate and Fe ions stimulated the rate of the production of ethylene from ACC by the transformed yeast, whereas 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 novel 1.2-kb mRNA that hybridized to the cDNA clone pAP4. We conclude that the cDNA sequence of the clone pAP4 encodes ACC oxidase