Identification of a tomato gene for the ethylene-forming enzyme by expression in yeast

Abstract

The ethylene-forming enzyme (EFE), which catalyzes the last step in the biosynthesis of the plant hormone ethylene, has never been purified and no molecular probes are available. Recently, a putative cDNA clone for tomato EFE (pTOM13) has been identified by inhibiting ethylene synthesis with an antisense gene expressed in transgenic plants. A direct test of its function has been made by expression of a pTOM13 gene in Saccharomyces cerevisiae. After cloning artefacts were discovered in the 5' region of the cDNA, a corrected cDNA (pRC13) was created by the fusion of the 5' end of a genomic clone to the 3' end of the cDNA and expressed in S. cerevisiae. Cultures of transformed yeast converted 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene, whereas control cells did not. This EFE activity displays similar characteristics to EFE found in plant tissue: it converts the trans isomer of the ACC analogue 1-amino-2-ethylcyclopropane-1-carboxylic acid to 1-butene in preference to the cis isomer, and it is strongly inhibited by cobaltous ions and 1,10-phenanthroline. Furthermore, information gained from the activity of effectors on yeast EFE activity supports the hypothesis that EFE is one of a group of hydroxylase enzymes.