Identification of a tomato gene for the ethylene-forming enzyme by expression in yeast.
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.
Hamilton
A J
AJ
Department of Physiology and Environmental Science, University of Nottingham School of Agriculture, Sutton Bonington, Loughborough, United Kingdom.
Bouzayen
M
M
Grierson
D
D
D000594:Amino Acid Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular / Q000379:methods*; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D005030:Ethylenes / Q000378:metabolism*; D017343:Genes, Plant; D007700:Kinetics; D008969:Molecular Sequence Data; D010944:Plants / Q000201:enzymology / Q000235:genetics*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*
D004271:DNA, Fungal; D005030:Ethylenes; D011994:Recombinant Proteins; D012313:RNA; C036216:ethylene; D000594:Amino Acid Oxidoreductases; C059460:1-aminocyclopropane-1-carboxylic acid oxidase