PLANT PHYSIOLOGY , Vol 108, Issue 3 1049-1057, Copyright © 1995 by American Society of Plant Biologists

DEVELOPMENT AND GROWTH REGULATION

Isolation and Expression of Three Gibberellin 20-Oxidase cDNA Clones from Arabidopsis

A. L. Phillips, D. A. Ward, S. Uknes, NEJ. Appleford, T. Lange, A. K. Huttly, P. Gaskin, J. E. Graebe and P. Hedden
IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Bristol, BS18 9AF, United Kingdom (A.L.P., D.A.W., N.E.J.A., A.K.H., P.G., P.H.)

Using degenerate oligonucleotide primers based on a pumpkin (Cucurbita maxima) gibberellin (GA) 20-oxidase sequence, six different fragments of dioxygenase genes were amplified by polymerase chain reaction from Arabidopsis thaliana genomic DNA. One of these was used to isolate two different full-length cDNA clones, At2301 and At2353, from shoots of the GA-deficient Arabidopsis mutant ga1-2. A third, related clone, YAP169, was identified in the Database of Expressed Sequence Tags. The cDNA clones were expressed in Escherichia coli as fusion proteins, each of which oxidized GA12 at C-20 to GA15, GA24, and the C19 compound GA9, a precursor of bioactive GAs; the C20 tricarboxylic acid compound GA25 was formed as a minor product. The expression products also oxidized the 13-hydroxylated substrate GA53, but less effectively than GA12. The three cDNAs hybridized to mRNA species with tissue-specific patterns of accumulation, with At2301 being expressed in stems and inflorescences, At2353 in inflorescences and developing siliques, and YAP169 in siliques only. In the floral shoots of the ga1-2 mutant, transcript levels corresponding to each cDNA decreased dramatically after GA3 application, suggesting that GA biosynthesis may be controlled, at least in part, through down-regulation of the expression of the 20-oxidase genes.

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