ISOLATION AND EXPRESSION OF 3 GIBBERELLIN 20-OXIDASE CDNA CLONES FROM ARABIDOPSIS

Using degenerate oligonucleotide primers based on a pumpkin (Cucurbita maxima) gibberellin (CA) 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 GA(12) at C-20 to GA(15), GA(15), and the C-19 compound GA(9), a precursor of bioactive GAs; the C-20 tricarboxylic acid compound GA(25) was formed as a minor product. The expression products also oxidized the 13-hydroxylated substrate GA(53), but less effectively than GA(12). 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 GA(3) application, suggesting that GA biosynthesis may be controlled, at least in part, through downregulation of the expression of the 20-oxidase genes.