Isolation and expression of three gibberellin 20-oxidase cDNA clones from Arabidopsis.
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.
Phillips
A L
AL
IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, United Kingdom.
Ward
D A
DA
Uknes
S
S
Appleford
N E
NE
Lange
T
T
Huttly
A K
AK
Gaskin
P
P
Graebe
J E
JE
Hedden
P
P
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D018744:DNA, Plant; D004926:Escherichia coli; D006899:Mixed Function Oxygenases / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction
D017931:DNA Primers; D018076:DNA, Complementary; D018744:DNA, Plant; D006899:Mixed Function Oxygenases; C089210:gibberellin, 2-oxoglutarate-oxygen oxidoreductase (20-hydroxylating, oxidizing)