MOLECULAR-CLONING AND PHOTOPERIOD-REGULATED EXPRESSION OF GIBBERELLIN20-OXIDASE FROM THE LONG-DAY PLANT SPINACH

Spinach (Spinacia oleracea L.) is a long-day (LD) rosette plant in whi ch stem growth under LD conditions is mediated by gibberellins (GAs). Major control points in spinach are the later steps of sequential oxid ation and elimination of C-20 of C-20-GAs. Degenerate oligonucleotide primers were used to obtain a polymerase chain reaction product from s pinach genomic DNA that has a high homology with CA 20-oxidase cDNAs f rom Cucurbita maxima L. and Arabidopsis thaliana Heynh. This polymeras e chain reaction product was used as a probe to isolate a full-length cDNA clone with an open reading frame encoding a putative 43-kD protei n of 374 amino acid residues. When this cDNA clone was expressed in Es cherichia coli, the fusion protein catalyzed the biosynthetic sequence GA(53)--> GA(44)--> GA(19)--> GA(20) and GA(19)--> GA(17). This estab lishes that in spinach a single protein catalyzes the oxidation and el imination of C-20. Transfer of spinach plants from short day (SD) to L D conditions caused an increase in the level of all GAs of the early-1 3-hydroxylation pathway, except GA(53), with GA(20), GA(1), and GA(8) showing the largest increases. Northern blot analysis indicated that t he level of GA 20-oxidase mRNA was higher in plants in LD than in SD c onditions, with highest level of expression in the shoot tips and elon gating stems. This expression pattern of GA 20-oxidase is consistent w ith the different levels of GA(20), GA(1), and GA(8) found in spinach plants grown in SD and LD conditions.