Feed-back regulation of gibberellin biosynthesis and gene expression in Pisum sativum L.

Treatment of tall and dwarf (3 beta-hydroxylase impaired) genotypes of pea (Pisum sativum L.) with the synthetic, highly active gibberellin (GA), 2,2-dimethyl GA(4), reduced the shoot contents of C-19-GAs, including GA(1), and increased the concentration of the C-20-GA, GA(19). In shoots of the slender (la cry(s)) mutant, the content of C-19-GAs was lower and GA(19) content was higher than in those of the tall line. Metabolism of GA(19) and GA(20) in leaves of a severe (na) GA-deficient dwarf mutant was reduced by GA treatment. The results suggest feedback regulation of the 20-oxidation and 3 beta-hydroxylation reactions. Feed-back regulation of GA 20-oxidation was studied further using a cloned GA 20-oxidase cDNA from pea. The cDNA, Ps074, was isolated using polymerase chain reaction with degenerate oligonucleotide primers based on pumpkin and Arabidopsis 20-oxidase sequences. After expression of this cDNA clone in Escherichia coli, the product oxidized GA(12) to GA(15), GA(24) and the C-19-GA, GA(9), which was the major product. The 13-hydroxylated substrate GA(53) was similarly oxidized, but less effectively than GA(12), giving mainly GA(44) with low yields of GA(19) and GA(20). Ps074 hybridized to polyadenylated RNA from expanding shoots of pea. Amounts of this transcript were less in the slender genotype than in the tall line and were reduced in GA-deficient genotypes by treatment with GA(3), suggesting that there is feed-back regulation of GA 20-oxidase gene expression.