Gibberellin 2-oxidation and the SLN gene of Pisum sativum

Two cDNAs encoding gibberellin 2-oxidases were isolated from maturing pea s eeds. The first, PsGA2ox1, was isolated by activity screening of a Lambda-Z AP cDNA library excised into phagemid form and expressed in Escherichia col i. The second, PsGA2ox2, was obtained initially as a PCR product using dege nerate primers designed according to conserved regions of plant 2-oxoglutar ate-dependent dioxygenases. E. coli heterologous expression products of PsG A2ox1 and PsGA2ox2 converted GA(1) to GA(8), as shown by HPLC-radiocounting , and gas chromatography-MS. PsGA2ox1 converted GA(20) to GA(29), but GA(20 ) was a poor substrate for the PsGA2ox2 expression product. Furthermore, Ps GA2ox1 converted GA(29) to GA(29)-catabolite at a low level of efficiency w hile PsGA2ox2 did not catalyse this step. A cDNA of PsGA2ox1 isolated from plants of genotype sin contained a single base deletion which was predicted to produce a truncated protein and gibberellin 2-oxidase activity could no t be demonstrated from this cDNA. A 10 bp size difference between the intro ns of the SLN and sin PsGA2ox1 genes was used to show co-segregation betwee n the SLN and sin phenotypes and the size of the PCR products. PsGA2ox1 tra nscripts were more abundant in cotyledons than in shoots, while the reverse was the case for PsGA2ox2. The expression patterns of the genes, together with the effects of the sin mutation, indicate that PsGA2ox1 plays a major role in GA(20) deactivation in both shoots and maturing seeds, while the Ps GA2ox2 gene might be important for GA1 deactivation in the shoot.