The gene encoding tobacco gibberellin 3β-hydroxylase is expressed at the site of GA action during stem elongation and flower organ development

Gibberellin 3 beta-hydroxylase catalyzes the final step in the biosynthetic pathway leading to the plant hormone gibberellin (GA) and, therefore, the in vivo localization of this enzyme should give a direct indication of the site of synthesis of bioactive GAs in plants. We have isolated a cDNA clone, Nty (Nicotiana tabacum GA 3 beta-hydroxylase), which encodes a putative GA 3 beta-hydroxylase, by RI-PCR using RNA from tobacco shoot apices. Functional analysis, using an NTY protein expressed in Escherichia coli, revealed that Nty encoded an active GA SP-hydroxylase. A high expression level of Nty was observed in shoot spices, flowers, roots, young internodes but not in leaves or seeds. We performed more detailed expression analyses using in situ hybridization and histochemical analyses of the GUS activity in transgenic tobacco plants carrying an Nty promoter::GUS fusion gene. These studies revealed that expression of Nty was restricted to specific regions, including actively dividing and elongating cells in the various organs; rib meristem and elongation zones of shoot apices, tapetum and pollen grains in developing anthers and root tips, which are consistent with the sites of GA action. it is proposed that GA actions depend on the modulation of endogenous bioactive GA levels through the regulation of GA 3 beta-hydroxylase expression in situ.