Molecular cloning of GA 2-oxidase3 from spinach and its ectopic expression in Nicotiana sylvestris

Previous work has shown that 13- hydroxylated gibberellins ( GAs) are predominant in the long- day ( LD) plant spinach ( Spinacia oleracea; GA(53), GA(44), GA(19), GA(20), GA(1), GA(8), and GA(29)). Also present in spinach are 2 beta- hydroxylated C-20- GAs: GA(97), GA(98), GA(99), and GA(110). Levels of the most abundant GA, GA(97), decreased when plants were transferred from short photoperiods ( SD) to LD. When [ 14 C] GA 53 was fed to spinach plants, more GA 53 was converted to GA 97 in SD than in LD, and more radioactive GA 20 was formed in LD than in SD. SoGA2ox3, encoding a GA 2- oxidase, was isolated from spinach. The recombinant protein converted only two C-20- GA precursors, GA(12) and GA(53), to their respective products, GA(110) and GA(97). GA2ox3 competes with GA20ox1 for their common substrate, GA(53). In SD, deactivation to GA(97) prevails, whereas in LD conversion to GA(20) is favored. Transcript levels of SoGA2ox3 were higher in shoot tips than in blades, petioles, and young leaves. Ectopic expression of SoGA2ox3 in the long- day plant Nicotiana sylvestris showed a range of dwarf phenotypes, such as reduced germination, short hypocotyl and stem, dark- green leaves, and late flowering, but normal flowers and seed production. The levels of GA(53) and GA(1) were 3- to 5- fold lower in transgenic plants than in wild type, whereas the levels of GA(97) and GA(110) increased 3- to 6- fold in transgenic plants. It is concluded that genetic manipulation of plant stature by increasing deactivation of precursors of active GA is more advantageous than increased deactivation of bioactive GA(1) itself.