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Molecular Cloning of GA 2-Oxidase3 from Spinach and Its Ectopic Expression in Nicotiana sylvestris

Lee, Dong Ju, Zeevaart, Jan A.D.
Plant physiology 2005 v.138 no.1 pp. 243-254
Spinacia oleracea, spinach, vegetable crops, plant proteins, oxygenases, complementary DNA, gene expression, gene overexpression, messenger RNA, recombinant fusion proteins, gibberellins, biosynthesis, biochemical pathways, Nicotiana sylvestris, tobacco, transgenic plants, photoperiod, Escherichia coli, phenotypic variation, plant morphology, seed germination, hypocotyls, root growth, plant biochemistry, plant genetics, plant physiology, nucleotide sequences, amino acid sequences
Previous work has shown that 13-hydroxylated gibberellins (GAs) are predominant in the long-day (LD) plant spinach (Spinacia oleracea; GA₅₃, GA₄₄, GA₁₉, GA₂₀, GA₁, GA₈, and GA₂₉). Also present in spinach are 2[beta]-hydroxylated C₂₀-GAs: GA₉₇, GA₉₈, GA₉₉, and GA₁₁₀. Levels of the most abundant GA, GA₉₇, decreased when plants were transferred from short photoperiods (SD) to LD. When [¹⁴C]GA₅₃ was fed to spinach plants, more GA₅₃ was converted to GA₉₇ in SD than in LD, and more radioactive GA₂₀ was formed in LD than in SD. SoGA2ox3, encoding a GA 2-oxidase, was isolated from spinach. The recombinant protein converted only two C₂₀-GA precursors, GA₁₂ and GA₅₃, to their respective products, GA₁₁₀ and GA₉₇. GA2ox3 competes with GA20ox1 for their common substrate, GA₅₃. In SD, deactivation to GA₉₇ prevails, whereas in LD conversion to GA₂₀ 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 GA₅₃ and GA₁ were 3- to 5-fold lower in transgenic plants than in wild type, whereas the levels of GA₉₇ and GA₁₁₀ 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 GA₁ itself.