Abstract
Satsuma mandarin (Citrus unshiu Marc.) has two GA 20-oxidase genes, CuGA20ox1 and CuGA20ox2, and the genomic sequence for CuGA20ox1 is shorter than that for CuGA20ox2, although the coding region of cDNA for CuGA20ox1 is slightly longer than that for CuGA20ox2. Southern blot analysis revealed that 12 Citrus cultivars examined and trifoliate orange [Poncirus trifoliata (L.) Raf.] have at least two types of GA 20-oxidase genes, such as CuGA20ox1 and CuGA20ox2 genes. CuGA20ox1 and CuGA20ox2 were differentially expressed in various tissues. CuGA20ox1 was expressed in almost all of the tissues investigated with relatively higher expression in vegetative than in reproductive tissues, whereas CuGA20ox2 was specifically expressed in flower buds just before anthesis. These distinct expression patterns of CuGA20ox1 and CuGA20ox2 imply that function of these two genes diverged in the process of evolution. The specific and relatively higher expression of CuGA20ox2 in flower buds would explain why GA-like activity was higher in Satsuma mandarin’s ovaries at anthesis. Transgenic Arabidopsis [Arabidopsis thaliana (L.) Heynh] plants ectopically expressing CuGA20ox1 or CuGA20ox2 were examined to elucidate the function of these two Satsuma mandarin genes. Phenotypic analysis revealed that both CuGA20ox1 and CuGA20ox2 caused elongated inflorescence but did not affect the timing of flowering in transgenic Arabidopsis as compared with wild-type controls. Ectopic expression of CuGA20ox1 and CuGA20ox2 significantly affected the levels of GA24 and GA34 on the non-13-hydroxylation pathway; GA24 decreased and GA34 increased. This observation indicates that both CuGA20ox1 and CuGA20ox2 accelerated the conversion of GA24, a substrate of a GA 20-oxidase, to GA9, a precursor of an active form of GA4. Likewise, on the early-13-hydroxylation pathway, ectopic expression of CuGA20ox1 significantly decreased GA19 and increased GA29 and GA8, inactive metabolites of 2-hydroxylation of GA20 and GA1, respectively, suggesting the activation of this biosynthetic pathway. CuGA20ox2 also had a tendency to activate the early-13-hydroxylation pathway although it increased only GA20 with a statistically significant difference. Taken together, we concluded that CuGA20ox1 and/or CuGA20ox2 activated both the early-13- and non-13-hydroxylation pathways for increasing active GAs, resulting in elongated inflorescences in transgenic Arabidopsis.
