Functional Analysis Of A 2OG-Fe (II) Oxidoreductase Involved In GA Biosynthesis And Interaction With ABA In Arabidopsis Thaliana

Seed germination is regulated by ABA and GA. ABA can inhibit germination and induce dormancy; GA stimulate seed germination and release dormancy. Recent studies have shown that the balance of abscisic acid (ABA) : gibberellin (GA) levels was the major factor. However, because GA and ABA signaling transduction are complicated, so we still know little about its regulation mechanism.In this study, using the T-DNA library we isolated one ABA mutant. Through genetic analysis of the mutatant we found its phenotype could be stable geneticed, so we named the mutant gai4 (gain function of ABA insensitive 4, gai4). Using TAIL-PCR technology we found that T-DNA was inserted into the promoter region of GAI4. In Arabidopsis, GAI4 encode a 2OG-Fe(II) oxidoreductase. Under normal conditions, the expression levels of GAI4 has been weakened. After the ultra-induced expression of estradiol, the expression levels of GAI4 was increased about 50 times. Through bioinformatics and biochemistry analysis we found that GAI4 maybe involved in the GA biosynthesis. We further analyzed its physiological function. Results showed that over-expression lines were insensitive to NaCl, H₂O₂ on germination and they did not need cold acclimation to promote germinate. Under red and far-red light, the over-expression lines were growing better than WT and gai4 on germination and hypotocyl length. These results gived us a clue that GAI4 was involved of GA biosynthesis. Using biochemistry analysis, we found that GAI4 coded one member of GA20oxs and it could use GA₁₂ as substrate to produce one new type of GA. Then we found GAI4 had higher expression in root by RT-PCR. Fluorescence co-localization result of GAI4::GFP fusion proteins using transient expression in protoplast of mesophyll cells showed GAI4 enzyme protein localized in cytoplasm. GAI4 its expression could induced by cold and red light. In gene expression aspects it could regulate the gene related to DELLA and ABA metabolic.In summary, we believe that GAI4 is involved in GA biosynthesis and can produce one new type of GA, then specially regulate the seed germination. Through the functional analysis of GAI4 can help us better understand the interactions between GA and ABA signal transduction.