Functional Analysis Of Cytokinin Oxidase/Dehydrogenase 3 In Rice(Oryza Sativa L.)

Cytokinins(CKs)are a kind of important signaling molecules in planta and plays a crucial role in many aspects of plant growth and development,including embryogenesis,root maintenance and budding of meristems,and vascular development.Currently,cytokinin oxidase/dehydrogenase(CKX)is considered to be the only way to degrade cytokinin in plants.There are 11 cytokinin oxidases in rice.Only OsCKX2,OsCKX4 and OsCKX9 have been reported,while the biological functions of other OsCKX genes have not been studied.In this work,we screened OsCKXs gene mutants and found that the OsCKX3 regulates rice leaf inclination.By means of phenotypic observation,expression pattern analysis,subcellular localization,in vitro enzymatic assays and histochemical observation,the function of OsCKX3 and the molecular mechanism of regulating leaf inclination were preliminarily elucidated.The major results are list as follows:(1)Phenotype analysis showed that osckx3 mutant exhibited a reduced leaf inclination phenotype at different stages of rice growth and development.The mutant also showed the phenotypes of abnormal root initiation,sword leaf broadening and grain size enlargement.The histochemical analysis of WT,osckx3 and 35S::OsCKX3 overexpression lines show that the change of leaf inclination was mainly caused by abnormal lamina joint that osckx3 mutant has a longer lamina joint and thicker adaxial cells;(2)Expression of OsCKX3 in different rice tissues was detected by real-time fluorescence quantitative PCR,and it was found that OsCKX3 was highly expressed in the lamina joint and relatively high in the root.GUS staining further verified the results of tissue expression,and significant GUS staining results were found in the lamina joint,principal root,root primordium,root hair primordium,flag leaf and young spike.Histochemical GUS staining showed that OsCKX3 was mainly located in parenchyma cells around the lamina joint.In the sections of mutants,overexpressed plants and wild-type,the overall length of parenchyma cells of mutants was significantly higher than that of wild-type and overexpressed mutants;(3)We detect the changes in the expression of OsCKX3 in rice seedlings treated with different forms of CKs.It was found that the OsCKX3 expression was significantly induced by tZ and iP.The subcellular localization showed that OsCKX3 was mainly located in an unknown organell in the cytoplasm.It was found that OsCKX3 expression was significantly induced 1 hour after brassinolide(BR)treatment.(4)In vitro enzyme activity of the recombinant OsCKX3 protein towards different substrates was assayed.It was found that the degradation efficiency of OsCKX3 protein towards tZ was highest than the other cytokinins we tested.The hormone profiling in wild type,mutant and overexpression line revealed that OsCKX3 showed stronger activity to t Z than other cytokinins.(5)The statistical analysis of agronomic traits in the field showed that the flag leaves of osckx3 mutant were wider and the grain length,grain width and 1000-grain weight were significantly increased compared to the wild-type plants.In summary,we revealed that OsCKX3 regulated the rice leaf inclination.OsCKX3 affected the cytokinin accumulation at the lamina joint which changed the number of parenchyma cells in the lamina joint,and thus affected the mechanical support force of the lamina joint on the leaf and changed the leaf inclination.Meanwhile,it was found that OsCKX3 was induced by exogenous BR.Our results elucidate the biological function of OsCKX3 in regulating the leaf inclination.Further work will be required to explore the crosstalk between cytokinin(CK)and brassinolide(BR)during rice leaf inclination.