Expression Profiles And Functional Analysis Of OsCKXs(Cytokinin Oxidase) In Rice

Rice is an important source of energy for human beings.The rice yield needs to be increased to meet the expanding food demands from the increasing population in the world.Cytokinins(CK)are a class of plant hormones that play an important role in many aspects of plant growth and development,including apical dominance,the formation and activity of shoot meristems,leaf senescence,nutrient mobilization,seed germination,and pathogen responses.The maintenance of cytokinin homeostasis in individual tissues,cells,and organelles is crucial for its proper function.CYTOKININ OXIDASE/DEHYDROGENASE(CKX)is responsible for cytokinin degradation and maintains the CK homoeostasis in plants.Previous studies show that loss-of-function mutant of OsCKX2 in plants significantly enhanced the grain number of rice.The mutant of OsCKX4 in rice shows showed shorter roots in comparison with wide type.The over-expression of AtCKX’s in Arabidopsis reduced the rosette size and seed number,and meanwhile,the double mutant atckx3/atckx5 produced more flowers and siliques.Our research focuses on all the OsCKXs family and investigated their expression profiles in different tissues and under different treatments,and further study their function in Arabidopsis and rice.Our results provide the preliminary data to explore the function of al the OsCKXs.The main research results are as follows:(1)We made the phylogenetic tree using the CKX proteins from Arabidopsis thaliana,Oryza sativa and Zea mays.(2)We overexpressed the individual OsCKX genes(OsCKXl to OsCKX11)under the control of the 35S promoter in Arabidopsis carrying the ARR5::GUS reporter.We found the ARR5::GUS expression was all reduced in all the 35S::OsCKXs transgenic plants,indicating the cytokinin contents were reduced and all the 11 members of OsCKXs can degrade cytokinins in plants.(3)Overexpression of different OsCKXs results smaller rosette leaves,longer root and less siliques.But for different CKX,the transgenic plants the reproductive development and vegetative growth of 35S::OsCKXs transgenic plants are different.This indicates that the substrate and enzyme activity of each OsCKXs may be different and thus affect the pheotype of the transgenic plants at different levels.(4)The spatiotemporal expression characteristics of 11 OsCKXs were detected by qRT-PCR.Most of the OsCKXs are mainly expressed in root and old tissues.(5)We conducted quantitative qRT-PCR analyses to investigate the responses of OsCKX genes to phytohormone treatments.In 6-BA-or tZ-treated plants,all the genes were up-regulated from two-fold to hundred-fold.This indicates that 11 OsCKXs are induced by cytokinin,every gene play an important role at different developmental stages and express in various tissues.(6)The agronomic traits of loss-of-function rice mutants of OsCKXs were investigated.Most mutants can show increased spike grain number,tiller number,reduced plant height,more seeds compared to the wild-type..