Function Analysis Of OsRopGEF7 In Rice Development

Acting as molecular switches,plant specific small G protein RAC/ROPs are involved in various of hormone signaling pathways and regulate plant development.Activator of small G protein RAC/ROPs,RopGEFs can activate RAC/ROP by exchanging inactive GDP-binding form for active GTP-binding form.Therefore,RopGEFs also function in small G protein RAC/ROPs mediated hormone signal transduction and plant development.Function of some RopGEFs has been well documented in Arabidopsis,such as RopGEF7 has been revealed to maintain the stem cell niche by auxin-dependent PLT pathway.RopGEF1 is involved in regulating pollen tube growth,RopGEF4 and RopGEF10 participate root hair growth by negatively regulating ABA signaling.But function of RopGEFs in rice is not yet clear.In this thesis,two rice RopGEFs,namely OsRopGEF7A and OsRopGEF7B,which are homolog of AtRopGEF7 were analyzed,the main results as follows:1、The expression pattern of OsRopGEF7A was analyzed by promoter-GUS and in situ.The hybridization results display that OsRopGEF7A is mainly expressed in root tip,crown root primordium and shoot apical meristem.In addition,the expression of OsRopGEF7A is rapidly induced by cytokinin.2、By analyzing down-regulated T-DNA insertion mutant Osropgef7-1,we found that seeding growth was delayed and significantly declined in the mutant.Exogenous CPPU or cytokinin could significantly inhibit the rice root initiation.Compared with wild type,Osropgef7-1 displayed hypersensitive response upon the cytokinin treatment.Upon low concentration of CPPU treatment,the number of crown root dramatically declined in the mutant.But over-expression lines displayed resistant to the cytokinin inhibitied effect.These results suggest that OsRopGEF7A regulates the rice crown root development by integrating into cytokinin related pathway.Phenotypes of Osropgef7-1 can be complemented by transforming the full length of OsRopGEF7A in mutant background,and the crown root phenotype was restored.Also we obtain knockout mutants by Cas9 technology,and the knockdown mutant showed retarded development and reduced crown root number which are in accordance with the T-DNA mutant.These results confirm that OsRopGEF7A functions in regulating crown root development by mediating cytokinin related pathway.3、we found that the expression of OsRR6 was reduced in the mutant,but up-regulated in the over-expression lines.OsRR6 is a negative regulator of cytokinin signaling pathways and regulate the development of rice root and shoot.Our results indicated that OsRopGEF7A was integrated into cytokinin signaling pathways by affecting the expression of OsRR6.4、OsRopGEF7 may also regulate the development apical meristem.Over-expression of OsRopGEF7A resulted in multiple apical meristems in one embryo and multiple plantlets from one seed,meanwhile shape of the apical meristem become more flat.Although the grain size become smaller in mutant,grain size increased significantly in over-expression lines compared to wild type.Cytokinin has been reported to play a prominent role in controlling the size of the apical meristem and seed development.Taken together,our results suggest that OsRopGEF7A may regulate the apical meristem and therefore affects seed size by cytokinin related pathways.5、qRT-PCR and OsRopGEF7B_Pro::Gus analyse showed that OsRopGEF7B is expressed in the root meristem and flower organs including the stigma,pollen and young panicle apical meristem.6、We identified a knockout T-DNA insertion mutant of OsRopGEF7B,phenotype analysis revealed that development of the mutant seedling is retarded,mutant plants produce abnormal floral organs and eventually seed setting rate declined dramatically.In conclusion,our results suggest that two homologous genes of RopGEF7 in rice have different functions,OsRopGEF7A may be integrated into cytokinin signaling pathway and regulates crown root development as well as shoot apical meristem and seed development.While OsRopGEF7B expresses in flower organ and may function in floral organ development.