Identification And Analysis Of Components Function In AtCAP1-regulated Root Hair Growth

Root absorbs water and nutrient for plant growth,and root hair of the root system plays major role.Root hair cell is a typical tip growth cell,and the easy way to be obtained for research and analysis,it has been recoganized as as the good model to explore the mechanism of cell growth and development.Our previous work characterized a receptor like kinase of Arabidopsis named as CAP1([Ca2+]cyt-associated protein kinase,CAP),which belongs to the family of CrRLK1Ls,can function in root hair growth by regulating cellular ammonium homeostasis.AtCAP1 deficient mutants showed shorter and less root hair in MS medium,while the abnormal root hair can be restored as wild type when the growth medium is free of ammonium.CAP1 regulates the gradients of Ca2+ and ROS in the tip of root hair cell to modulated root hair growth.However,the detailed upstream and downstream components of CAP1 are still not know,in order to further understanding the function of CAP1,components in CAP1 regulated pathway were analysed by suppressor screening and Y2H methods.By EMS mutagenesis screening,suppressors with long root hair were screened and obtained.Previously,our team have got several lines.By hybridization and self-crossing with capl-1,three lines(18,Y and S62)were finally decided for further study,segregation ratio of 3(short root hair):1(long root hair)were showed in the F2 generation,which suggesting the three suppressors were controlled by single recessive gene in according to Gregor Mendel's genetic law.After gDNA sequencing and MutMap analysis with the BC2F2,seven genes were identified for these three suppressors,they are 18(AT1G193xx),Y(AT1G580xx,AT1G601xx),S62(At5G192xx,At5G202xx,At5G184xx,At5G196xx).To further make sure which will interact with CAP1,both genetic and biochemical methods were used.T-DNA insertion mutants of these genes were ordered,double mutants were constructed,and their root hair growth were analyzed.Complementation were also conducted by transforming these putative genes into the suppressors.Further relationship with CAP1 will be verified by BiFC-Split,co-location detection.Finally,AT1G193xx was decided on further study,experiments for this gene showed the probabilities of interaction with CAP1.AT1G193xx encodes protein of RRAx,which belongs to the GTs(GTs,glycosyltransferases)family.RRAx participates in the process of O glycosylation of cell wall extensins,which is the key step in the formation of cell wall.Interestingly,mutant of RRAx is also defective in root hair growth.The double mutant rrax cap1-1 partially restored the short root hair phenotype of cap1-1,suggesting RRAx may function in CAP1 regulated root hair development.For another putative interacting protein(Pollole e 1;17)that obtained by the Y2H,BiFC experiment and root hair phenotype experiment suggesting that there is no interaction between POE1;17 with CAP1.In conclusion,at least one potential suppressor was obtained,further study of the suppressor will be helpful in understanding CAP 1 function.