The Regulation Of A Protein Kinase CIPK8 On Plasma Membrane Na~+/H~+ Antiporter SOS1 In Arabidopsis Thaliana

Plants have to endure various environmental stresses during their growth,such as soil salinization,desertification and extreme temperatures.Due to seawater flowing backward the area of saline land in coastal areas is increasing year by year,which has seriously affected the performance of crops.Most of the crops are salt sensitive plants,making their growth and development on salinization soils slowed down or even died.In order to resist high salinity environment,plants increased exclusion of Na+ and compartmentalization of Na+into vacuoles by Na+/H+ antiporters on the plasma membrane.So far,there are two pathways for the Na+/H+ antiporters SOS1 in Arabidopsis,including SOS1+SOS2+SOS3 and SOS1+SOS2+CBL10 involved by CIPK/CBL.However,there are few reports on the involvement of other members of the CIPK/CBL family.In our study,exploring the responses of CIPK/CBL family members AtCIPK8 and AtCBL5 under Salt Stress.The main results are in details.1.Through the phenotypic analysis of sos1 and sos2 mutants under salt stress and the analysis of the relationship between CIPK family proteins,we concluded that AtCIPKS and AtCEPK24 were closely related,and speculated that they might participate in the SOS pathway.2.The expression pattern analysis of AtCIPK8 and GUS tissue staining showed that the expressions of AtCIPK8 in root and flower was higher than other tissues.QRT-PCR analysis proved that AtCIPK8 could be up-regulated by salt stress at transcriptional level;subcellular localization showed that AtCIPK8 was located in cytoplasm.3.Phenotypic analysis showed the sos2 cipk8 double mutant displayed significantly higher sensitivity than either of the single mutant sos2 or cipk8 under salt stress.Overexpression AtCIPK8 lines displayed increase growth compared with the wild type under salt stress.The overexpression of AtCIPK8 in sos2 mutant lines significantly increased the salt tolerance of sos2 mutant.This demonstrated that AtCIPK8 can participate in the response of Arabidopsis to salt stress,due to AtSOS2 is powerful,it has not observed.4.Germination analysis showed that cipk8 mutant was sensitive to Na+,and the germination rate of cipk8 mutant displayed reduced compared with wild type under salt stress.AtCIPK8 restorer lines could restore germination rate of cipk8 mutant.This demonstrated that AtCIPK8 was involved in the germination stage of Arabidopsis.5.To identify potential target of AtCIPK8,protein interactions between AtCIPKS and all 10 AtCBLs in Arabidopsis were tested by yeast two-hybrid assay.Co-transformation with AtCBL1,AtCBL5,AtCBL10 and AtCIPK8 were combination able to grown on selective media.The yeast functional analysis showed that AtCIPK8 could not regulate AtSOSl in transgenic yeast,but the salt tolerance of AXT3K strain co-transformed with AtCBL5 or AtCBL10 and AtSOSl increased significantly.Bimolecular fluorescence complementation(BiFC)and firefly luciferase complementation imaging(LCI)assay proved that AtCIPK8 interace with AtCBL5 or AtCBL10.AtCBL5 interacts with AtSOS2 regulate AtSOS1 in transgenic yeast.6.The expression pattern analysis of AtCBL5 and GUS tissue staining showed that the expressions of AtCBL5 in flower and silique was higher than other tissues,but not in root,seedling stage only in cotyledon and hypocotyl.Subcellular localization showed that AtCBL5 was located in cytoplasm.7.Phenotypic analysis showed that cbl5 mutant did not show significant obvious growth phenotypes under salt stress.Overexpression AtCBL5 lines displayed increase growth compared with the wild type but slightly increase with cipk8 mutant under salt stress.Overexpression AtCBL5 lines in sos2 cipk8 mutants did not increased salt tolerance,but significance in sos2 mutant.This demonstrated that AtCBL5 can participate in the response of Arabidopsis to salt stress through interaction with AtCIPK8 and AtSOS2.8.Germination analysis showed that cbl5 mutant was sensitive to Na+,and the germination rate of cbl5 mutant displayed reduced compared with wild type under salt stress.AtCBL5 restorer lines could restore germination rate of cbl5 mutant.This demonstrated that AtCBL5 was involved in the germination stage of Arabidopsis.Growth of overexpression AtCBL5 lines were increased in cipk8 and sos2 mutants but not in double mutant under salt stress.This demonstrated that AtCBL5 interacts with AtSOS2 or AtCIPK8 during germination stage.