Regulation Mechanism Of Plasma Membrane Na~+/H~+ Antiporter Of Sesuvium Portulacastrum L.

Plant exhibited several symptoms such as growth inhibition, development acceleration, senescence or even to death under salt stress. In order to adapt to salinity,plant will take a series of physiologic and biochemistric mechanisms including restricting Na+ absorption, accumulating some osmotic solutes. The cation/proton antiporters(CPA)regulating ion homeostasis play important role in salt tolerance. The plasma membrane Na+/H+ antiporter SOS1(salt overly sensitive 1) transported excessive Na+ in the cytosol out of the cell to reduce the Na+ toxicity. Sesuvium portulacastrum, which is a mangrove plant grow in the seaside, has a distinguished ability to grow under severe salinization,and becomes a good material to study salt tolerance. Four SOS1 related genes were isolated from the halophyte Sesuvium portulacastrum in this study; their functions were researched using the Saccharomyces cerevisiae and Arabidopsis ectopic expressing system. The study was aimed to understand the salt tolerance pathway of Sesuvium portulacastrum, and provide theoretical basis for discovering plant salt-tolerant mechanism. The main results are performed as follows:1 The SpSOS1 gene was isolated from the halophyte Sesuvium portulacastrum using the homology cloning and RACE methods. Phylogenetic tree analysis showed that the SpSOS1 protein belonged to the SOS1 subfamily of the monovalent cation/proton antiporters superfamily, and the amino acid sequence shared higher similarity with AtSOS1(61.53%), OsSOS1(60.58%), ThSOS1(60.39%), the proteins from Arabidopsis thaliana, Oryza sativa, Thellungiella halophila, respectively. The expression of SpSOS1 was induced in roots other than in leaves and stems under salt stress. Subcellular localization showed that SpSOS1 was a plasma membrane protein. The SpSOS1 gene could compensate the salt sensitivity of the Saccharomyces cerevisiae mutant AXT3 K,the yeast strain transformed with SpSOS1 gene had tolerant to Na+, K+ and Li+. Compared with the sos1 mutant plants, the SpSOS1 overexpressing plants could complement salt sensitivity and grow well under salt treatment.2 An H+-ATPase gene SpAHA1 was cloned from Sesuvium portulacastrum using RACE technique. The SpAHA1 expression exhibited a similar pattern with SpSOS1 gene in the roots under salt stress. The SpAHA1 protein localized in the plasmalemma. The AXT3 K strain co-transformed with SpSOS1 and SpAHA1 grew better than transgenic strain with single SpSOS1 or SpAHA1 under salinity. The Na+ content in the yeast cells co-expressing SpSOS1 and SpAHA1 was significantly lower than that transformed with single gene. The plasmalemma vesicles were isolated and the H+-ATPase activity andNa+/H+ exchange activity were measured in the transgenic yeast strain and untransformed strain. The strain co-transformed with two genes showed the highest H+-ATPase activity and Na+/H+ exchange activity. These results indicated that the plasma membrane H+-ATPase SpAHA1 could provide energy for the plasmalemma Na+/H+ antiporter SpSOS1 excluding Na+.3 A series of 3’-deleted truncation mutants of SpSOS1 gene were obtained by PCR methods. We analyzed the functions of these mutants using yeast ectopic system, the results indicated that the amino acids from 986-1060 constituted the auto-inhibitory domain and 882-986 constituted the activity domain in SpSOS1. The activity of Sesuvium portulacastrum SpSOS1 was regulated positively in the presence of AtSOS2 alone or AtSOS2/AtSOS3(AtCBL4) complex. Interestingly, there existed other phosphorylated sites in SpSOS1 excepted for DSPS domain and that might be located in the amino acids from 1005-1045. In the CBL family, only CBL10 protein could interact with AtSOS2 and regulate positively SpSOS1 except for CBL4(AtSOS3) protein, the regulated sites might also be located in the amino acids from 1005-1020 by AtSOS2/AtCBL10 complex.4 A SpCIPK8 gene encoded a polypeptide of 447 amino acid belonging to CIPK family and a SpCBL10 gene encoded a polypeptide of 252 amino acid belonging to CBL family in the CBL-CIPK regulated network were isolated from S. portulacastrum using the homology cloning and RACE methods. Bioinformation analysis showed that the SpCIPK8 protein has 77.63% sequence identity with AtCIPK8 protein from A. thaliana,and SpCBL10 protein has 68.65% sequence identity with AtCBL10 protein from A.thaliana. The SpCIPK8 gene and SpCBL10 gene were all up-regulated in roots by Real-time PCR methods. Subcellular localization of SpCIPK8-GFP and SpCBL10-GFP proteins were studied in the tobacco leaves mediated by Agrobacterium tumefaciens, the results showed that SpCIPK8 protein was localized to the cytoplasm while SpCBL10 protein was predominantly targeted to the plasma membrane. Yeast two-hybrid test illustrated that SpCIPK8 could interact with SpCBL10 in vivo, SpCIPK8 could interact with the full length SpCBL10 protein and its N-terminal deletion mutants, but could not interact with its C-terminal deletion mutants, SpCBL10 could interact with full length SpCIPK8 and the C-terminal 147-amino acid region. Bimolecular fluorescence complementation(BiFC) analysis showed that SpCIPK8-SpCBL10 complex localized at the plasma membrane in planta.5 The full length SpCIPK8 could not regulate SpSOS1 in transgenic yeast, but the salt tolerance of the AXT3 K strain co-transformed with the SpCIPK8-306 mutant onlyremaining kinase domain and SpSOS1 gene increased significantly. SpCIPK8-306K38 N,a Lys38-to-Asn mutation in the kinase domain, lost the kinase activity and could not regulate SpSOS1. The only phosphorylated sites in SpSOS1 regulated by SpCIPK8-306 were DSPS domain. The activity of SpSOS1 could regulate positively by SpCIPK8/SpCBL10 complex, which was similar with SpCIPK8-306. These illustrated that we found a new salt tolerance pathway in Sesuvium portulacastrum L.: the SpCBL10 protein interacted with the protein kinase SpCIPK8 and then phosphorylated the DSPS domain in SpSOS1 to regulate the Na+/H+exchange activity of SpSOS1.