The Molecular Genetical And Biochemical Study Of Receptor-like Cytoplasmic Kinase STRK1 In Salt Stress Regulation In Rice

Soil salinization is the major environmental factor limiting rice(Oryza sativa L.)growth and productivity. It causes ion imbalance, water deficit, oxidative damage,which result in macromolecular damage, growth inhibition and even death of plants.Receptor-like kinases(RLKs) in plant, is located in the cell membrane, include an extracellular domain, a transmembrane domain, and an intracellular Ser/Thr protein kinase domain. The extracellular domains of RLKs perceive a wide range of environmental stress signals, resulting in phosphorylation of target protein by transphosphorylation of the intracellular kinase domains, and initiating physiological and biochemical adaptive response to reduce or eliminate the hazards. Thus, RLKs play important role in response to environment cues. Receptor-like cytoplasmic kinases(RLCKs) belong to RLKs superfamily without extracellular or transmembrane domain. Although number of RLCKs were reported to be regulation by abiotic stresses, only a few RLCKs have been functionally characterized to be involved in abiotic stress response and the mechanisms of RLCKs response to abiotic stress remain unclear. In this study, a quantitative real-time PCR analysis was employed to examine the expression of salt induced RLKs genes selected based on chip data. Six RLKs were found to be induced by salt, and we generated over-expression transgenic rice plants for these genes. T2 seedlings of the RLKs overexpression lines were used to identity the phenotypes under salt stress conditions. A RLCK gene, STRK1(salt tolerance receptor-like cytoplasmic kinase 1), has been found to positively regulate salt tolerance in rice plants, and the molecular mechanism of salt tolerance regulation of STRK1 in rice was studied. The main results are as follows:(1) Overexpression of the STRK1 gene in rice increases tolerance to salt stress at the seedling and reproductive stage. While the STRK1-RNAi plants are sensitive to salt stress. Compared with the wild-type, the STRK1-overexpressing plants had less content of MDA and relative ion leakage, indicating that STRK1 can reduce membrane damage caused by salt stress. Under salt stress conditions, there was no significant difference in the panicle number between STRK1 transgenic and wild-type plants. But the STRK1-overexpressing plants had higher spikelet fertility and grain yield than wild-type plants, whereas the STRK1-RNAi plants had lower spikelet fertility and grain yield than wild-type plants. These results suggest thatoverexpression of the STRK1 in rice improved the grain yield under salt stress conditions.(2) STRK1 belonged to RLCK, but the subcellular localization assay shows that STRK1 is located in the plasma membrane. Substitution of three cysteine residues in the N-terminal region of STRK1 to alanine(STRK1-C5,10,14A-YFP) resulted in cytoplasmic localization of the STRK1 protein, suggesting that STRK1 may be anchored to the plasma membrane by palmitoylation. The transgenic rice plants overexpressing STRK1-YFP showed higher tolerance to salt stress compared with the wild-type plants. The transgenic rice plants overexpressing STRK1-C5,10,14A-YFP showed no obvious phenotypes compared with the wile-type plants. These results indicated that plasma membrane localization is required for STRK1 to function in salt signal transduction. The expression of STRK1 was induced by Na Cl and H2O2 treatments. GUS staining of STRK1pro::GUS transgenic plants shows that the STRK1 is predominantly expressed in young root, leaf vein, stem, 4-d-old seedling, leaf sheath and young spikelet.(3) The yeast two hybrid and Bimolecular Fluorescence Complementation assay show that the STRK1 interact with the proteins of CATs family(Cat A、Cat B and Cat C)in rice. β-Galactosidase assays shows that the interaction of STRK1-Cat C is stronger than the STRK1-Cat A and STRK1-Cat B interactions. Furthermore, Pull-down and Co-immunipreciptation assays were employed to verify the interaction between STRK1 and Cat C. STRK1 phosphorylated Cat C and activated the activity of Cat C in vitro. Cat C phosphorylation by STRK1 was enhanced upon STRK1 activation with Na Cl treatment. The activity of Cat C is positively correlated with the phosphorylation level of Cat C.(4) Under salt stress conditions, the CAT activity was enhanced and the H2O2 concentration was decreased in shoots and roots of STRK1-overexpressing plants compared with those of wild-type plants. In contrast, the CAT activity was reduced and H2O2 concentration was increased in shoots and roots of salt-treated STRK1-RNAi plants compared with those of wild-type plants. These results indicate that STRK1 improves salt tolerance through regulation of H2O2 homeostasis by modulating of CAT activity.(5) To investigate the effect of STRK1 on oxidative stress, the STRK1 transgenic and wild-type plants were subjected to methyl viologen(MV) and H2O2 treatments.Under MV stress conditions, STRK1-overexpressing plants show higher CAT activity,seedling height and chlorophyll content compared with wild-type plants, whereas theSTRK1-RNAi plants is opposite. Under H2O2 stress conditions, the leaves of STRK1-overexpressing plants accumulate less H2O2 than that of wild-type, whereas heavier accumulation of H2O2 is observed in STRK1-RNAi leaves than wild-type leaves. These results indicate that STRK1 increase oxidative stress tolerance result from a enhanced ROS-scavenging ability.