Functional Characterization Of BdCIPK31 In Response To Abiotic Stress

Since plants are sessile organisms,therefore,their growth and development are more easily affected by the environmental conditions.With the continuous changes in the global environment and the expansion of human activities,the negative impact of abiotic stresses on the normal growth of plants is becoming increasingly serious,bringing a huge loss in environmental protection and agricultural production.Plants have established a complex mechanism to adapt to environmental changes.The stress response mechanisms include stress signal perception,transduction,and physiological effects.The Ca2+ signaling pathway is one of the most important signaling pathways in plants,and extensivelty participates in stress signal transduction.In Ca2+ signaling pathway,calcineurin B-like protein(CBL)and CBL-interacting protein kinase(CIPK)play essential roles in Ca2+ signal tranduction and decoding.As a kind of plant specific protein kinase families,CIPKs have been involved in the processes of plant growth and development,nutrient uptake,and stress response.However,the functional roles of CIPKs in plant reponse to abiotic stresses have not been fully understood.Brachypodium distachyum is a model plant in the Pooideae subfamily,which has a close evoluationary relationship with wheat and sorghum.To date,no CIPK gene from B.distachyon has been functionally characterized.In the present study,we cloned and characterized a Bd CIPK gene,designated Bd CIPK31.Further analyses on the stress-tolerance function of Bd CIPK were performed at both transcriptional as well as physological and biochemical levels.The main results are as follows:1)The expression of Bd CIPK31 gene under different stress treatments was examined by using quantitative reverse-transcription PCR technique.The results showed that the expression of Bd CIPK31 gene was decreased by PEG6000,Na Cl,H2O2,cold,or ascisic acid(ABA)treatments,indicating that the Bd CIPK31 expression responded to osmotic,high salinity,oxidative,and low temperature stresses,and ABA was involved in this response.2)The promoter sequence of the Bd CIPK31 gene was obtained and analyzed.Many cis-elements related to stress-response were found in the Bd CIPK31 promoter region,including three ABA-reponsive elements but no DRE/CRT binding elements.These findings indicated that the transcriptional regulation of the Bd CIPK31 gene might be mainly through the ABA-dependent pathway.A 2 kb fragement of the Bd CIPK31 promoter region was cloned to construct the Promoter Bd CIPK31::GUS vector and then the construct was transformed into tobacco plants.The transcriptional acitivity of the Bd CIPK31 promoter was observed by histochemical staining and evaulated by GUS activity assay.The result showed that the transcriptional activity of the Bd CIPK31 promoter was decreased under osmotic,salt,or ABA treatments.3)Transgenic tobacco plants overexpressing Bd CIPK31 gene were generated and subjected to different treatments.The results showed that overexpression of Bd CIPK31 improved plant tolerance to drought,salt,or low temperature.The plant sensitivity to exogenous ABA was also elevated by Bd CIPK31.Further investigations revealed that although Bd CIPK31 enhanced drought and salt tolerance only in presence of ABA,overexpression of Bd CIPK31 does not affect the synthesis of endogenous ABA.4)Overexpression of Bd CIPK31 promoted stomatal closure under drought and ABA treatments,indicating that Bd CIPK31 plays a positive role in ABA-mediated stomatal movement.Bd CIPK31 also elevated the transcript level of guard cell K+ efflux channel gene TORK1,suggesting that Bd CIPK31 may enhance stomatal closure through regulating guard cell K+ efflux.5)Transgenic tobacco overexpressing Bd CIPK31 accumulated more Na+,K+,and Ca2+ than the controls under high salinity.The transcript levels of ion transporter genes such as Nt SOS1,Nt NHX2,NKT1,and Nt CAX2 were also higher in the transgenic plants,indicating that Bd CIPK31 participates in the compartmentalization of Na+,K+,and Ca2+ in plants.Furthermore,Bd CIPK31 overexpression prevented salt-shocked K+ efflux in the roots,indicating that Bd CIPK31 can promote K+ uptake.These findings suggested that Bd CIPK31 functions postively in maintaining plant intracellular ion homeostasis under salt stress.6)Overexpression of Bd CIPK31 improved the activities of antioxidative enzymes such as catalase,peroxidase,superoxide dismutase,and glutathione S-transferase in transgenic tobaccos under drought,salt,or cold stresses.Bd CIPK31 overexpressing also enhanced accumulation of antioxidants such as ascorbic acid and anthocayanin under the same conditions.As results,the transgenic tobacco plants retained less reactive oxygen species and suffered milder membrane damage than the controls did.The enhanced oxidative stress tolerance confirmed this conclusion.7)Overexpression of Bd CIPK31 positively regulated the synthesis of osmolytes such as proline,soluble sugar,and polyamine in the transgenic tobaccos under drought or high salinity,indicating that Bd CIPK31 could adjust the celluar osmotic pressure to cope with the osmotic stress caused by abiotic stresses.8)The expression levels of stress-related transcription factors such as Nt ABF1,Nt ABF2,Nt RD26,and Nt DREB3 were significantly elevated by Bd CIPK31 under drought,salt,or cold treatment,and the results showd that transcriptional regulation mode of Bd CIPK31 under drought or salt stress was different to the mode under low temperature stress.In addition,stress-defence genes such as Nt ERD10 C,Nt ERD10 D,Nt LEA5,and Tob LTP1 were also elevated by Bd CIPK31 under different stress conditions,indicating that Bd CIPK31 could positively regulate the transcription of stress-related genes through ABA-dependent and ABA-independent pathways.In conclusion,our study demonstrated that Bd CIPK31 plays a positive role in plant response to abiotic stresses through ABA and Ca2+ signaling pathway.