Molecular Mechanisms Of Calcium Signaling Proteins Osclo5 And Osrbohi To Regulate Abiotic Stress Tolerance In Rice

Under environmental stresses,calcium signal is produced in plant cells,and calcium receptor proteins can recognize and transduce calcium signal,resulting in the change of downstream genes expression,which leads to plant stress resistance.In plants,the functions and signal transduction mechanisms of typical calcium receptor proteins like CaM/CML,CDPK,CBL-CIPK have been well studied.However,the mechanisms of calcium receptor proteins,such as caleosins with one EF-hand motif and NADPH oxidases with two EF-hand motifs,in Ca2+signal transduction and stress response remain to be elucidated.In rice,the caleosin family contains eight members(OsClo1?OsClo8)and the NADPH oxidase family contains nine members(OsRbohA?OsRbohI).Nevertheless,the functions and molecular interaction mechanisms of OsClo5 and OsRbohI in abiotic stress are still unclear.Therefore,in this project,we studied the biochemical activities of OsClo5 and OsRbohI,screened the candidate proteins interacting with them,and further identified the functions and new interaction mechanisms of OsClo5 and OsRbohI under abiotic stress.The results will deepen the understanding of the concept and mechanism of plant calcium receptor proteins.The main results are as follows:OsClo5 was localized in the endoplasmic reticulum and nucleus in rice protoplasts,and had calcium-binding and calcium-dependent phospholipid-binding properties.OsClo5 was highly expressed in plumule,coleoptile,stem nodes,anthers and pericarp tissues,and was induced by salt,drought and ABA.OsClo5 negatively regulates tolerance to ABA and salt stress in rice seedlings.Protein interaction experiments demonstrated that OsClo5 interacted with itself to form a homodimer or with OsDil9-5 to form a heterodimer,and the EF-hand motif of OsClo5 was essential for the interactions of OsClo5 with itself and OsDi 19-5.Y1H,EMSA and dual-luciferase reporter assays identified OsDi19-5 to inhibit the transcription of salt stress-related genes,OsUSP and OsMST,by binding to the TACART cis-element in their promoters.In addition,OsClo5 enhanced the inhibitory effect of OsDi 19-5 in the tobacco transient system.Transgenic experiments showed that OsClo5 and OsDi19-5 cooperatively inhibited the transcription levels of target genes OsUSP and OsMST,thus negatively regulating the response of rice seedlings to salt stress.Finally,an OsClo5-OsDi19-5-OsUSP/OsMST salt stress signal transduction pathway was identified.OsRbohI was located on the plasma membrane of rice protoplasts.OsRbohI was significantly induced by heat stress,and positively regulates tolerance to heat stress in rice.Forty-nine candidate proteins that might interact with OsRbohI were screened out by yeast two-hybrid cDNA library,including OsPERK1,OsATG8a and OsANN1.Specifically,Y2H and BiFC assays indicated that OsANN1 could also interact with other members of the OsRboh family.The tobacco transient transformation system confirmed that OsANN1 positively regulates the activity of OsRbohI and promotes OsRbohI to produce more H2O2,and analyzed the molecular mechanism of OsRbohI activated by OsANN1 regulating rice heat resistance.To sum up,this study deepened our understanding of the molecular mechanism of caleosins and plant NADPH oxidase in stress response in rice,and these new findings may be applied to molecular breeding for stress tolerance in rice and other crops.