Functional Study Of Rice OsCBL Family And OsCIPK11 In Abiotic Stress

Calcium ion(Ca2+)is an ubiquitous second messenger that plays a crucial role in regulating plant growth and in mediating various adaptive responses under environmental stresses.Specificity of a signaling response can exploit spatial information and temporal dynamics.Many signaling modules have been identified to participate in Ca2+ signal decoding,transmission and processing.The plant-specific CBL(calcineurin B-like protein)proteins are a group of Ca2+ sensors.They contain EF-hand motif which make them Ca2+-binding,and interact with a family of protein kinase referred to CIPKs(calcineurin B-like(CBL)protein interaction protein kinase).The forming CBL-CIPK protein kinase complex then will phosphorylate and activate the downstream functional proteins.In Arabidopsis,26 CIPKs form a signaling network with 10 CBL calcium sensor proteins.So far,many CBL and CIPK genes have been identified involving plant responses to abiotic streses.Rice(Oryza sativa)is the main staple food for more than 50%of the world’s population and also is a powerful model system for genomic research of monocotyledon species.Developing its resistant ability to environmental stresses is always one focus of the rice genetic improvement.Consisting of 10 CBLs and 34 CIPKs,the complex CBL-CIPK calcium signaling pathways have also been found in rice.For better understanding the molecular mechanism of CBL-CIPK signaling and identifying new rice stress-related genes,the first part of this study started with the spatial localization of rice OsCBLs.For the first time the subcellular localization of all 10 members in this rice Ca2+ binding protein family has been systematically studied.The information obtained is important foundation for subsequent functional study of OsCBLs and OsCIPKs,and also for understanding how the CBLs-CIPKs pathway is regulated by Ca2+.Afterwards,SOS3/AtCBL4 homologous OsCBLs and one OsCIPK drought-sensitive mutant were selected to study and explore the role of CBL-CIPK pathways in the regulation of salt tolerance and drought tolerance in rice.Finally,a stable genetic transformation material containing Ca2+ indicator YC3.6 protein was first time generated in a cooperative manner,which laid the foundation for subsequent observation and analysis of Ca2+ signals in rice.The main results of the study are as follows:1.Full-length CDS of 10 OsCBLs were amplified from rice Nipponbare cDNA.Transformation vectors have been generated for each OsCBL,and later used for the subcellular localization in rice protoplasts and tobacco epidermis.The results showed that OsCBL1 was localized on plasma membrane;OsCBL2,3,6 localized only on vacuolar membrane;OsCBL4,5,7,and 8 shared a similar expression pattern which was on plasma membrane,in cytoplasm and in nucleus;OsCBL10 localized on vacuolar membrane and in nucleus;OsCBL9 showed a poor expression driven by 35S promoter.OsCBL9 was found to localize on vacuolar membrane and endomembrane system including Golgi apparatus.2.SOS pathway is conserved in rice.OsCBL4 is a rice ortholog of SOS2/AtCBL4.In this study,functional exploration was performed on OsCBL5,7,8,another three homologues of OsCBL4.These results indicated that OsCBL5,7,8 share a similar function with OsCBL4,and could play a positive role in regulating salt tolerance of rice by participating in the SOS pathway.3.Tissue specific expression analysis showed that the expression patterns of OsCBL4,5,7,and 8 have their own characteristics.The expression level of one single gene in different tissues or organs is significantly different.There is also big difference in Ca2+ binding ability among OsCBL4,5,7,and 8.The differences in the specificity among OsCBL4,5,7,and 8 indicate that rice may contain a much more precise regulation mechanism for the salt stress response through more complex SOS signaling networks.4.oscipk11 is found to be quite sensitive to osmotic stress and drought stress comparing to WT plant.When treated under the same stress conditions,the OsCIPK11 RNAi lines showed a similar sensitive phenotype like oscipk11.The OsCIPK11 over-expression lines showed an opposite insensitive phenotype.These results suggest OsCIPK11 play a role in rice response to osmotic and drought stress5.Yeast drop test study showed that OsCIPK11 interact with vacuolar membrane localized OsCBL2,3 and 6.It is demonstrated that OsCIPK11 can be recruited to the vacuolar membrane to function by interacting with OsCBL2,3,and 6.6.Using OsCIPK11 as the bait protein,six positive clones named as IP6,15,19,29,31,32 respectively have been found by screening the split-ubiquitin based yeast two hybrid membrane system library.Follow-up work will mainly focus on IP6 and IP29.Sequence analysis revealed that IP6 and IP29 are homologous to Arabidopsis VHA-c"subunit and VHA-c subunit,respectively.It is speculated that OsCIPK11 is localized to the vacuolar membrane through its interaction with OsCBL2,3,6 and regulates the activity of V-ATPase by phosphorylating OsVHA-c/c".7.A stable genetic transformation rice containing the Ca2+ indicator protein YC3.6 was first time generated.It was found that the UBQ10 promoter can stably drive the highly efficient expression of YC3.6 in all rice tissues.This indicates that the UBQ10 promoter is more effective to avoid expression silencing and is more suitable for YC3.6 expression in rice than the 35S promoter.8.The transgenic material was used in subsequent experiments to study the Ca2+ signal changes in rice root tips under hyperpolarization/depolarization cycles or glutamate treatment.The Ca2+ signatures generated have been compared with in Arabidopsis roots.The results showed that under the same treatment,the parameters of Ca2+signatures were very different between two species.Compared with Arabidopsis,the maximum signal amplitude of rice Ca2+ signature is usually lower but with a significantly increased duration.This shows major difference in the mechanism of Ca2+ signature generating in rice and Arabidopsis.