Dissecting The Molecular Mechanism Of An Arab Idopsis CPK Gene In Aba And Ros Signaling Pathways

Calcium signaling constitutes a highly integrated signal network,which plays a vital role in plant growth.Calcium signals in plants can be decoded by calcium binding proteins and transfer to downstream components(e.g.transcription factors,N ADPH,etc).Calcium binding proteins in plants can be divided into four groups,one of them is Ser/Thr protein kinase-Calcium-dependent protein kinase(CDPK/CPK),which only exists in plants and protists.ABA(Abscisic acid)and ROS(Reactive oxygen species)play vital roles in plant response to stresses.Biotic and abiotic stresses have negative effect on plant growth and development.So it is important to investigate the potential molecular mechanism of CPK genes regulating ROS and ABA.In a previous study,we found that transient express of the constitutively activated(ca)form of At CPKa can induce ROS accumulation and cell death in N.benthamiana.Real-time quantitative RT-PCR(q RT-PCR)and bimolecular fluorescence complementation(Bi FC)assays demonstrated that there is an interaction between At CPKa and At Rboh D.It was also found that cpka-1 mutant is insensitive to ABA during germination.O verexpression lines of At CPKa have a relatively weak phonotype of sensitive to ABA,which indicates that At CPKa may be involved in regulation of ABA signalling.Bi FC assay identified that there are interactions between At CPKa and b ZIP transcription factor s,clade A.Phenotypic assay of double mutants cpka-1abi5-1 showed a stronger insensitivity to ABA than the abi5-1 single mutant.Further,through prokaryotic induction and expression of fusion proteins of At CPKa-GST,At CPKaca-His,At Rboh D-DN(N-terminal cytoplasmic region)-FLAG and At ABI5-FLAG,and in vitro kinase assay,we found that At CPKaca can phosphorylate At Rboh D-DN and At ABI5.However,At CPKa showed kinase activity only in the presence of Ca²⁺,which indicates that At CPKa is a calcium-dependent protein kinase.In addition,At CPKa has auto-phosphorylation activity,however At CPKaca doesn't,which indicates that auto-phosphorylation activity may relay on calcium binding domain of At CPKa.Then we performed genetic experiment,and the result showed that leaf chlorosis was still observed in inducible expression of At CPKaca in rbohd background,which indicates that At CPKa exerts its function not solely through phosphorylating the At Rboh D.Since a mb SUS assay found that At CPKa can interact with At Rboh B,we initiated to constructed double mutants of rbohbrbohd,aiming to further investigate signal transduction pathway of At CPKa regulating ROS.