Research On The Molecular Mechanism Of CPKa Regulating ABA Response And ROS Accumulation In Arabidopsis

Calcium is an important ubiquitous second messenger in both plants and animals.Calcium-dependent protein kinases?CDPKs/CPKs?,being specific to plants and protists,can usually bind to calcium and phosphorylate substrates,so as to regulate plant response to multiple abiotic and biotic stresses.There are a total of 34 CPK genes in Arabidopsis?Arabidopsis thaliana?,however,only some of them have been functionally characterized with molecular mechanisms illuminated.In this study,we found that CPKa,a member of CPK family in Arabidopsis not only regulates plant response to stress hormone ABA?abcisic acid?,but also modulates reactive oxygen species?ROS?accumulation.qRT-PCR assay showed that CPKa was expressed in a variety of organs and tissues and,its transcription profile did not significantly change under multiple abiotic stress treatments.Analysis of promoter activity through GUS reporter gene demonstrated that CPKa was highly and specifically expressed in shoot apical meristem?SAM?and guard cells.CPKa was a membrane-associated protein and it was localized at plasma membrane,endoplasmic reticulum?ER?membrane and tonoplast.The constitutively active form of CPKa retained to be associated with membranes,while a point mutant form of CPKa,i.e.CPKacaG2 A was localized mainly in nuclei but not membrane.Then,we found that both mutants of CPKa,cpka-1 and cpka-2,were insensitive to ABA during seed germination,however,CPKa-overexpressing lines had no ABA insensitive phenotype,compared to wild type?WT?.In vivo biomolecular fluorescence complementation?BiFC?analysis revealed that CPKa interacted with six members of bZIP-type transcription factors involved in ABA signaling pathway.We found that double mutant cpka-1abi5-1 was more insensitive to ABA than single mutant abi5-1,in that the former had a higher germination rate than abi5-1 on half strength MS medium containing 5 ?M ABA.Moreover,the two mutants of CPKa were more insensitive to drought stress but abi5-1 had a drought-sensitive phenotype.However double mutant cpka-1abi5-1 had a phenotype similar to wild type upon dehydration stress.Further,we identified that a hypersensitive response?HR?-like cell death symptom was induced by transiently expressing CPKaca in N.benthamiana.Quantification of some physiological indices related to cell death showed a significantly higher accumulation in leaf discs expressing CPKa than those expressing the control GFP,such as relative conductivity,DNA fragmentation,malondialdehyde?MDA?content and the H₂O₂ content.In contrast,expression of CPKacaD202 A,which had a substitution of D to A at position 202 in the kinase domain of CPKaca,did not induce any cell death,which suggests that CPKaca plays its function in cells through its kinase activity.qRT-PCR profiling showed that some ROS-and defense-related genes were induced by expressing CPKaca in N.benthamiana leaves,with the results partially confirmed by dual-LUC reporter system.In vivo BiFC analysis discovered interactions between CPKa and NbRBOHA,NbRBOHB as well as AtRBOHD.In addition,after silencing NbRBOHA and NbRBOHB,respectively,through virus-induced gene silencing?VIGS?in N.benthamiana,the symptom of PCD and ROS accumulation caused by expressing CPKaca was significantly attenuated,which indicates CPKaca-induced ROS accumulation and cell death depend on RBOHA and RBOHB in N.benthamiana.At last,a strong inhibition of seed germination and root growth was observed when CPKaca was inducibly expressed in Arabidopsis by using estradiol induced system.On the contrary,this phenotype was not observed when CPKacaD202 A expression was induced.Furthermore,when CPKaca was expressed in rbohd background,obvious retardation of seed germination and root elongation was still observed,which suggests CPKa may still exert its function through phosphorylating other RBOH enzymes such as AtRBOHF.Overall,our study not only identified a novel CPK gene with dual function,but also laid a solid foundation for understanding its mechanism and exploring possible application in the near future.