Exploration Of The Mechanisms Of Two Calcium-associated Protein Kinases Regulating ABA Signaling Transduction In Arabidopsis

Various abiotic and biotic stresses,such as high salt,drought and extreme temperatures,as well as pathogens,affect the growth,development and yield,quality of crop plants.In the long-term defense against stresses,plants have evolved a variety of complex and effective signal networks to reduce the damage and adapt to environmental changes.The research on the regulation of calcium(Ca²⁺)and plant hormone ABA?abscisic acid?signaling has also been gradually increased.Previous studies have shown that the Ca²⁺and ABA-related signaling networks are involved in a variety of biotic and abiotic stresses,such as extreme temperatures,wounding,osmotic stress,drought,hormone stimuli and immune responses.However,as the large number of proteins involved,the molecular mechanisms of many members including kinases and transcription factors lack study,which poses obstacles for the application of agricultural technology such as molecular breeding.In this study,Arabidopsis was used as the model plant to explore two members of CPK?CALCIUM-DEPENDENT PROTEIN KINASE?and CIPK?CBL-INTERACTING PROTEIN KINASE?families.The spatiotemporal expression profiles of these two gene were detected by fluorescence real-time quantitative PCR?qRT-PCR?under various stresses and hormone treatments.The substrate transcription factors were identified by yeast two-hybrid,bimolecular fluorescence complementation?BiFC?and confirmed by co-immunoprecipitation.The phosphorylation and phosphorylation sites of the two transcription factors were determined by in vitro phosphorylation assay.The dual luciferase reporter system?Dual-LUC?and qRT-PCR were used to examine the effects of phosphorylation on transcriptional activity in tobacco and Arabidopsis mutants and transgenic overexpression plants,respectively.The phenotypes of the relevant mutants and overexpression plants under ABA and drought treatments were also compared.The results showed that,the expression of CPK6 is induced by ABA and dehydration.Two cpk6 T-DNA insertion mutants are insensitive to ABA during seed germination and root elongation of seedlings,in contrast,overexpression of CPK6 showed the opposite phenotype.Moreover,CPK6-overexpressing lines showed enhanced drought tolerance.Study on interaction showed that,CPK6 strongly interacts with core ABA signaling-related transcription factors ABF3?ABA-RESPONSIVE ELEMENT-BINDING FACTOR 3?,ABI5?ABA INSENSITIVE 5?and AREB3?ABA-RESPONSIVE ELEMENT BINDING FACTOR 3?in vivo and in vitro.But the interaction between CPK6 and ABF2/4 was weak.Subsequent in vitro phosphorylation assay and mass spectrometry identification of phosphorylation sites showed that CPK6 phosphorylates ABF3 and ABI5,with the sites of phosphorylation confirmed with site-directed mutagenesis of serine and threonine residues in the transcription factor proteins through a kinase assay.Analysis of target genes of ABF3and ABI5 with dual luciferase reporter system in tobacco and qRT-PCR performed in Arabidopsis lines showed that the CPK6-mediated phosphorylation enhances TF's transcriptional activity.Phenotypic analysis of cpk6 abf3 or cpk6 abi5 double mutants during seed germination and root growth showed that CPK6 and ABF3/ABI5 have slight additive effect in the ABA signaling pathway.In addition,loss-of-function mutants of CIPK26 are hypersensitive to ABA.qRT-PCR showed that the expression of CIPK26 is rapidly induced by various stresses.The cipk26-1mutant plants showed enhenced drought tolerance.CIPK26 interacts with and phosphorylats AREB3 at the residue of Ser21.Moreover,CIPK26-mediated phosphorylation of Ser21 in AREB3 is necessary for its transcriptional activity.Germination assay of cipk26 and areb3 mutants showed that AREB3 promotes seed dormancy,while CIPK26 show the opposite.Protein stability experiments showed that the degradation of CIPK26 and AREB3 through ubiquitin-dependent pathway is promoted by ABA.Besides,CIPK26 enhances the degradation of AREB3 by phosphorylating Ser21.Subsequent protein interaction analysis reveals that AREB3 interacts with two E3 ubiquitin ligases,SKP1B and HOS1,suggesting the degradation of AREB3 may be regulated by SKP1B and HOS1.Taken together,the evidences show that CPK6 positively regulates ABA signaling and drought tolerance through phosphorylating ABFs/AREBs,which reveals the molecular mechanism of calcium-dependent Ser/Thr protein kinase in ABA signaling.In addition,researches on CIPK26 show that,the phosphorylation of CIPK26 activates ABA-induced transcriptional activity of AREB3,which in turn promotes the degradation of AREB3,thereby fine-tuning the ABA signaling pathway in the early stage of seed dormancy.