Molecular Mechanism Of Receptor-like Protein Kinase CRPK1 In Regulating Plant Response To Cold Stress

As a kind of important environmental factor,low temperature adversely affects the growth,development,metabolism and distribution of plants.During evolution,plants have acquired sophisticated mechanisms to protect against cold stress.Under cold stress,changes in fluidity of the plasma membrane in plant cells may serve as the primary sensor of cold stress.However,the precise mechanism by which plants sense and transducer the cold signal remains elusive.Up to date,the CBF/DREB1(C-repeat-binding factor/drought responsive element binding protein)-dependent cold signal pathway has been extensively studied.These CBF genes are rapidly induced by cold stress,and the induced CBF proteins bind to CRT/DRE DNA regulatory elements in the promoters of cold-regulated(COR)genes,which in turn positively regulates plant response to cold stress.Previous studies have been mainly focused on the transcriptional regulation of CBFs,and a series of transcription factors have been identified.Nevertheless,little is known about the mechanism of the translational and post-translational regulation of CBF proteins.Moreover,overexpression of CBF genes inhibits the plant growth,and how plants subtly fine-tune cold stress response and growth is still unknown.Through a genetic screen for protein kinase mutants,we isolated a receptor-like protein kinase mutant with enhanced freezing tolerance.This mutant is a null mutant of a novel receptor like kinase CRPK1(cold-responsive protein kinase1).The cold-induced CBF expression level in the mutant was slightly higher than in the wild type;however,the expression of the CBF target genes was significantly up-regulated compared to the wild type.Wild-type CRPK1 genes can fully rescue the phenotype of the crpk1-1 mutant,as well as the expression of CBFs and their target genes.These results demonstrate that CRPK1 participates in the cold response dependently on the CBF signaling pathway.By confocal microscopy and cell fractionation assay,we observed that CRPK1 was localized on the plasma membrane.In vitro and in vivo phosphorylation assays showed that CRPK1 was an active kinase,and cold stress could activate its kinase activity.Furthermore,a kinase-dead form CRPK1K69E failed to show either auto-phosphorylation or phosphorylation of 14-3-3?,and failed to rescue freezing tolerance of crpk1-1.These results suggest that the kinase activity of CRPK1 is crucial for its function in cold response.By screening its interacting proteins using yeast two-hybrid assay,we identified 14-3-3 proteins as CRPK1 interacting proteins,which was confirmed by pull-down and Co-IP assays.In vitro and in vivo phosphorylation assays showed that the one presentative 14-3-3 protein,14-3-3?,can be phosphorylated by CRPK1 under cold stress.We also identified four phosphorylation sites,S70,S112,S193 and T214.When these four amino acids were mutated to Ala(a non-phosphorylated inactive form,14-3-3?4A),the phosphorylation signals of 14-3-3? by CRPK1were almost abolished.Similar to the crpkl mutant,the 14-3-3k? double mutant showed tolerant phenotype to freezing stress,which was rescued by the wild type 14-3-3? but not 14-3-3?4A mutant form,indicating that function of 14-3-3? in regulating plant freezing tolerance requires cold-mediated CRPK1 phosphorylation.Interestingly,we found that 14-3-3? protein was translocated from cytoplasm to the nucleus after cold treatment.Experiments in vitro and in vivo proved that the 14-3-3?,interacted with CBF proteins,and promoted the degradation of CBF1/3 proteins in nuclei.Further study showed that the translocation of 14-3-3s was dependent on its phosphorylation by CRPK1,and the unphosphorylated form 14-3-34A can neither translocate into the nuclear nor promote CBF1/3 protein degradation.Furthermore,genetic analysis showed that CRPK1 and the 14-3-3s act the upstream of CBFs to modulate cold response.In summary,the plasma membrane-localized receptor like protein kinase CRPK1 is a negative regulator in the cold response.Cold stress activates CRPK1,which phosphorylates 14-3-3 proteins in the cytoplasm,thereby triggering 14-3-3s to transduce into the nuclei.In the nucleus,phosphorylated 14-3-3s promote the 26S proteasome-mediated degradation of CBFs to regulate cold response.Our study demonstrates that CRPK1-14-3-3 module transduces the cold signal from the plasma membrane to the nucleus.The CRPK1-14-3-3 module mediated negative feedback regulation helps plant avoid excessive cold response,and balances plant growth and resistance to stress.This study will shed more light on our understanding of molecular mechanism underlying plant perception and response to cold stress.