In Depth Study Of CBFs Transcription Factors In Regulating Cold Acclimation In Arabidopsis

As a major abiotic stress,low temperature not only affects plant growth and development,but also limits the geographical distribution of plants.In order to cope with cold stress,plants have evolved complex and sophisticated regulatory networks,of which the most widely studied is the CBF-dependent pathway.When plants sense the cold signal,the expression of CBFs is rapidly induced,which in turn activates the downstream COR gene expression and enhances plant tolerance to freezing stress.In Arabidopsis,the CBF/DREB1(C-repeat binding factor/dehydration-responsive element binding factor 1)transcription factors contain three members,namely CBF1/DREB1B,CBF2/DREB1C and CBF3/DREB1A,which are closely arranged in tandem on Arabidopsis chromosome 4.Overexpression of CBFs in plants significantly enhances the freezing tolerance.However,due to the fact that CBFs are located in tandem on the chromosome,it is impossible to generate cbfs homozygous mutants by recombination segregation,which limits the further study on the function of CBFs.In addition,most of the current researches on CBFs mainly focus on their transcriptional regulation.It has been reported that transcription factors,such as ICE1,CAMTA3,MYB15 and EIN3,directly regulate CBFs gene expression.In recent years,researchers have also found that E3 ligases(HOS1,COP1,and SIZ1),and protein kinases(SnRK2.6/OST1 and MPK3/6)can modulate ICE1 protein stability or transcriptional activity,thereby regulating the expression of CBFs.However,little is known about the post-translational modification of CBFs.Previous report showed that the cell membrane-located protein kinase CRPK1 can be activated by cold stress,which in turn phosphorylates downstream 14-3-3 proteins.The phosphorylated 14-3-3 proteins shuttle from the cytosol to the nucleus to interact with CBF1/3 and promote the degradation of CBF1/3 via the 26S proteasome pathway.The recent report revealed that the cold-activated SnRK2.6 phosphorylates downstream BTF3L,which enhances the interaction between BTF3L and CBFs and stabilizes CBFs.However,how CBFs are modulated by cold stress and whether they are regulated by other post-translational modifications remain further study.To examine the contribution of CBF genes in plant responses to cold stress,we generated homozygous cbf1 cbf3,cbf1 cbf2,cbf2 cbf3 double mutants and cbfs triple mutants by CRISPR/Cas9 technology.Phenotypic analysis showed that there was no significant difference between cbfs and wild type(WT)shoot in vegetative and reproductive growth,while the root length of cbfs was shorter than that of WT.However,under chilling stress,the shoot growth of cbfs mutant was significantly larger than that of WT,and the root length difference was reduced,which indicates that the response of cbfs mutant to chilling stress is significantly reduced.In addition,the results of freezing experiments showed that there was no significant difference between cbfs and WT under non-acclimated conditions,while cbfs showed extremely freezing sensitive phenotype after cold acclimation.Besides,cbf1,2,cbf1,3 and cbf2,3 double mutant also showed different degrees of freezing sensitive phenotype,indicating that CBFs are necessary for cold acclimation of plants.Further RNA sequencing analysis showed that the expression of 134 genes was regulated by CBFs,of which 112 genes were up-regulated and 22 genes were down-regulated by CBFs.This part of study not only reveals the essential functions of CBFs in plant cold signaling,but also provides ideal materials for the genetic dissection of components in CBF-dependent cold signaling.In this study,the CBFs protein expression pattern in WT under cold stress was determined by anti-CBF antibodies,which indicated that the CBFs protein was not accumulated under normal temperature,but accumulated under chilling stress.The CBFs protein was gradually degradated when the chilling stress treated exceed 9 h.To observe the tissue localization of CBFs proteins,constructs of CBFs fused with GUS gene driven by their native promoters were generated(pCBFs::CBFs-GUS)and transformed into Arabidopsis WT.GUS staining showed that CBFs proteins were expressed in Arabidopsis roots,hypocotyls,and veins.By replacing GUS with GFP,the expression of CBFs proteins in roots was closely observed by confocal.CBFs were observed to be expressed in the stele of root maturation zone.CBF2 and CBF3 were also expressed in root elongation zone and meristem zone.In addition,CBF3 was also found to accumulate in the nuclei of guard cells.These results indicated that CBFs expressed in plant vascular tissues.In order to investigate the post-translational regulation of CBFs proteins,three members of the SnRK2 protein kinase family,SnRK2.2,SnRK2.3 and SnRK2.6,were found to interact with CBF1,CBF2 and CBF3,respectively.Both luciferase complementation assay(LCI)and bimolecular fluorescence complementation assay(BiFC)demonstrated that three SnRK2 proteins interacted with CBFs in vivo.The pull-down assay showed that SnRK2s directly interacted with CBFs.In vitro phosphorylation assay indicated that CBFs were phosphorylated by SnRK2s.The phosphorylation of SnRK2.6 to CBFs enhanced their protein stability and transcriptional activity.Immunoblot analysis showed that CBFs protein stability in ost1-4 mutant was significantly reduced compared to the WT under chilling stress.In addition,the degradation of CBF proteins in ost1-4 CBFs-Myc-PER8 plants was faster than that in CBFs-Myc-PER8 plants.Furthermore,in vitro and in vivo degradation assays also demonstrated that CBF2S196A,which can not be phosphorylated by SnRK2.6,was more unstable than CBF2WT.The transient expression assay in tobacco,EMSA and ChIP assays indicated that CBFs transcriptional activity was enhanced by SnRK2.6,while the transcriptional activity of CBF2S196A was significantly reduced.Previously reprot has shown that SnRK2.6 positively regulate plant freezing tolerance,thus,we further dissected the freezing phenotypes of snrk2.2 snrk2.3 double mutant.The results showed that snrk2.2 snrk2.3 double mutant showed decreased freezing tolerance compared with the WT both under non-acclimated and cold acclimated conditions.In addition,qRT-PCR showed that cold-induced expression of CBF downstream genes in snrk2.2 snrk2.3 double mutant was significantly decreased.These results indicated that SnRK2.2 and SnRK2.3 also positively regulate plant freezing tolerance,as SnRK2.6 does.Since SnRK2s plays a key role in the ABA signaling pathway,we asked whether CBFs were also involved in plant ABA response.The results showed that the expression of CBFs was induced by ABA.In cbfs triple mutant,the expression of CBF downstream genes,which are also responsive to ABA,was significantly decreased.The phenotypic analysis further demonstrated that cbfs mutant was insensitive to ABA and hypersensitive to drought stress.These results indicate that CBFs act as positive regulators in ABA signaling pathway.Taken together,this study demonstrates the essential functions of CBFs in chilling stress response and cold acclimation.Protein kinases SnRK2.2,SnRK2.3 and SnRK2.6 positively regulate plant freezing tolerance by phosphorylating CBFs,and enhancing CBFs protein stability and transcriptional activity.These results not only prove the key role of CBFs in plant response to low temperature,but also unravel a molecular mechanism by which CBFs proteins are regulated,which shed more light on understanding cold signaling pathway in plants.