Mechanisms Of Cold Stress Responses Regulated By Protein Kinase BIN2 In Arabidopsis

Cold stress is one of the most important environmental factors that limit plant growth,development,and geographic distribution.Due to the effects of El Nino,global warming and et al.,extreme weather occurs more often and cold stress becomes more serious,so it is significant for us to find out how plants respond cold stress.Through their long history,plants have evolved complicated mechanisms to adapt to cold stress,and one of the most crucial strategies is cold acclimation.This is a process that plants increase their tolerance to cold stress by pre-exposing to low but non-lethal temperature.Extensive studies have shown that CBF/DREB1 transcription factors play critical roles in cold acclimation.In Arabidopsis genome,there are three CBFs in tandam located on chromosome 4.Their abundance will increase dramatically during cold stress,and they can induce the COR genes,which will increase protective substances and change the membrane composition to protect plants from cold injuries.Several studies have shown that plant hormones can regulate plants cold responses by affecting CBF pathway.For instance,EIN3,an important transcription factor in ethylene signalling,could bind to CBF promoters directly and negatively regulates the CBF expression,thereby affecting plant freezing resistance.JAZ1,an important negative regulator of jasmonic acid signalling,decreases the expression of CBFs by inhibiting the transcriptional activity of ICE 1,and the freezing tolerance of plants can be enhanced by adding JA.Brassinosteroid is an important phytohormone,which affects plant growth and development.Researchers had found that the plant cold tolerance would enhance when treated with BR,but the molecular mechanism is not clear.In order to understand how BR signalling affects plant cold responses,in this study we started with performing phenotypic screening using BR signalling related mutants.First,we focused on BIN2,an important negative regulator in the BR signalling.The phenotypic experiments indicated that the triple mutant of BIN2 and its two homologs BIL1/2,bin2-3 bill bil2 showed increased freezing tolerance compared with the wild type,and overexpression of BIN2 would weaken the freezing tolerance of Arabidopsis,which suggests that BIN2 plays a negative role in plant cold responses.In BR signaling,BIN2 phosphorylates and inhibits two important downstream transcription factors,BZR1 and its homolog BES1.In this study,we found that during cold stress,BZR1 protein would dephosphorylate rapidly,and this dephophorylation reached a peak at around 3 h after cold treatment.Freezing phenotype experiments showed that the gain-of-function mutant bzr1-1D and bes1-D both showed increased freezing tolerance,suggesting that BZR1 plays a positive role in cold stress response.These results demonstrate that BZR1 activated by cold can enhance plant-freezing tolerance.Previous ChIP-chip studies have shown that BZR1 and BES1 can bind to the promoters of CBF1 and CBF2.We confirmed these results using ChIP and EMSA experiments.The transient activation experiments showed that BZR1 could induce the expressions of CBFs.Consistent with these results,the expression of CBFs and the downstream COR genes in the bzr1-1D mutant was higher than the wild type under cold stress.These results indicate that BZR1 is activated during cold stress,and then induces the expression of CBFs to enhance the freezing tolerance of plants,while BIN2 negatively regulates plant cold responses by inhibiting the cold-activation of BZR1.In addition,we found that BIN2 protein interacted with ICE 1 through yeast two-hybrid system,and confirmed their interaction by pull-down and Co-IP experiments.In vitro phosphorylation assays and in vivo phos-tag assays demonstrated that BIN2 phosphorylated ICE1 and promoted ICE 1 degradation under cold stress,which resulted in decrease of cold-induced CBFs.Finally,we used CRISPR/Cas9 to construct ice1-3 bin2-3 bill bil2 mutant,performed a serious of freezing experiments,and confirmed that BIN2 regulated plant-freezing tolerance at least partially through ICE1.In summary,this study indicats that BIN2 can inhibit the cold-activation of BZR1 during cold stress,thus inhibiting the expression of CBF genes and their downstream targets to reduce the plant freezing tolerance.Addtionally,BIN2 can also phosphorylate ICE1 and promote its degradation to down-regualate CBFs and plant freezing tolerance.Moreover,this study reveals that the kinase activity of BIN2 is decreased rapidly in the early stage of cold stress,but restored in the later stage.This suggests that during early stage of cold stress,by some mechanisms unknown,plants can inhibit the kinase activity of BIN2 and enhance freezing tolerance.In the later period,the BIN2 kinase activity is recovered to attenuate excessive cold responses.In conclusion,this study proves that BIN2 is an important switch of plant cold responses,which extends our understanding on the plants responses to cold stress.