Molecular Mechanism Of Freezing Tolerance Regulated By Cytokinin Type-B Response Regulators In Arabidopsis

Plants adapt to the fluctuating environment by integrating signals that are relayed by multiple en-dogenous phytohormone pathways.Cold stress,as one of the widespread abiotic stresses,not only greatly limits the geographical distribution of plants,but also affects the production of crops.Therefore,in-depth study of plant response to cold stress and tolerance mechanism is of great signaficance.Meanwhile,cytokinin plays a crucial role in regulating plant freezing tolerance,but the underlying mechanism remains poorly understood.Previous studies have found that cytokinin metabolic enzyme CKXs and type-A ARRs,as negative regulators of cytokinin signaling,play a positive role in cold stress.In this study,the loss-of-function mutants of the histidine kinases AHK2/AHK3 and phosphorylation proteins AHP1-5 are freezing toler=ance in Arabidopsis thaliana,indicting that they play a negative role in cold stress.Furthermore,Ara-bidopsis type-B response regulators(ARRs),the key output component of cytokinin signaling,were found to be negative factors in cold stress by freezing tolerance assays of arr1 arr12,arr1 arr11 arr12,arr1 arr10 arr 12 mutants.By detecting the signal intensity of TCS-GFP and the transcriptional activity of ARR1,it was found that the transcriptional activity of type-B ARR was suppressed under cold stress.Further,BiFC(Bimolecular fluorescence complementation)and Co-immunoprecipitation(Co-IP)as-says revealed that the attenuation of the interaction between ARR1 and AHP proteins at low temperature accounts for the reduced transcriptional activity of ARR1.Then we performed a genome-wide transcriptome analysis and identified a group of cold respon-sive(COR)genes regulated by type-B ARRs,including several Lipoxygenase(LOX)genes,which en-code enzymes catalyzing the synthesis of fatty acid hydroperoxides,which can serve as important sub-strates for jasmonic acid(JA)biosynthesis.Moreover,type-B ARRs were found to negatively regulate plant freezing tolerance by directly binding to the promoters of LOX genes and downregulating their expression.Consistent with this finding,the freezing tolerance of the arr1 arr11 mutant was partially suppressed by the lox2 mutant.Previous studies revealed that a well-known transcription factor in JA signaling pathway MYC2 can also regulates LOX2 expression.MYC2 interacted with ARR1,ARR11,and ARR12 in yeast two-hybrid assays.Pull-down assays further verified that MYC2 interacted directly with ARR1,ARRI 1 and ARR12 proteins in vitro.Co-IP assays demonstrated that MYC2 and ARR1/ARR11/ARR12 were in the same protein complex in plants.The freezing assays revealed that the myc2 mutants showed a freezing sensitive phenotype,indicating that MYC2 is a positive regulator in freezing tolerance.Mean-while,the freezing-tolerant phenotype of arrl arr12 was partially suppressed by the myc2 mutation.Using Dual-LUC assays and competition EMSA assays,we found that MYC2 interacted with ARR1 to interfere with its DNA-binding ability.In addition,we found that CKX3,SAG13,SAG29 and ZAT10 genes were also ARR1 targets by EMSA and ChIP assays.Freezing tolerance assays also showed that they were positive regulators of cold stress response.In summary,cytokinin signaling component type-B ARRs are important negative factors in freez-ing tolerance in Arabidopsis.Type-B ARRs can regulate the transcription expression of COR genes,including LOX2,type-A ARRs,CKX3,SAG13,SAG29 and ZAT10.Additionally,by physically interac-tion,the type-B ARR-MYC2 module regulates the expression of LOX2 to fine-tune freezing tolerance.This study reveals the molecular functions of three type-B ARRs in regulating freezing tolerance,and uncovers a negative regulatory mechanism for the fine-tuning of cold responses,thereby providing in-sight into how plants balance stress tolerance and growth for better survival during unfavorable condi-tions.