Molecular Mechanism Of BpMADS11 Transcription Factor On Regulating Drought Tolerance In Betula Platyphylla

Birch(Betula platyphylla)is an excellent tree species mainly grown in the southwest andnorthern regions of China.Since the drought is severe in the northern region of China,it is particularly important to gain insight into the molecular mechanisms of birch response to drought stress.Among them,it is important to study the drought tolerance of birch through the regulatory role of transcription factors.In this study,we constructed a three-layer hierarchical regulatory network of birch in response to drought stress with an algorithm based on a partial correlation coefficient,in which BpMADS11 was identified as a key regulatory transcription factor at the top layer,and further investigated the molecular mechanism of the MADS family transcription factor BpMADS11 in response to drought tolerance in birch,and obtained the following findings:1.Construction of a three-level hierarchical regulatory network in response to drought stress in birchWild-type birch was treated with PEG(simulating drought stress)for 0 h,2 h,4 h,6 h and 9 h,and the material was taken for RNA-seq sequencing analysis.A total of 1799 differentially co-expressed genes were obtained,and GO enrichment analysis showed that a large number of genes were enriched in stress-related biological processes.Based on a biased correlation coefficient algorithm,a three-level hierarchical gene regulatory network was constructed using differentially co-expressed genes,and the key regulatory transcription factor BpMADS11 was screened out from the top-level regulatory network to investigate the possible involvement in drought stress resistance in birch.2.Characterization and expression pattern analysis of BpMADS11BpMADS11 is located on chromosome 8 of birch,with a full-length CDS sequence of 501 bp, encoding 166 amino acids,and the protein localized in the nucleus.RT-q PCR showed that BpMADS11 was expressed mainly in roots and leaves and was induced by Na Cl,ABA,JA and drought stress.Promoter-driven GUS staining experiments showed that drought stress activated BpMADS11 promoter activity to promote BpMADS11 expression in leaves and roots of birch.3.BpMADS11 positively regulates drought tolerance in birchOverexpression and knockout vectors of BpMADS11 were constructed and genetically transformed into birch.The transgenic strain of BpMADS11 overexpression(BpMADS11-OE)and mutant of the gene(bpmads11~#)were obtained.Phenotypic observations and biomass measurements were performed on overexpressed,gene edited mutant and wild type(WT)birch plants.The results showed that BpMADS11-OE plants had slow growth and root elongation under drought stress,while the mutant plants showed the opposite phenotype.Physiological measurements showed that BpMADS11-OE plants could reduce membrane peroxidation and increase H2O2 accumulation by increasing CAT and POD enzyme activities in birch,thereby improving the ability of BpMADS11-OE plants to resist drought stress.4.BpMADS11 directly regulates the expression of BpERF61BpERF61 is a target gene of BpMADS11 in the three-level hierarchical regulatory network of birch in response to drought stress.RT-q PCR results showed that BpERF61 was significantly highly expressed in the BpMADS11 overexpression strain compared to WT plants,while its expression was significantly down-regulated in the gene editing mutant.It was further verified that BpMADS11 can directly bind the CAr G-box element on the BpERF61 promoter from in vivo and in vitro by yeast one-hybrid,Ch IP-q PCR and EMSA experiments.Dual luciferase assay showed that BpMADS11 could promote the transcription of BpERF61.Altogether,BpMADS11can positively regulate the expression of BpERF61 by directly binding to the CAr G-box element on the BpERF61 promoter.5.Analysis of drought resistance of BpERF61The full-length CDS sequence of BpERF61 is 927 bp,encoding 308 amino acids,and the protein is localized in the nucleus.BpERF61 was significantly induced by drought stress to be expressed in roots and leaves.BpERF61 overexpression(BpERF61-OE)and gene edited mutant(bperf61~#)transgenic strains were obtained and phenotypic observations and physiological indicators were performed.The results showed that the phenotype of BpERF61-OE was similar to the phenotype of BpMADS11-OE after drought stress,and BpERF61-OE was also able to enhance drought tolerance by increasing POD and CAT activities to clear the accumulation of ROS in birch.However,the gene editing mutant plants showed the opposite phenotype.These results suggest that BpERF61 also has excellent drought tolerance as a target gene of BpMADS11.6.Screening and identification of BpERF61 target genesA total of 202 genes that may be regulated by BpERF61 were obtained in birch by joint Ch IP-seq and RNA-seq analysis.Further analysis of these genes showed that many were related to stress resistance,including BpERD15 and BpPOD51.In addition,it was verified that BpERF61directly positively regulates the expression of BpERD15 and BpPOD51 by RT-q PCR,yeast single hybridization,Ch IP-q PCR,EMSA and dual luciferase assays.7.Mechanism of BpMADS11 phosphorylationDesphosphorylation modification of BpMADS11 protein was induced after drought stress.The BpMADS11 and BpPP2C2 interaction was identified by yeast two-hybrid assay,and further confirmed that BpMADS11 can interact with BpPP2C2 by BiFC,GST pull-down and dual luciferase assay.Moreover,BpPP2C2 dephosphorylated BpMADS11 protein and promoted the transcription of BpERF61 in vivo and in vitro.Knockdown BpPP2C2 plants in overexpressing BpMADS11 showed reduced resistance compared with overexpressing BpMADS11 plants.Therefore,BpMADS11 and BpPP2C2 interacted to dephosphorylate BpMADS11 under drought conditions and promoted the expression of the target gene BpERF61 by binding to the CAr G-box element.Meanwhile,BpERF61 positively regulates expression of BpERD15 and BpPOD51 by binding to their promoters to increase POD and CAT activities in birch,thereby reducing the accumulation of ROS and improving drought tolerance in birch.