Study On Molecular Mechanism Of PtrbZIP12 On Regulating Drought Tolerance In Populus Trichocarpa

Plant growth is irreversible,and it is easily affected by abiotic stresses,such as salt,heavy metals,waterlogging and drought.Drought is one of the main abiotic stress factors that affect the normal growth and development of plants.Drought stress affects plant phenotype,physiology and biochemistry,expression of plant protein coding genes and other levels.Analyzing the response of plants to drought stress from different levels is of great significance to the research of plant drought resistance and the breeding and screening of new plant varieties with drought tolerance.In this study,the molecular mechanism of PtrbZIP12regulating drought stress in Populus trichocarpa was studied and the following results were obtained.（1）PtrbZIP12 was cloned in P.trichocarpa.Analysis of the sequences showed that the full-length coding sequence of PtrbZIP12 was 690 bp.This gene encoded 229 amino acids.Structural analysis shows that PtrbZIP12 protein has N-x7-R/K motif,which is the unique DNA-binding domain of bZIP protein.Phylogenetic analysis found that PtrbZIP12 belongs to the S subfamily of bZIP family.Besides,the results of subcellular localization showed that PtrbZIP12 was localized in the nucleus,and had the characteristics of transcription factor expression.（2）qRT-PCR results showed that drought stress simulated by PEG₆₀₀₀treatment could induce the expression of PtrbZIP12 gene.Sequence analysis of promoter of PtrbZIP12 gene showed that there were many cis-acting elements related to stress.Therefore,a fusion expression vector driven by the promoter of PtrbZIP12 gene for GUS gene was constructed for transient transformation of Populus tomentosa.Results showed that the activity of the promoter of PtrbZIP12 gene was enhanced after PEG₆₀₀₀stress.（3）Over-expression（OE）and CRISPR/Cas9 knockout mutant（ptrbzip12）strains of PtrbZIP12 gene were obtained respectively.After 4%PEG₆₀₀₀stress treatment,it was found that compared with WT,OE strain enhanced its tolerance to drought stress,while ptrbzip12showed sensitivity to drought stress.The phenotype of earth-cultured seedlings under drought stress was consistent with that in tissue-cultured seedlings.The result of measuring physiological index before and after stress showed that PtrbZIP12 gene could increase superoxide dismutase（SOD）activity and superoxide dismutase（POD）activity,increase proline（Pro）content,and reduce hydrogen peroxide（H₂O₂）,relative conductivity and malondialdehyde（MDA）content,thereby reducing cell death and membrane lipid peroxidation of P.trichocarpa and further improve that drought resistance of P.trichocarpa overexpressing PtrbZIP12 gene.While ptrbzip12 has the opposite effect.（4）Transcriptome sequencing and differential expression gene analysis were carried out on wild-type and PtrbZIP12 overexpressed Populus tomentosa strains under normal watering and PEG₆₀₀₀stress for 12 h.For control,1972（1462 up,510 down）DEGs were identified in OE vs WT.After PEG₆₀₀₀stress,1896（1097up,799down）DEGs was identified in OE vs WT,There are 539 overlapping genes in control and stress.The GO functional annotation analysis of the overlapping differential genes revealed that GO terms significantly enriched in DEGs were related to abiotic stresses,including ROS clearance,ABA synthesis,and expression of stress-resistant related genes.（5）In order to find the target gene that PtrbZIP12 directly binds to drought resistance,the yeast one-hybrid（Y1H）test was used to verify PtrbZIP12 and four candidate target genes related to stress resistance（Ptr DHN,Ptr POD,Ptr LEA and Ptr RD22）.The results showed that PtrbZIP12 could bind to the promoter regions of Ptr DHN and Ptr POD genes.The results of yeast single hybrid（Y1H）and Gel retardation assay（EMSA）further showed that PtrbZIP12gene could bind to ABRE element.Chromatin immunoprecipitation（Ch IP）and Dual-Luciferase test showed that PtrbZIP12 gene could specifically bind to ABRE element in the promoter region of Ptr DHN gene,thus directly regulating the expression of Ptr DHN gene.The results of Dual-Luciferase test further verified that PtrbZIP12 gene could be combined with the promoter of Ptr POD gene,thus directly regulating the expression of Ptr POD gene.Then,eight Ptr DHN overexpression（Ptr DHN-OE）and three Ptr POD gene overexpression（Ptr POD-OE）were obtained,respectively.The SOD activity,POD activity and H2O2 content of WT,Ptr DHN-OE and Ptr POD-OE strains were measured under normal growth conditions and PEG₆₀₀₀stress conditions.The results showed that compared with WT,Ptr DHN and PTRDPOD genes could increase the activities of superoxide dismutase（SOD）and peroxidase（POD）,and reduce the content of hydrogen peroxide（H₂O₂）,so as to reduce the cell death of P.trichocarpa,and further improve the drought resistance of Ptr DHN-OE and Ptr POD-OE in P.trichocarpa.（6）The interacting protein PtrbZIP3 of PtrbZIP12 were screened by yeast two hybrid（Y2H）.Further,it was verified by bimolecular fluorescence complementation（Bi FC）test that PtrbZIP12 protein interacted with PtrbZIP3 to form heterodimer.The results of Dual-Luciferase test showed that the co-expression of PtrbZIP12 and PtrbZIP3 could improve the transcription level of Ptr DHN gene.（7）The qRT-PCR results showed that PEG₆₀₀₀,ABA and Na Cl stress could induce the expression of PtrbZIP3 gene.PtrbZIP3 overexpression（OE）and CRISPR/Cas9 knockout mutant（ptrbzip3）strains were obtained,respectively.After drought stress of PEG₆₀₀₀,compared with WT,OE strains increased its tolerance to drought stress,while ptrbzip3 showed sensitivity to drought stress.The results of physiological indexes measurement before and after stress showed that PtrbZIP3 gene could increase superoxide dismutase（SOD）activity and peroxidase（POD）activity,increase proline（Pro）content,and reduce hydrogen peroxide（H₂O₂）,relative conductivity and malondialdehyde（MDA）content.Under the PEG₆₀₀₀stress,the stomatal aperture of OE was the smallest,followed by WT,and the stomatal aperture of ptrbzip3 plant was the largest.Compared with WT,the expression of Potri.005G001600.1 and Potri.008G070400 in OE plant was induced,and the expression in mutant was reduced.Thus,PtrbZIP3 can reduce the cell death and lipid peroxidation of P.trichocarpa,and then improve the drought resistance of P.trichocarpa over-expressing PtrbZIP3 gene,but knocking out PtrbZIP3 gene has the opposite effect.In conclusion,on the one hand,under drought stress,PtrbZIP12 can regulate the expression of Ptr DHN and Ptr POD by combining with the promoters of Ptr DHN and Ptr POD,and improve the drought resistance of transgenic plants by scavenging ROS,reducing cell death and lipid peroxidation.On the other hand,co-expression of PtrbZIP12 and PtrbZIP3 can also improve the transcription level of Ptr DHN,improving the drought resistance of transgenic plants of PtrbZIP12.