The Molecular Mechanism Underlying Regulating Drought Tolerance Of The Poplar WRKY Transcription Factor PtrWRKY75

Poplar is one of the main tree species used for afforestation and production in the north of China.However,most areas in the north of China are short of water,and poplars often suffer from abiotic stress such as drought,which seriously affects their growth and development.WRKY transcription factors have been widely studied in recent years,which play an important role in plant response to abiotic stress.However,there are few studies on the woody plants,especially in the role of WRKY transcription factors in drought stress response in plants,.Therefore,it is of great significance for the large-scale cultivation and production of poplars to further analyze and study the mechanism of WRKY transcription factors in regulating drought stress tolerance of poplars and improve their tolerance to drought stress.Based on previous research in the laboratory,this study systematically analyzed the WRKY transcription factor family using Arabidopsis thaliana and Populus trichocarpa as the experimental material,and overexpressed PtrWRKY75 gene,which was up-regulated in response to drought stress,in Arabidopsis thaliana and triploid white poplar(Populus tomentosa)by genetic engineering technology to further explore its biological functions under drought stress.The main results are as follows:1.According to reports,100 members of WRKY transcription factor family members were found in poplar.Neighboring method was used to construct phylogenetic tree,the result showed that the WRKY transcription factor family of poplar can be divided into three subfamily(I,II,III).Chromosome location analysis showed that 100 members of the WRKY transcription factor family are distributed on 18 of the19 chromosomes of poplar.By analyzing the conserved domain of WRKY transcription factor family members,WD conserved domain was found,including WRKY sequence and CX₄–₇-CX₂₃–₂₈-HX₁–₂-H or CX₄–₇-CX₂₃–₂₈-HX₁–₂-C zinc finger structures.Group I contained two WRKY sequences and CX₄–₇-CX₂₃–₂₈-HX₁–₂-H zinc finger structure.Group II mostly contained one WRKY sequence and CX₄–₇-CX₂₃–₂₈-HX₁–₂-H zinc finger structure.Members of Group III contained a WRKY sequence and a CX₄–₇-CX₂₃–₂₈-HX₁–₂-C zinc finger structure.Through the analysis of promoter elements,it was found that WRKY transcription factor genes have multiple response elements related to growth and development,hormonal response,biological and abiotic stress,and infer their functional diversity during plant growth and development.2.In this study,PtrWRKY75,a member of the WRKY transcription factor family,was cloned from Populus trichocarpa.Through tissue localization analysis,it was found that the PtrWRKY75 gene was mainly expressed in the leaves,and it also responded to dehydration and salicylic acid treatment.PtrWRKY75 gene was preliminarily determined to play a role in drought stress.The expression vector PtrWRKY75::e GFP was constructed,and the subcellular localization of PtrWRKY75 was observed by tobacco injection method.And it was found that GFP green fluorescence only appeared in the nucleus,indicating that PtrWRKY75 protein was located in the nucleus,which was in line with the characteristics of PtrWRKY75 as a transcription factor.3.The PtrWRKY75 was transformed into wild type(col-0)and mutant(wrky75)Arabidopsis thaliana by inflorescence infection,and overexpressed ox PtrWRKY75 plants were obtained.Wild type(col-0),mutant(wrky75),reversion(wrky75/PtrWRKY75)and overexpressed(ox PtrWRKY75)strains were treated with mannitol in MS solid medium,respectively.It was found that ox PtrWRKY75 had higher seed germination rate and longer roots compared to wild plants under both treatments.The soil drought experiment of each genotype of Arabidopsis thaliana showed that the overexpressed plant had stronger drought resistance,and by measuring the photosynthetic rate,total chlorophyll content and chlorophyll fluorescence of each genotype of plants,it was found that the overexpressed plants had higher photosynthetic rate,chlorophyll content and chlorophyll fluorescence,which were about 2,1.8 and 1.2times of WT plants,respectively.In conclusion,overexpression of the PtrWRKY75 gene can improve drought tolerance in Arabidopsis thaliana.4.PtrWRKY75 gene was transformed into triploid white poplar genome by Agrobacterium-mediated leaf disc method,and overexpressed plants were detected by PCR and q RT-PCR.The photosynthesis-light response curve,stomatal conductance-light response curve and transpiration rate-light response curve of WT and transgenic plants were measured,and it was found that the stomatal conductance and leaf transpiration rate of transgenic plants were significantly lower than that of WT plants,while the photosynthesis of transgenic and WT plants showed no significant difference.Therefore,the transient water use efficiency(WUEi).The short-and long-term drought experiments were carried out on WT and transgenic plants,and the phenotypic analysis showed that transgenic plants had a better growth conditions.Through the measurement of the related physiological indexes,it was found that the transgenic plants had higher photosynthetic rate,relative water content of leaves,stem elongation rate and lower stomatal conductance,transpiration rate and relative electrical conductivity of leaves under drought conditions.In addition,the biomass accumulation of transgenic plants was about 35%and 46%higher than that of wild-type plants,respectively.The results showed that the transgenic plants had a better growth advantage and a stronger drought tolerance under drought conditions.5.The exogenous salicylic acid(SA)induced the expression of PtrWRKY75,so the leaves of wild-type and transgenic plants were treated with SA.Through reactive oxygen species staining and stomatal sensitivity tests,it was found that the accumulation of H2O2 in transgenic plants was higher than that in wild-type plants,stomatal closure of transgenic plants was faster than that of WT plants,and stomatal aperture of transgenic plants was smaller than that of WT plants.These results suggested that PtrWRKY75promoted SA-induced stomatal closure via H2O2 production and then improved drought tolerance of plants.The content of salicylic acid in WT and transgenic plants was further determined under normal and dehydration conditions,and it was found that PtrWRKY75 gene could increased the content of salicylic acid under dehydration conditions.Furthermore,the expression of PtrPAL1,a key salicylic acid synthesis gene in overexpressed plants,was significantly up-regulated by q RT-PCR.In addition,EMSA,transient transformation analysis and dual luciferase reporter assay were performed to verify the binding of PtrWRKY75 and the promoter of PtrPAL1,promote the expression of PtrPAL1.Therefore,WRKY75promoted the production of SA through the PAL1 pathway,and SA promoted the production of H2O2,induced stomatal closure and improved drought tolerance of plants.In summary,we studied the function of PtrWRKY75 gene,a member of the WRKY transcription factor family,in Arabidopsis thaliana and poplar,and found that under drought stress,PtrWRKY75 gene could improve plant drought tolerance without affecting plant biomass.In addition,we also revealed the molecular mechanism of PtrWRKY75 regulating drought tolerance in poplars.The PtrWRKY75 gene in poplar could promote the biosynthesis of salicylic acid(SA)by activating the transcription of PtrPAL1,thereby promoting the accumulation of H2O2,causing stomatal closing of plant leaves,reducing transpiration,improving plant water use efficiency and drought resistance.The study provided a theoretical basis for the study of drought tolerance in woody plants.At the same time,this paper lay a foundation for cultivation of new drought-resistant poplar varieties and poplar plantation development in drought area.