Molecular Mechanism Of Transcription Factor PagMYB73 In Regulation Of Salt Tolerance In Populus Alba × P.glandulosa

MYB transcription factor family is one of the largest transcription factor families in plants and can participate in multiple biological processes.Previous studies have mostly focused on the role of poplar R2R3-MYB transcription factor in the regulation of synthesis of anthocyanins,lignin and secondary walls.However,researches on R2R3-MYB genes in response to salt stress are limited in poplar.In this study,84K poplar was used as material and transcriptome sequencing was used to analyze the salt stress response of poplar R2R3-MYB family genes.It was found that the expression level of PagMYB73(MW793352)was significantly increased under salt stress,but its function has not yet been reported in related studies.This study analyzed the biological function of PagMYB73 gene and its regulatory mechanism in response to salt stress.The results are as follows:(1)207 R2R3-MYB family members were identified from poplar,and phylogenetic analysis,structural analysis,gene tandem and segmental duplication events analysis,as well as gene collinearity analysis between different species were performed.Based on the data of transcriptome profiling,24 salt stress responsive R2R3-MYB genes were identified.(2)PagMYB73 protein was localized in the nucleus and had no transcriptional activation activity in yeast.Through yeast one-hybrid experiment,it was found that PagMYB73 protein could bind to E-box element.The promoter of PagMYB73 gene contained eight abscisic acid response elements.PagMYB73 gene was expressed in stems,roots,leaves and buds,and the expression level decreased sequentially.In addition,PagMYB73 was gradually up-regulated in the early stage of stress and then gradually recovered in different tissues under salt and osmotic stresses.(3)The developed transgenic 84K poplar plants overexpressing or suppressing PagMYB73 were used for salt stress tolerance analysis.The results showed that the salt tolerance of transgenic poplar overexpressing PagMYB73 was significantly improved,while the PagMYB73 inhibition lines displayed opposite effects.Further investigation found that PagMYB73 gene could improve salt stress tolerance of plants by enhancing the scavenging ability of reactive oxygen species and the accumulation of proline under salt stress.(4)556 differentially expressed genes were identified from the transgenic 84K poplar overexpressing the PagMYB73 gene,including many up-regulated genes related to stress resistance.Such as genes encoding glutathione S-transferase,early-responsive to dehydration stress protein,late embriogenesis abundant protein,oxidoreductase,heavy metal transporter,potassium transporter protein,and peroxidase.(5)Yeast one-hybrid experiment,co-transformation experiment of the effector vector and the reporter vector,and chromatin immunoprecipitation assay proved that PagMYB73 can directly bind to the E-box elements of the promoters of downstream stress resistance genes PagLEA51,PagGSTU22 and PagGSTU19.(6)Yeast two-hybrid experiments and bimolecular fluorescent complimentary experiment proved that PagMYB73 protein can form homodimers with itself and interact with PagHNHEl and PagMYB74 proteins.In conclusion,salt stress can induce the expression of PagMYB73 gene,and the induced PagMYB73 gene can improve the salt stress tolerance of plants in two ways.On the one hand,PagMYB73 can increase the plant's ability to remove excessively accumulated reactive oxygen species by increasing the activities of SOD and POD under salt stress or by combining with the E-box on the promoters of downstream target genes PagGSTU22 and PagGSTU19 to up-regulate their expression.On the other hand,PagMYB73 can regulate the osmotic balance of plants by increasing the accumulation of proline under salt stress or by combining with the E-box on the promoter of the downstream target gene PagLEA51 to up-regulate its expression.This study identified an important salt stress responsive gene and analyzed its molecular mechanism to improve plant salt stress tolerance,providing a reference for using molecular biology and genetic transformation technology to improve stress resistance of plants.