Molecular Mechanism Of Transcription Factor PsnHDZ01 In Regulation Of Drought And Salt Tolerance In Populus Simonii × Populus Nigra

Plants are often affected by adverse environment during their growth and development.In order to survive and adapt to the environment,plants have evolved a variety of signal transduction pathways and complex physiological and biochemical mechanisms in response to stress.HD-ZIP is a plant specific transcription factor,which is widely involved in plant development and stress response.Previous studies on HD-ZIP family genes were mostly limited to leaf development,flowering time,adventitious root development,stem elongation and tillering,etc.However,there are few reports on the drought and salt tolerance function of poplar HD-Zip family genes.In this study,we took Populus simonii×P.nigra as the research object to preliminarily explore the response mechanism of PsnHDZ01 under stress.The results are as follows:（1）A total of 63 HD-ZIP family members were identified from the poplar genome,which were randomly distributed on 19 chromosomes,and all conserved Homeobox and HALZ domains.Based on gene structure and phylogenetic relationships,these members were divided into four subfamilies,which were rich in segmental duplication events and underwent strong purifying selection,and also had diverse colinear evolutionary relationships.Among 63 HD-ZIP family gene,40 differentially expressed genes（DEGs）were identified by RNA sequencing under salt stress,of which PsnHDZ01 was significantly up-regulated in roots,stems and leaves.（2）Subcellular localization analysis showed that PsnHDZ01 was a nuclear localization protein.The PsnHDZ01 gene expression has tissue specificity and mainly expressed in the stem.The analysis of spatiotemporal expression patterns showed that PsnHDZ01 could be induced by salt,drought,ABA and Me JA in different tissues,and reached the peak at 24 h.Yeast One-hybrid determined that PsnHDZ01 could specifically bind to the anti-stress cis-acting elements,including TA1（TAATTA）,TA2（AATATTATT）,HBS1（AAGAAG）,Myb-P2（CCTACC）,DRE1（ACCGAC）and ERD（TGTCA）.（3）The PsnHDZ01 promoter was cloned,and the transgenic poplar were obtained by the agrobacterium-mediated method.The results of GUS staining showed that the promoter had transcriptional activity.PsnHDZ01 promoter contains numerous elements related to growth and development,abiotic stress,and hormone synthesis.Two stress-resistance related proteins PsnHDZ06 and Psn ABF3 were identified by Yeast One-hybrid,which could specifically bind to the PsnHDZ01 promoter and regulate the expression level of PsnHDZ01 gene as upstream regulators.（4）Transgenic poplar over-expressing/suppressing PsnHDZ01 were obtained by the agrobacterium-mediated method.Under salt stress,the transgenic poplar over-expressing PsnHDZ01 showed better growth state,significant increases of POD and SOD activities,and chlorophyll and proline concentration,but decreases of H₂O₂and MDA accumulation,and decreases of electrical conductivity and water loss rate when compared to the non-transgenic poplar and transgenic poplar suppressing PsnHDZ01.（5）A tatal of 464 differentially expressed genes（DEGs）were identified from the transgenic poplar over-expressing PsnHDZ01 by RNA-Seq,of which 335 were up-regulated and 129 were down-regulated.GO classification analysis showed that these DEGs were mainly involved in biological processes such as metabolism,biology and stress response.Yeast One-hybrid,luciferase activity verification and Ch IP experiments showed that PsnHDZ01 could bind to promoters of Psn WRKY51,Psn SRK2C and Psn WRKY70 to regulate the expression levels of these genes.（6）Self-activation experiments showed that PsnHDZ01 has self-activating activity,and the self-activating region is located at the C-terminus.Eight potential proteins interacting with PsnHDZ01 were selected from poplar c DNA library by Yeast Two-hybrid.Verification from Yeast Two-hybrid and Bi FC indicated that PsnHDZ01 could interact with PsnHMP20,PsnHDZ13,PsnHDZ50 and itself to form dimer.Transgenic poplar over-expressing/suppressing PsnHDZ50were obtained by the agrobacterium-mediated method.Transgenic poplar over-expressing/suppressing PsnHDZ50 and transgenic poplar over-expressing/suppressing PsnHDZ01 showed similar state both in phenotype and physiological indexes,indicating that PsnHDZ50 and PsnHDZ01have synergistic interaction relationship.In summary,this study identified a significant stress resistance gene PsnHDZ01 from the HD-ZIP family of poplar,preliminarily defined the stress resistance mechanism and signal transduction of this gene,and provided a basis for improving the complex stress response network of poplar.The clues provide important theoretical support and genetic resources for poplar stress resistance breeding in the future.