Functional studies on PdbRAV2 transcription factor mediates positive regulation of drought tolerance in Populus davidiana × P. bolleana

• PdbRAV2 enhances drought tolerance by promoting ROS scavenging in poplar. • ●PdbRAV2 directly binds to the 'CAACA' and 'CACCTG' elements in the promoter regions of antioxidant genes ( PdbSODs, PdbPODs, PdbCAT2 ) and positively activates their expression. • ●The PdbRAV2 transcription factor is succinylated at lysine K361 in response to drought stress, enhancing its DNA-binding affinity to stress-responsive cis-elements. • PdbRAV2 interacts with PdbWRKY2, boosting its transcriptional activity. • ●Co-transformation of PdbRAV2 and PdbWRKY2 confers significantly stronger drought resistance compared to single-gene transformations. The RAV (related to ABI3 and VP1) transcription factor, which belongs to APETALA2/ethylene response factor (AP2/ERF) family plays a crucial role in plant responses to environmental stress. In this study, we identified a PdbRAV2 transcription factor acting as a positive regulator in drought tolerance. RT-qPCR and GUS staining revealed that PdbRAV2 was induced by drought stress in leaves, roots, and stems. Subcellular localization showed that the PdbRAV2 protein was localized in the nucleus. Drought analysis demonstrated that overexpression of PdbRAV2 in Populus davidiana × P. bolleana (Shanxin poplar) enhanced drought resistance by promoting ROS scavenging,while the knockout lines generated using the CRISPR/Cas9 system exhibited reduced drought tolerance. Further investigation demonstrated that PdbRAV2 directly bound to the promoters of PdbSODs,PdbPODs and PdbCAT2 , and positively activated their expression. Co-immunoprecipitation (Co-IP), yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) identified and confirmed PdbWRKY2 interacting with PdbRAV2 and enhanced its transcription. Additionally, the PdbRAV2 transcription factor was succinylated at lysine K361 under drought stress. Dual luciferase assays showed that after mutating the site, the PdbRAV2 transcription factor had a reduced ability to bind ‘CAACA’ and to ‘CACCTG’. These findings could offer valuable insights into new genetic resources for the molecular breeding of poplar.