The miR396d–PagGRF20– PagXTH5 module regulates salt tolerance in poplar

Salinity is a major environmental constraint that limits biomass production and perturbs cell-wall biogenesis, yet how cell-wall remodeling regulates salt tolerance in perennial trees remains largely unexplored. In this study, we revealed that the miR396d-GROWTH REGULATING FACTOR 20 (PagGRF20)-XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE 5 (PagXTH5) regulatory module orchestrates salt tolerance in poplar. Despite the general growth-promoting function of the GRF family, PagGRF20 acts as an atypical member that negatively modulates both stress tolerance and leaf expansion. Transgenic poplars with miR396d overexpression or paggrf20 knockout exhibited improved growth and reduced stress symptoms under salt stress. Concurrent with increased salt tolerance, these transgenic plants exhibited reduced H2O2 accumulation and elevated activities of ROS scavenging enzymes. Combining transcriptome data, ATAC-seq and DAP-seq analyses, we identified PagXTH5, a xyloglucan endotransglucosylase/hydrolase and negative regulator of salt tolerance, as the direct target of the miR396d-PagGRF20 module. We further reveal that PagGRF20 recruits the ethylene-response factor PagRAP2.3 to synergistically activate PagXTH5 expression. In conclusion, this study elucidated the mechanism underlying the miR396d-PagGRF20-PagXTH5 regulatory axis. By balancing growth with stress responses, this module offers a promising genetic strategy for breeding salt-resilient bioenergy poplar by optimizing the trade-off between biomass accumulation and stress survival.