The PtNACA1-EXPA6 module negatively regulates salt tolerance by disrupting ionic and ROS homeostasis in Populus trichocarpa

Soil salinization poses a serious threat to plant growth and ecological stability. Expansins (EXPs) mediate cell wall loosening, yet their transcriptional regulation and roles in woody plants under salt stress remain unclear. In this study, we cloned PtEXPA6 from Populus trichocarpa and identified the NAC transcription factor PtNACA1 as its upstream regulator. DNA pull-down coupled with mass spectrometry, EMSA, Y1H, LRA, and VIGS showed that PtNACA1 specifically binds to an ACG-containing region of the PtEXPA6 promoter and promotes its expression; silencing of PtNACA1 also reduced PtEXPA6 expression in P. trichocarpa leaves. Genetic transformation experiments showed that transient overexpression of PtNACA1 in P. trichocarpa leaves increased PtEXPA6 expression, whereas stable overexpression in 84 K poplar increased PagEXPA6 expression and root-tip cell wall extensibility. Under NaCl stress, PtNACA1 -overexpressing plants accumulated more Na + and showed altered Na + and K + fluxes. Osmotically induced root cell wall contraction and relaxation were accompanied by reversible changes in xylem and petiole Na + flux, supporting an association between wall loosening and Na + transport. Moreover, PtNACA1 bound the SOS3 promoter in vitro, and PtNACA1 -overexpressing plants showed weaker induction of SOS2 and SOS3 , lower antioxidant enzyme activities and gene expression, stronger DAB staining indicative of H 2 O 2 accumulation, higher malondialdehyde content and electrolyte leakage, and greater inhibition of photosynthesis and growth than controls. Collectively, our findings identify a PtNACA1- EXPA6 module that increases salt sensitivity in poplar by linking expansin-mediated cell wall remodeling to disrupted ionic and ROS homeostasis.