WRKY71 regulates lignin biosynthesis genes, promoting apoplastic barrier formation and enhancing salt exclusion in roots

• Precise staging of root apoplastic barrier development . Using histochemical staining for lignin and suberin, this study achieves precise division of the three developmental stages of the apoplastic barrier in Arabidopsis thaliana roots, and clearly defines the distance of each stage from the root tip. It fills a gap in the accurate temporal and spatial characterization of root apoplastic barrier development. • Identification of key transcription factor AtWRKY71. Through large-scale sampling of roots at the three developmental stages for transcriptome sequencing, the key transcription factor AtWRKY71, which is involved in root apoplastic barrier formation, is successfully screened. This provides a critical target gene for deciphering the molecular regulatory network of apoplastic barrier development. • Unveiling the dual-regulation mechanism of AtWRKY71 in salt tolerance . AtWRKY71 expression is induced by NaCl stress. It exerts a dual regulatory role: directly binding to the promoter of At4CL3 to positively regulate its expression (promoting S-type lignin monomer synthesis and enhancing Casparian strip formation) and binding to the promoter of AtCDEF1 to negatively regulate its expression (reducing AtCDEF1-mediated suberin degradation and increasing suberin content in the apoplastic barrier). • Establishing the link between AtWRKY71 and root apoplastic barrier-mediated salt exclusion . This study clarifies that AtWRKY71 enhances the functional integrity of the root apoplastic barrier through its dual regulatory effect on At4CL3 and AtCDEF1 , thereby significantly improving the salt exclusion ability of Arabidopsis thaliana . It provides a novel molecular mechanism for understanding plant salt tolerance related to root structural barriers. The Casparian strip (CS) and suberin form the apoplastic barrier, which blocks solute entry into the vascular stele under high-salt conditions. Here, transcriptome analysis of Arabidopsis roots during three stages of apoplastic barrier development (undifferentiated, differentiating, and mature) revealed differential expression of the transcription factor gene WRKY71 . We generated WRKY71 -overexpression (OE) and WRKY71- complementation (COM) lines. Metabolomics analysis showed that WRKY71 promotes the biosynthesis of S-type lignin monomers. Compared to wild type and wrky71 plants, WRKY71- OE and COM plants showed enhanced apoplastic barrier formation and less Na + accumulation under 100 mM NaCl treatment, exhibiting better salt-exclusion ability during germination and seedling growth. Yeast one-hybrid assays revealed that WRKY71 interacts with the lignin biosynthesis genes 4CL3 and CUTICLE DESTRUCTING FACTOR 1 ( CDEF1 ). WRKY71 promotes 4CL3 expression and inhibits CDEF1 expression, as determined by dual-luciferase reporter assay. To further investigate the functions of 4CL3 and CDEF1 , we generated the corresponding OE lines. Under 100 mM NaCl treatment, 4CL3 -OE plants showed enhanced salt tolerance through increased CS formation, whereas CDEF1 -OE plants showed increased salt sensitivity due to inhibited suberin formation. Our identification of the WRKY71– 4CL3 / CDEF1 module sheds light on the role of the apoplastic barrier in salt tolerance.