A regulatory module integrates lipid metabolism and cell wall remodeling to establish the seed coat palisade barrier.

Seed physical dormancy is a crucial adaptive trait conferred by the water-impermeable seed coat, particularly its specialized palisade layer. Although the structural components of this barrier, including the cuticle and cell wall polysaccharides, are well characterized, the molecular mechanisms coordinating their formation and remodeling remain poorly understood. Here, using single-nucleus RNA sequencing of Medicago truncatula, we identify a regulatory module essential for palisade layer formation and the establishment of physical dormancy. We show that MtKNOX4 interacts with cytoplasmic MtSAW1/2 proteins and facilitates their nuclear recruitment, thereby enabling the formation of functional nuclear MtKNOX4-MtSAW1/2 complexes. These complexes activate the expression of downstream target genes, including MtATT1 (encoding a fatty acid ω-hydroxylase belonging to the CYP86A subfamily of cytochrome P450 enzymes) and MtPAE8 (encoding a pectin acetylesterase). Thus, the MtKNOX4-MtSAW1/2 module coordinates two parallel barrier-forming pathways involving lipid polyester remodeling and pectin deacetylation, thereby reinforcing seed coat impermeability. Loss-of-function mutations in these regulators disrupt palisade layer integrity and impair physical dormancy. Collectively, our results reveal a regulatory module in which MtKNOX4-mediated nuclear recruitment of MtSAW1/2 links transcriptional regulation of lipid metabolism and cell wall remodeling to seed coat barrier formation, providing potential targets for tuning hard-seededness in legume crops.