The PagGRF20-PagMYB4 regulatory module coordinates wood formation in poplar

Despite decades of research on NAC-MYB master regulators, the transcription factor (TF)-mediated network governing wood development remains fragmented, hindering a systems-level understanding of secondary xylem formation. In this study, we identified GROWTH REGULATING FACTOR 20 (PagGRF20) as a key regulator within the poplar wood formation network. Compared to wild-type (WT) poplars, the PagGRF20-overexpressing lines showed a 22.1%-23.9% increase in secondary xylem thickness, a 37.7%-43.3% increase in cell wall thickness, and higher cellulose and xylan content. These phenotypic changes coincided with upregulation of genes involved in cellulose and xylan biosynthetic pathways. Conversely, PagGRF20-RNAi lines displayed opposite phenotypic traits. Molecular analyses revealed that PagGRF20 binds to the TGT[C/T]AGA cis-regulatory elements in the promoters of wood polymer biosynthesis genes, modulating polysaccharide production by activating the glycosyltransferase IRREGULAR XYLEM 14-LIKE (PagIRX14L). Crucially, overexpression of PagIRX14L in poplar increased the xylan content and decreased lignin levels, suggesting that PagGRF20-mediated transcriptional activation of PagIRX14L significantly contributes to the observed shifts in cell wall composition. Additionally, the R2R3-MYB repressor PagMYB4 physically interacts with PagGRF20, forming a transcriptional complex that suppresses lignin biosynthesis genes, including LACCASE 11 (PagLAC11), potentially altering the balance of cell wall components and affecting cellulose and xylan accumulation. Finally, we built a three-layer, PagGRF20-centered regulatory network to uncover the mechanisms and target genes underlying wood formation. Our results suggest that the PagGRF20-PagMYB4 module coordinates secondary cell wall (SCW) biosynthetic pathways, providing novel insights into engineering wood with enhanced polysaccharide deposition and low lignin content.