Phylostratigraphic analysis revealed that ancient ohnologue <i>PtoWRKY53</i> innovated a vascular transcription regulatory network in <i>Populus</i>

The secondary vascular system represents a hallmark innovation in woody plant evolution. While contemporary studies have decoded key molecular networks governing its development, the evolutionary trajectory of these regulatory systems remains elusive. We performed integrated phylostratigraphic and transcriptomic profiling across six Populus species spanning five phylogenetic clades to decipher the evolutionary dynamics of regulatory genes in the secondary vascular system. This analysis revealed that over 51% of regulatory genes originated from the most ancient phylostratum (PS1). The observed 65% gene expansion in the PS1 was driven by whole-genome duplication (WGD). Through phylotranscriptomic and gene co-expression network analysis, we found that dosage-unconstrained ohnologues from three PS1 families, MYB domain protein, NAC domain containing protein, and WRKY DNA-binding protein, have significant xylem-specific expression. We identified an ohnologue, PtoWRKY53, which forms a new transcription regulatory network in xylem via neofunctionalization. Transgenic analyses revealed that PtoWRKY53 transcriptionally represses PtoCESA3, thereby inhibiting xylem secondary wall biosynthesis and reducing poplar height (26.39%) and stem diameter (18.36%). Our study highlights the importance of interactions between ancient genes and WGD in generating morphological innovations of the secondary vasculature in Populus.