PHYTOCHROME-INTERACTING FACTOR3 (PIF3) orthologs orchestrate stem elongation and wood formation in Populus.

Phytochrome-interacting factors (PIFs) have been established as negative regulators of vascular patterning and xylem differentiation in the herbaceous plant Arabidopsis thaliana, however, PIFs' regulatory role in secondary growth in woody species remains unclear. Here, we examined the expression patterns and involvement of PtoPIF3.1 and PtoPIF3.2 during stem growth and secondary xylem development in Populus tomentosa. Overexpression of either PtoPIF3.1 or PtoPIF3.2 significantly enhances both longitudinal stem growth and radial wood development. Conversely, CRISPR-generated Ptopif3.1 and Ptopif3.2 mutants exhibit reciprocal phenotypic defects. Exogenous auxin application partially restores the phenotypes of Ptopif3.1 and Ptopif3.2 mutants, and the auxin biosynthesis-deficient mutant Ptoyuc8 exhibits developmental abnormalities similar to those observed in Ptopif3 mutants. Further analysis revealed that PtoPIF3s directly bind to and activate expression of PtoYUC8 and cell expansion-related genes PtoEXPA1.1/1.2, while modulating cambial division and secondary xylem development marker genes (PtoWOX4, PtoANT, PtoCYCD3s, and PtoHB7/8) through auxin-mediated signaling. Together, our findings establish PtoPIF3.1/3.2 as key regulators that coordinate stem elongation and secondary growth in Populus, highlighting the functional divergence of PIF homologs between herbaceous and woody species.