The Jumonji C (JMJ-C) domain-containing gene family regulates epigenetic and developmental processes in plants. We identified 55 JMJ-C genes in Populus alba × Populus glandulosa using HMM and BLASTp analyses. Chromosomal mapping revealed an asymmetric distribution with conserved synteny. Phylogenetic reconstruction revealed that PagJMJ genes segregate into five evolutionarily conserved subfamilies, exhibiting classification patterns identical to those of Arabidopsis thaliana and Populus trichocarpa. Synteny analysis indicated a closer relationship with P. trichocarpa than with A. thaliana. Motif and promoter analyses highlighted subfamily-specific features and diverse cis-elements, particularly light-responsive motifs. Expression profiling revealed tissue-specific patterns, with key genes enriched in roots, vascular tissues, and leaves. Developmental analysis in cambium and xylem identified four expression clusters related to wood formation. Co-expression analysis identified six key PagJMJ genes (PagJMJ6, 29, 34, 39, 53, and 55) strongly associated with wood formation-related transcription factors. ChIP-qPCR analysis revealed that key genes co-expressed with PagJMJ genes were marked by H3K4me3 and H3K9me2 modifications. These findings provide insights into the evolutionary and functional roles of PagJMJ genes in poplar vascular development and wood formation.

N⁵-methylcytosine (m⁵C) RNA methylation plays an essential role in gene regulation, yet its functions in woody plant development remain poorly understood. This study systematically identified 12 putative tRNA-specific methyltransferase 4 (TRM4) homologs (PagTRM4s) within the P. alba × P. glandulosa genome. Phylogenetic and synteny analyses revealed high evolutionary conservation across Arabidopsis and rice. Structural and domain analyses suggested functional conservation among allelic pairs. Expression profiling showed that PagTRM4B-a maintained stable expression in xylem at different developmental stages. PagTRM4B-a overexpression in poplar resulted in decreased plant height and stem diameter, modified wood composition, and increased RNA m⁵C levels. Histological analysis showed that overexpression enhanced xylem cell expansion and secondary xylem formation without affecting cambial activity. Moreover, the expression levels of secondary cell wall (SCW) biosynthesis genes were significantly down-regulated in PagTRM4B-a-OE transgenic plants. This study offers new insights into the epigenetic control of secondary growth in trees and highlights PagTRM4B-a as a potential target for influencing wood formation in woody plants.