Identification of the RNA m5C methyltransferase genes in <i>Populus alba</i> × <i>Populus glandulosa</i> and the role of <i>PagTRM4B</i> in 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 (<i>PagTRM4s</i>) within the <i>P. alba</i> × <i>P. glandulosa</i> genome<i>.</i> 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 <i>PagTRM4B-a</i> maintained stable expression in xylem at different developmental stages. <i>PagTRM4B-a</i> 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 <i>PagTRM4B-a-OE</i> transgenic plants. This study offers new insights into the epigenetic control of secondary growth in trees and highlights <i>PagTRM4B-a</i> as a potential target for influencing wood formation in woody plants.