Genome‐Wide Splicing Landscapes Across Poplar Cultivars Reveal a GBF3 ‐Mediated Post‐Transcriptional Switch Modulating Monolignol Biosynthesis

Black poplars (Populus deltoides, P. nigra, and their hybrids) represents economically and ecologically vital hardwood taxa characterised by rapid biomass accumulation, superior wood properties, and wide adaptability. However, the post-transcriptional regulatory mechanisms orchestrating xylem development in these perennial trees remain poorly understood. Here, we systematically profiled transcriptome and alternative splicing (AS) dynamics in the developing xylem across five representative cultivars: P. euramericana 'Zhonglin46' (Pe1), P. euramericana 'Guariento' (Pe2), P. nigra 'N179' (Pn1), P. deltoides 'Danhong' (Pd1), and P. deltoides 'Nanyang' (Pd2). RNA-seq analysis of 20 independent xylem libraries revealed 9,981 differentially expressed genes (DEGs) and 6,638 alternatively spliced genes across 10 pairwise comparisons. Functional enrichment indicated that transcriptional variations primarily coordinates cell wall biosynthesis and cytoskeleton organisation, whereas AS preferentially tunes nucleic acid binding and metabolic processes. Regulatory network modelling positioned key AS events upstream of core xylogenesis cascades. Crucially, we identified the G-box binding factor GBF3 as a master splicing-regulated hub during secondary vascular differentiation. An intron retention event in GBF3 yields a truncated isoform with aberrant subcellular localisation and abolished DNA-binding capacity, which impairs its transcriptional repression of key lignin biosynthetic genes (PAL2, CAD1, and CCoAOMT2). Collectively, these high-resolution transcriptional and post-transcriptional landscapes illuminate the regulatory architecture of wood formation and provide a valuable genomic resource for trait improvement in woody perennials.