PBU Concentration-Dependent Regulation of Callus Differentiation in Eucalyptus urophylla × E. grandis: Integrated miRNA and Metabolomic Insights

PBU (N-phenyl-N′-thiazolylurea) promotes callus induction and adventitious bud differentiation in eucalyptus, but the miRNA-mediated regulatory mechanisms underlying these effects remain unclear. In this study, calli of Eucalyptus urophylla × E. grandis clone DH32-29 with distinct phenotypes were cultured at four PBU concentrations (0, 0.1, 1 and 5 mg L−1) and analyzed by small-RNA sequencing, targeted metabolomics, and qRT-PCR validation. A total of 114 common differentially expressed miRNAs were identified, targeting 610 mRNAs. Functional enrichment analysis revealed that these targets were predominantly associated with lignin metabolism, phenylpropanoid metabolism, biotin metabolism, tryptophan metabolism, protein processing in the endoplasmic reticulum, and galactose metabolism. Metabolomic profiling detected 3029 metabolites, with differential metabolites enriched in the ABC transporter pathway, galloyl sugar biosynthesis, and cofactor biosynthesis. Key miRNA families, including miR164, miR165/166, and miR396, exhibited PBU concentration-dependent expression patterns and were predicted, based on in silico target prediction and qRT-PCR co-expression, to be potentially associated with target genes involved in lignin biosynthesis, ROS-related metabolism, and cytokinin homeostasis; these regulatory relationships remain to be experimentally validated. Among the tested concentrations, 1 mg L−1 PBU was the dosage associated with the strongest reprogramming of secondary metabolism and with metabolic signatures suggestive of better preserved redox homeostasis; future work will build on this reference dataset with quantitative regeneration phenotyping and direct redox measurements to confirm this candidate optimum. These findings provide new insights into PBU-mediated in vitro regeneration in eucalyptus and offer a molecular basis for optimizing regeneration systems in E. urophylla × E. grandis. These findings provide new insights into the miRNA-metabolite regulatory network underlying phenylurea-mediated callus differentiation in woody plants.