Objective Based on quasi-targeted metabolomics technology, this study aimed to investigate the major metabolite types and contents of two Eucalyptus urophylla clones exhibiting significant branching differences, aiming to provide a metabolomic reference and theoretical foundation for breeding superior narrow-crown E. urophylla varieties.

Method Four-month-old tissue-cultured seedlings of E. urophylla clones 'ZQUC14' (A, wide-crown) and 'LDUD26' (B, narrow-crown) were used as materials. After decapitation treatment, exogenous gibberellin (GA₃) was applied. Liquid chromatographymass spectrometry (LC-MS) was employed to comparatively analyze metabolite profile differences before and after decapitation, and under GA₃ treatment. Inter-sample correlation analysis and principal component analysis (PCA) were performed to assess metabolic differences among groups. Differential metabolites were screened and subjected to pathway enrichment analysis.

Result A total of 1, 028 metabolites were identified across the four treatment groups, of which 31.91% were differential metabolites, primarily belonging to amino acid derivatives, flavonoids, and carbohydrates. Significant differences in metabolites within the same tissue were observed between the two clones after decapitation, with changes in amino acid derivatives and flavonoids being particularly notable. Exogenous GA₃ treatment further intensified these differences. Furthermore, clone B (narrow-crown) exhibited fewer metabolic pathways affected by decapitation and GA₃ compared to clone A (wide-crown). However, both clones showed significant alterations in endogenous hormone redistribution patterns following the treatments.

Conclusion This study revealed the core differential metabolites and key metabolic pathways in the responses of narrow-crown (B) and wide-crown (A) E. urophylla clones to decapitation and exogenous GA₃. It clarified that hormone redistribution is a critical shared response characteristic, laying the groundwork for elucidating the metabolic mechanisms underlying GA₃ -regulated branching in E. urophylla.