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Tree peony (Paeonia suffruticosa), as a popular ornamental plant worldwide, has a unique floral fragrance, and it is important in the pollination, ornamental, food, and fragrance product industries. However, the underlying molecular mechanisms for the synthesis of floral fragrance terpenoids in tree peony are not well understood, constraining their exploitation. P. suffruticosa ‘Oukan’ produces strong floral fragrance terpenoids with high ornamental value and excellent stress resistance and is considered a valuable model for studying tree peony floral fragrance formation. Based on transcriptome data analysis, the PsHMGR1 and PsTPS1 genes associated with floral terpene synthesis were cloned. Then, PsHMGR1 and PsTPS1 were functionally characterized by amino acid sequence analysis, multiple sequence alignment, phylogenetic tree construction, qRT-PCR, and transgenic assay. PsHMGR1 contains two transmembrane structures and a conserved HMG-CoA_reductase_class I domain, and PsTPS1 belongs to TPS-a subfamily. The qRT-PCR analysis showed that the expression levels of PsHMGR1 and PsTPS1 increased and then decreased at different flower development stages, and both were significantly higher in flowers than in roots, stems, and leaves. In addition, the linalool content in PsHMGR1 transgenic lines was significantly higher than that of WT. Germacrene D, which was not found in WT, was detected in the flowers of PsTPS1 transgenic lines. These results indicate that PsHMGR1 and PsTPS1 promote terpene synthesis in plants and provide ideas for the molecular mechanism of enhancing terpene synthesis in tree peony floral fragrance. Full article