Volatile terpenes constitute a predominant class of floral scent emitted by Paeonia lactiflora. Despite their ecological and economical significance, the genetic blueprint of the underlying biosynthetic pathway remains poorly elucidated. Although a few terpene synthase (TPS) genes have been reported, the broader network of genes orchestrating terpene production in P. lactiflora is still largely unresolved. In this study, we attempted to address this gap by exploring the terpene biosynthetic pathway genes in P. lactiflora ‘Zifengyu’. β-caryophyllene, geraniol, citronellol, and 1, 8-cineole were identified as the dominant floral terpenes, and catalytic functions of key proteins—terpene synthase (PlTPS), Nudix hydrolase (PlNUDX), and prenyltransferase (PlPT) were comprehensively characterized. Briefly, biochemical analyses revealed that six of the nine identified PlTPS proteins utilized diverse prenyl diphosphates to generate both monoterpenes and sesquiterpenes, while their products specificity were determined by plastidic or cytosolic localizations in planta. In particular, PlTPS4, PlTPS5, and PlTPS9 catalyzed the production of β-caryophyllene, 1, 8-cineole, and geraniol, respectively. Besides, two amino acid residues were found to drive catalytic activity and product profiles in PlTPS4 and PlTPS5. Markedly, PlNUDX hydrolyzed GPP and NPP to yield geraniol and nerol thereby providing a plastid-independent pathway for monoterpene biosynthesis, and prenyltransferases were further functionally characterized to clarify the supply of prenyl diphosphates feeding into volatile terpenes. Collectively, these findings not only provide a mechanistic framework for understanding floral terpene biosynthesis in P. lactiflora but also reveal alternative metabolic routes that enrich its volatile profiles that could be utilized in scent improvement of ornamental plants.

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