Integrated Aroma Profiling and Functional Characterization of LcTPS24 Uncover Its Role in Monoterpene Biosynthesis in Litchi Fruit

Litchi (Litchi chinensis) is a commercially valuable subtropical fruit prized for its unique floral–fruity aroma, which largely determines consumer preference and commercial quality. Terpenoids serve as the core volatile compounds that shape the characteristic litchi fruit aroma. Despite their importance, the volatile profile of litchi fruit and the key genes underlying its aroma formation remain poorly understood. Herein, we determine the gas chromatography–mass spectrometry (GC-MS)-based metabolomic profiling of litchi cultivars and functionally characterize a key litchi terpene synthase gene, LcTPS24, a candidate selected on the basis of previously reported TPS-family expression patterns and gene–metabolite associations. GC-MS profiling of four commercially significant cultivars, i.e., Feicui (FC), Guiwei (GW), Jingganghongnuo (JGHN), and Lufennuo (LFN), at three developmental stages revealed that terpenes predominate the volatile organic compound (VOC) spectrum, accounting for up to 94% of total volatiles in JGHN. Monoterpenes represent the principal fraction in GW (98%), JGHN (81%), and FC (75%), while comprising 44% in the LFN cultivar. Phylogenetic analysis classified LcTPS24 within the TPS-b subfamily of angiosperm monoterpene synthases. Biochemical assays performed through prokaryotic expression and HS-SPME-GC-MS revealed that recombinant LcTPS24 exclusively utilizes geranyl pyrophosphate (GPP) rather than farnesyl pyrophosphate (FPP), producing ten monoterpene products: α-thujene, α-pinene, (+)-sabinene, (−)-β-pinene, α-phellandrene, α-terpinene, 2-carene, p-cymene, γ-terpinene, and (+)-4-carene. In JGHN fruit, virus-induced gene silencing was associated with reduced LcTPS24 transcript abundance and lower monoterpene accumulation, providing evidence consistent with a contribution to fruit monoterpene biosynthesis. Electronic-tongue measurements were analyzed separately to examine treatment-associated variation in juice sensor responses, although these measurements did not directly assess volatile composition or aroma perception. This study functionally validates the role of LcTPS24 in terpenoid volatile biosynthesis and provides genetic resources and mechanistic insights for molecular breeding of high-aroma litchi cultivars.