Evolutionary and functional analyses of terpene synthases involved in floral scent of Rhododendron fortunei

Abstract

Rhododendrons are prized ornamentals, yet most cultivars lack fragrance. This study investigates the molecular basis of floral scent in the fragrant wild species Rhododendron fortunei by integrating metabolomic, genomic, transcriptomic, and biochemical approaches. The floral scent of R. fortunei is dominated by monoterpenes and sesquiterpenes, with 3-carene and β-pinene as the major constituents. Comparative genomics revealed that fragrant Rhododendron species harbor a larger number of terpene synthase (TPS) genes than non-fragrant germplasms, with the two fragrant species R. fortunei and R. ovatum exhibiting distinct expansion patterns. Four highly expressed TPSs, RfTPS09, RfTPS20, RfTPS36, and RfTPS65, localize to plastids or the cytosol and collectively catalyze the formation of approximately 60% of the floral terpenoid volatiles, with distinct substrate preferences. Notably, all five TPS-f members are tandemly arranged on chromosome 13, among which RfTPS09 exhibits the highest floral expression and harbors a specific MYB-binding element. RfMYB27 and RfMYB29 directly bind the RfTPS09 promoter and activate its transcription, yet promoter cis-element variation, rather than MYB expression levels, likely drives its high expression. Importantly, RfTPS09 deviates from canonical kaurene synthases (KS) function by producing both mono- and sesquiterpenes, suggesting neofunctionalization toward floral scent production via structural divergence and regulatory rewiring. Collectively, these findings elucidate the enzymatic, evolutionary and transcriptional mechanisms underlying floral fragrance in Rhododendron, and provide a foundation for scent improvement in ornamental plants.