Fine-tuned terpene synthase gene expression, functional promiscuity, and subcellular localization: implications for the evolution of complex floral volatile bouquet in Caladenia orchids.

Chemically mediated floral volatile signals are crucial for pollinator attraction across angiosperms. However, beyond model plant systems, the molecular mechanisms underpinning their tissue-specific biosynthesis, regulation, and emission are still poorly understood. In this study of a food-deceptive insect-pollinated orchid (Caladenia denticulata), we elucidated the molecular basis of α-pinene biosynthesis-the major floral volatile emitted by this species and diverse lower abundance monoterpenes and sesquiterpenes. To achieve this, we combined comparative transcriptomics between active glandular trichome-rich sepal tips and labellum and non-active remaining flower tissues, floral volatile headspace profiling, phylogenetic analysis of a multigene family, and protein functional assays. We found (i) multiple branch points of the terpene synthase (TPS) biosynthetic pathway were highly expressed and coordinately upregulated in the active floral tissues compared to non-active ones, (ii) the monoterpene synthase CdTPS-b3 underpinning α-pinene biosynthesis and a bona fide promiscuous TPS CdTPS-b4 that may contribute to the diverse array of low-abundance mono- and sesquiterpenes found in its flowers, and (iii) dual localization (plastid and cytosol) of CdTPS-b3 and CdTPS-b4. Our findings highlight metabolic pathway specialization at multiple TPS pathway branch points supporting the biosynthesis and emission of α-pinene in C. denticulata flowers that are implicated in its generalist pollinator attraction. Furthermore, the complexity of diverse floral terpenes in Caladenia is likely mediated by finely tuned TPS gene expression, functional promiscuity, and subcellular localization. We predict that the combination of these three mechanisms underpin the evolution of multiple deceptive pollination strategies in Caladenia.