RNA-seq analysis of floral volatile gene expression in Cypripedium henryi in response to pollination treatments
Floral scent serves as a primary mechanism for pollinator attraction, acting as a driver for reproductive isolation and speciation. Because shifts in the composition of floral Volatile Organic Compounds (VOCs) significantly influence a plant's attractiveness, and because the production of these compounds is metabolically costly, plants often reduce scent emissions following successful pollination. This resource-saving strategy is particularly expected in long-lasting fragrant flowers, such as those of the deceptive orchid genus Cypripedium. To investigate potential post-pollination effects on the floral scent of Cypripedium henryi, four pollination treatments were applied to flowers approximately two days post-anthesis (n = 3 replicates per treatment, 12 flowers total): • Treatment P: Removal of both pollinia. • Treatment G: Deposition of a donor flower's pollinium onto the stigma. • Treatment GP: Simultaneous removal of pollinia and deposition of a donor pollinium. • Treatment K (Control): No artificial manipulation. Floral scent was sampled from each flower immediately before pollination treatment and again 48 hours later. Following the final scent collection, three specific tissue types per flower were harvested for RNA analysis: the labellum, staminode, and lateral petal. The resulting 36 RNA samples were extracted and sequenced to facilitate de novo transcriptome assembly. We quantified transcripts and performed differential expression analysis to compare the pollination treatments against the control group, as well as to identify variation across the different tissue types. Following gene annotation, we focused our investigation on candidate genes within the terpene and fatty acid biosynthesis pathways, which are responsible for producing the characteristic volatiles of C. henryi. Finally, the expression levels of these candidate genes were evaluated in direct relation to the volatile levels identified in the scent analysis to provide a comprehensive view of the molecular regulation of post-pollination scent emission.