Nymphaea is a key genus of the ANA grade (Amborellales, Nymphaeales, and Austrobaileyales) of basal flowering plants, which serve as a key model to study the early evolution of floral traits. In this study, we comprehensively investigated the emission, biosynthesis, and biological function of the floral scent in a night-blossoming waterlily Nymphaea prolifera. The headspace volatile collection combined with GC–MS analysis showed that the floral scent of N. prolifera is predominately comprised by methylated benzenoids including anisole, veratrole, guaiacol, and methoxyanisole. Moreover, the emission of these floral benzenoids in N. prolifera exhibited temporal and spatial pattern with circadian rhythm and tissue specificity. By creating and mining transcriptomes of N. prolifera flowers, 12 oxygen methyltransferases (NpOMTs) were functionally identified. By in vitro enzymatic assay, NpOMT3, 6, and 7 could produce anisole and NpOMT5, 7, 9, produce guaiacol, whereas NpOMT3, 6, 9, 11 catalyzed the formation of veratrole. Methoxyanisole was identified as the universal product of all NpOMTs. Expression patterns of NpOMTs provided implication for their roles in the production of the respective benzenoids. Phylogenetic analysis of OMTs suggested a Nymphaea-specific expansion of the OMT family, indicating the evolution of lineage-specific functions. In bioassays, anisole, veratrole, and guaiacol in the floral benzenoids were revealed to play the critical role in repelling waterlily aphids. Overall, this study indicates that the basal flowering plant N. prolifera has evolved a diversity and complexity of OMT genes for the biosynthesis of methylated benzenoids that can repel insects from feeding the flowers. These findings provide new insights into the evolutional mechanism and ecological significance of the floral scent from early-diverged flowering plants.

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