Diverse O-methyltransferases catalyze the biosynthesis of floral benzenoids that repel aphids from the flowers of waterlily Nymphaea prolifera

Abstract

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.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31872140, 31470693, 31100503), the Natural Science Foundation of Zhejiang Province (LY18C160006), the Zhejiang Provincial Key Laboratory of Characteristic Aquatic Vegetable Breeding and Cultivation Open Fund Project (KL-2022-01, KL-2022-02), and the Project for Science and Technology in Jinhua City (Grant No. 2019-2-002). The authors thank TopEdit (www.topeditsci.com) for its linguistic assistance during the preparation of this manuscript.

Author contributions

Y.J., F.C., and J.F. conceived the project. G.L., W.Z., M.Y., M.F., Q.W., X.Y., and H.C. performed the experiments and analysed the data. L.W., Y.X., L.S., C.Y., and D.Y. managed N. prolifera plantations. G.L., Y.J., and F.C. wrote the manuscript.

Data availability

All data generated or analysed in this study are included in this published article and its supplementary information files.

Conflict of interest

The authors declare no competing financial interest.

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