Epiphyllum oxypetalum, a renowned ornamental species in Cactaceae, releases attractive fragrance during its infrequent, transient, and nocturnal blooms. However, the floral fragrance composition and biosynthesis remain largely unexplored. Employing volatilomics, transcriptomics, and biochemistry, we systematically characterized the composition, emission dynamics, and biosynthesis of the floral scent of E. oxypetalum. The floral scent composition of E. oxypetalum was highly dynamic. Starting after 8 p.m. local time, volatile emission increased 200-fold within 6 h. At full bloom, geraniol accounted for 72.54% of the total emission, followed by benzyl alcohol (12.96%) and methyl salicylate (3.75%). These scents predominantly originated from petals and sepals. Transcriptomic analysis and inhibition assays using pathway-specific inhibitors revealed that the mevalonate pathway was the precursor source for geraniol biosynthesis. Functionally characterized cytosol-localized geraniol synthase EoTPSa1 was the key enzyme responsible for geraniol biosynthesis. Together, these findings pinpoint a cytosolic biosynthetic route for the major scent volatile geraniol in E. oxypetalum. Our study provides new insights into the emission dynamics and biosynthesis of E. oxypetalum floral scents. In particular, we demonstrate a distinctive mevalonate pathway-based geraniol biosynthetic pathway, which may hold potential for the development of novel perfume products.

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