CYP79D73 Participates in Biosynthesis of Floral Scent Compound 2-Phenylethanol in Plurneria rubra

Plumeria (Plumeria rubra), well known for its brightly colored and fragrant flowers, emits a number of floral volatile organic compounds (VOCs). Plumeria flowers emit a total of 43 VOCs including nine phenylpropanoids/benzenoids, such as 2-phenylethanol (2PE), benzaldehyde, 2-phenylacetaldehyde (PAld), (E/Z)-phenylacetaldoxime (PAOx), benzyl nitrile (BN), and 2-phenylnitroethane (PN). To identify genes and pathways involved in the production of the major compound 2PE, we analyzed the plumeria floral transcriptome and found a highly expressed, flower-specific gene encoding a cytochrome P450 family 79D protein (PrCYP79D73), which catalyzed the formation of (E/Z)-PAOx. Feeding experiments with deuterated phenylalanine or deuterated (E/Z)-PAOx showed that (E/Z)-PAOx is an intermediate in the biosynthesis of 2PE, as are two nitrogen-containing volatiles, BN and PN, in plumeria flowers. Crude enzyme extracts from plumeria flowers converted L-phenylalanine to (E/Z)-PAOx, PAld, 2PE, BN, and PN. The biosynthesis of these compounds increased with addition of PrCYP79D73-enriched microsomes but was blocked by pretreatment with 4-phenylimidazole, an inhibitor of cytochrome P450 enzymes. Moreover, overexpression of PrCYP79D73 in Nicotiana benthamiana resulted in the emission of (E/Z)-PAOx as well as PAld, 2PE, BN, and PN, all of which were also found among plumeria floral VOCs. Taken together, our results demonstrate that PrCYP79D73 is a crucial player in the biosynthesis of the major floral VOC 2PE and other nitrogencontaining volatiles. These volatiles may be required for plant defense as well as to attract pollinators for the successful reproduction of plumeria.