Abstract
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 nitrogen-containing volatiles. These volatiles may be required for plant defense as well as to attract pollinators for the successful reproduction of plumeria.