Three cytochrome P450 fromNicotiana attenuataplay key roles in triterpene biosynthesis

Abstract Pentacyclic triterpenoids, recognized for their natural bioactivity, display complex spatiotemporal accumulation patterns within the ecological model plant, Nicotiana attenuata . Despite their ecological significance, the underlying biosynthetic enzymes and functional attributes of triterpenoid synthesis in N. attenuata remain unexplored. Three multifunctional cytochrome P450 monooxygenases (NaCYP716A419, NaCYP716C87, NaCYP716E107) from N. attenuata were shown to oxidize the pentacyclic triterpene skeleton as evidenced by heterologous expression in Nicotiana benthamiana . NaCYP716A419 catalyzed a consecutive three-step oxidation reaction at the C28 position of β-amyrin/lupeol/lupanediol, yielding the corresponding alcohol, aldehyde, and carboxylic acid. NaCYP716C87 hydroxylated the C2α position of β-amyrin/lupeol/lupanediol/erythrodiol/oleanolic acid/betulinic acid, while NaCYP716E107 hydroxylated the C6β position of β-amyrin/oleanolic acid. Three CYP716 enzymes are highly expressed in flowers and respond to induction by ABA, MeJA, SA, GA 3 , and abiotic stress treatments. Using VIGS technology, we revealed that silencing of NaCYP716A419 affects the growth and reproduction of N. attenuata , suggesting the ecological significance of these specialized metabolite biosynthetic steps. One-sentence summary Three CYP716 enzymes diversify N. attenuata’s triterpenoid sector with potential roles in growth and development.