A single oxidosqualene cyclase produces the seco-triterpenoid α-onocerin

8,14-seco-triterpenoids are characterized by their unusual open C-ring. Their distribution in nature is rare and scattered in taxonomically unrelated plants. The 8,14-seco-triterpenoid α-onocerin is only known from the evolutionarily distant clubmoss genus Lycopodium and leguminous genus Ononis, which makes the biosynthesis of this seco-triterpenoid intriguing from an evolutionary standpoint. In our experiments with Ononis spinosa, α-onocerin was detected only in the roots. Through transcriptome analysis of the roots, an oxidosqualene cyclase, OsONS1, was identified that produces α-onocerin from squalene-2,3;22,23-dioxide, when transiently expressed in Nicotiana bethaminana. In contrast, in Lycopodium clavatum two sequential cyclases LcLCC and LcLCD, are required to produce α-onocerin in the N. benthamiana transient expression system. Expression of OsONS1 in the lanosterol synthase knock-out yeast strain GIL77, which accumulates squalene-2,3;22,23-dioxide, verified the α-onocerin production. A phylogenetic analysis, predicts that OsONS1 branches off from specific lupeol synthases and does not group with the known L. clavatum α-onocerin cyclases. Both the biochemical and phylogenetic analysis of OsONS1 suggest convergent evolution of the α-onocerin pathways. When OsONS1 was co-expressed in N. benthamiana leaves with either of the two O. spinosa squalene epoxidases OsSQE1 or OsSQE2, α-onocerin production was boosted, most likely because the epoxidases produce higher amounts of squalene-2,3;22,23-dioxide. Fluorescence Lifetime Imaging Microscopy (FLIM) analysis demonstrated specific protein-protein interactions between OsONS1 with both O. spinosa squalene epoxidases, and co-expression of OsONS1 with the two OsSQEs suggests that OsSQE2 is the preferred partner of OsONS1 in planta. Our results provide an example of convergent evolution of plant specialized metabolism.