Genomic, transcriptomic, and metabolomic analyses reveal convergent evolution of oxime biosynthesis in Darwin's orchid.

Abstract

Angraecum sesquipedale, also known as Darwin's orchid, possesses an exceptionally long nectar spur. Charles Darwin predicted the orchid to be pollinated by a hawkmoth with a correspondingly long proboscis, later identified as Xanthopan praedicta. In this plant-pollinator interaction, the A. sesquipedale flower emits a complex blend of scent compounds dominated by diurnally regulated oximes (R₁R₂C=N-OH) to attract crepuscular and nocturnal pollinators. The molecular mechanism of oxime biosynthesis remains unclear in orchids. Here, we present the chromosome-level genome of A. sesquipedale. The haploid genome size is 2.10 Gb and represents 19 pseudochromosomes. Cytochrome P450 encoding genes of the CYP79 family known to be involved in oxime biosynthesis in seed plants are not present in the A. sesquipedale genome nor in the genomes of other members of the orchid family. Metabolomic analysis of the A. sesquipedale flower revealed a substantial release of oximes at dusk during the blooming stage. By integrating metabolomic and transcriptomic correlation approaches, flavin-containing monooxygenases (FMOs) encoded by six tandem-repeat genes in the A. sesquipedale genome are identified as catalyzing the formation of oximes present. Further in vitro and in vivo assays confirm the function of FMOs in the oxime biosynthesis. We designate these FMOs as Orchid Oxime Synthases 1-6. The evolutionary aspects related to the CYP79 gene losses and neofunctionalization of FMO-catalyzed biosynthesis of oximes in Darwin's orchid provide new insights into the convergent evolution of biosynthetic pathways.