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

Abstract

<p><em>Angraecum sesquipedale</em>, 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 <em>Xanthopan praedicta</em>. In this plant-pollinator interaction, the <em>A. sesquipedale</em> 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 <em>A. sesquipedale</em>. The haploid genome size is 2.10 Gb and represents 19 pseudochromosomes. Cytochrome P450 encoding genes of the <em>CYP79</em> family known to be involved in oxime biosynthesis in seed plants are not present in the <em>A. sesquipedale</em> genome nor in the genomes of other members of the orchid family. Metabolomic analysis of the <em>A. sesquipedale</em> 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 <em>A. sesquipedale</em> genome are identified as catalyzing the formation of oximes present. Further <em>in vitro</em> and <em>in vivo</em> 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 <em>CYP79</em> gene losses and neofunctionalization of FMO-catalyzed biosynthesis of oximes in Darwin’s orchid provide new insights into the convergent evolution of biosynthetic pathways.<br></p>