The fern CYPome: Fern-specific cytochrome P450 family involved in convergent evolution of chemical defense

Abstract

Plant natural products encompass an enormous chemical diversity bearing witness to great molecular innovation that occured throughout land plant evolution. Cytochrome P450 monooxygenases (CYPs) catalyze a wide variety of monooxygenation reactions essential to the metabolic repertoire of plants natural products. Ferns constitute the second largest group of vascular plants and hold a significant phylogenetic position in land evolution, lying sister to seed plants. To date, CYP diversity has not been described for this taxon and pathway discovery in ferns in general is scarce, despite possessing a rich diversity of natural products. We analysed over 8000 available fern CYPs, classifing and characterizing the landscape of this super-enzyme group. Fern CYPs are dominated by fern-specific families (∼60%), with the largest family – CYP981 – constituting approximately 15% of all predicted fern CYPs in the dataset. The abundancy and dynamics of the CYP981 family suggest a position equivalent to the CYP71 family present in seed plants, with potential roles in natural product biosynthesis. Ferns are the evolutionary oldest group to biosynthesize cyanogenic glycosides; amino acid-derived defense compounds. We show that CYP981F5 from the highly cyanogenic fern Phlebodium aureum catalyzes the conversion of phenylacetonitrile to mandelonitrile, an intermediate step in cyanogenic glycoside biosynthesis. The fern CYPome provides an important platform to further understand evolution of metabolite biosynthesis throughout the plant kingdom, and in ferns specifically.

Sequence similarity networks bear out hierarchical relationships of green cytochrome P450

“…In several instances, one clan was nested within another clan (Figure S2), speaking for a shared evolutionary history. In agreement with previous reports (Nelson and Werck-Reichhart, 2011;Thodberg et al, 2021), clan 71 is the largest. Clan 71, in our analyses, also encompasses sequences that have been annotated as clans 72, 85, 710 and 711.…”

Plant cytochrome P450 plasticity and evolution

“…Mining of the 1kP transcriptomes and genomes of early land plants revealed many taxaspecific families (Figure 1; supplemental data 1 and 2), some of which show great abundance, such as the fern-specific CYP981 family (clan 71), which is the richest CYP family in this taxon (Thodberg et al, 2021). Other CYP families are shared between a few taxa (supplemental data 1).…”

“…Only very few CYPs from taxa-specific CYP families in early land plants have been functionally characterized. These include CYP788A1 from the lycophyte S. moellendorffii (Weng et al, 2010), CYP964A1 and CYP970A14 from the moss Calohypnum plumiforme (Mao et al, 2020), and CYP981F5 from the fern Phlebodium aureum (Thodberg et al, 2021). Notably, these CYPs enable the species to produce compounds already known from angiosperms through similar or alternative metabolic routes (discussed further below).…”

“…First, EcCYP706C55 converts phenylacetaldoxime to phenylacetonitrile, which is subsequently hydroxylated by EcCYP71B103 to form the a-hydroxynitrile mandelonitrile. In the fern P. aureum, CYP981F5 was recently identified to hydroxylate phenylacetonitrile in cyanogenic glucoside biosynthesis (Thodberg et al, 2021). P. trichocarpa and Fallopia sachalinensis also produce phenylacetonitrile, which is released upon herbivore attack (Irmisch et al, 2014;Yamaguchi et al, 2016).…”

Recruitment of distinct UDP‐glycosyltransferase families demonstrates dynamic evolution of chemical defense within Eucalyptus L'Hér

“…The functional characterization of a CYP79A, CYP706C, and CYP71B in different Eucalyptus species shows that the three-CYP system is conserved within the subgenus Symphyomyrtus, thereby requiring a total of four biosynthetic enzymes for prunasin production. Recent work suggests that a four-step pathway for prunasin biosynthesis is also operating in the cyanogenic fern Phlebodium aureum (Thodberg et al, 2021), demonstrating that this biosynthetic configuration is not unique to Eucalyptus.…”