PhD defence: Hunting for Ancestral Genes - Elucidating Metabolic Pathways in Ferns – University of Copenhagen

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PhD defence: Hunting for Ancestral Genes - Elucidating Metabolic Pathways in Ferns

PhD defence by Sara Thodberg

Cyanogenic glucosides (CNGlcs) are a class of plant specialized metabolites found throughout the plant kingdom. These defence compounds work in a two-component system, where upon tissue disruption by herbivores, an enzymatic degradation is initiated to liberate toxic hydrogen cyanide gas. The biosynthetic pathway of CNGlcs has been widely studied in gymnosperms, mono- and dicot-plants. In the species studied, the pathway consists of two or three cytochromes P450 (CYPs), and a UDP-glycosyltransferase (UGT).
CNGlc occurs as mono-, di- or even tri-glucosides, however only (UGTs) able to attach the first sugar moiety have been characterized.

In almond (Prunus dulcis) the abundance of the cyanogenic di-glucoside amygdalin, causes the bitter tasting phenotype. A library-guided search against Prunus genomic and transcriptomic resources identified five UGT candidates. Two UGT94s were successfully characterized to attach a glucose to the cyanogenic monoglucoside prunasin, forming amygdalin. Transcript expression analysis by qPCR of the entire amygdalin pathway in P. dulcis, revealed that the bitter vs sweet almond phenotype is due to differential expression of the CYPs.
Ferns are the evolutionary oldest plant group known to synthesize CNGlcs. With the rise of initiatives such as the OneKP, it is now possible to investigate pathway evolution in ferns. However, an evolutionary conundrum arises, as known CNGlc biosynthetic CYPs were not identified in the oneKP database. To narrow the search for CYP candidates, comparative transcriptomic analysis of two CNGlc-containing fern species, Phlebodium aureum(PAU) and Pteridium aquilinum(PAQ) was conducted. Characterization of CYP981F5 from PAU by transient expression revealed its involvement in CNGlc biosynthesis. This represents the first CYP to be characterized from ferns. Broad transcriptome mining for flavoproteins, identified a flavin monooxygenase (FMO) that is 99% conserved between PAU and PAQ, despite the split of the species occurring more than 40 mya. This FMO was characterized and found catalyze the N-hydroxylation of phenylalanine to the corresponding oxime. This oxime synthase hereby named FOS1, is the first enzyme outside the CYP79 family to catalyze this reaction. This work shows that the emergence of CNGlc biosynthesis in ferns is independent of the CNGlc pathway present in gymnosperms and angiosperms.

Professor Birger Lindberg Møller
Associate Professor Elizabeth H.J. Neilson

Assessment committee
Professor Daniele Werck, University of Strasbourg, France
Professor Sarah O’Connor, John Innes Centre, UK
Associate Professor Fernando Geu-Flores (Chair)