Reconfigured cyanogenic glucoside biosynthesis in Eucalyptus cladocalyx involves a cytochrome P450 CYP706C55

Hansen, C.C., Sørensen, M., Veiga, T.A.M., Zibrandtsen, J.F.S., Heskes, A.M., Olsen, C.E., Boughton, B.A.ORCID: 0000-0001-6342-9814, Møller, B.L. and Neilson, E.H.J. (2018) Reconfigured cyanogenic glucoside biosynthesis in Eucalyptus cladocalyx involves a cytochrome P450 CYP706C55. Plant Physiology, 178 (3). pp. 1081-1095.

Abstract

Cyanogenic glucosides are a class of specialized metabolites widespread in the plant kingdom. Cyanogenic glucosides are α-hydroxynitriles, and their hydrolysis releases toxic hydrogen cyanide, providing an effective chemical defense against herbivores. Eucalyptus cladocalyx is a cyanogenic tree, allocating up to 20% of leaf nitrogen to the biosynthesis of the cyanogenic monoglucoside, prunasin. Here, mass spectrometry analyses of E. cladocalyx tissues revealed spatial and ontogenetic variations in prunasin content, as well as the presence of the cyanogenic diglucoside amygdalin in flower buds and flowers. The identification and biochemical characterization of the prunasin biosynthetic enzymes revealed a unique enzyme configuration for prunasin production in E. cladocalyx. This result indicates that a multifunctional cytochrome P450 (CYP), CYP79A125, catalyzes the initial conversion of l-phenylalanine into its corresponding aldoxime, phenylacetaldoxime; a function consistent with other members of the CYP79 family. In contrast to the single multifunctional CYP known from other plant species, the conversion of phenylacetaldoxime to the α-hydroxynitrile, mandelonitrile, is catalyzed by two distinct CYPs. CYP706C55 catalyzes the dehydration of phenylacetaldoxime, an unusual CYP reaction. The resulting phenylacetonitrile is subsequently hydroxylatedby CYP71B103 to form mandelonitrile. The final glucosylation step to yield prunasin is catalyzed by a UDP-glucosyltransferase, UGT85A59. Members of the CYP706 family have not been reported previously to participate in the biosynthesis of cyanogenic glucosides, and the pathway structure in E. cladocalyx represents an example of convergent evolution in the biosynthesis of cyanogenic glucosides in plants.

Cyanogenic glucosides are a class of specialized metabolites found widespread in the plant kingdom. They are present in diverse taxa, including species in the genera Pteridium (monilophyte), Taxus (gymnosperm), Sorghum (monocot), Manihot, Trifolium, Prunus, and Eucalyptus (eudicots; Vetter, 2017). Upon tissue disruption, such as would be caused by herbivory, the cyanogenic glucoside is hydrolyzed by a specific β-glucosidase that cleaves off the sugar moiety. The resulting cyanohydrin dissociates, spontaneously or catalyzed by an α-hydroxynitrile lyase, resulting in the release of toxic hydrogen cyanide. This process is known as cyanogenesis (Gleadow and Møller, 2014). Their ability to release hydrogen cyanide makes cyanogenic glucosides chemical defense molecules against generalist herbivores and pathogens (Gleadow and Woodrow, 2002). Cyanogenic glucosides also have been shown to constitute a source of reduced nitrogen that is mobilized by endogenous turnover pathways (Pičmanová et al., 2015; Bjarnholt et al., 2018; Schmidt et al., 2018a).

The biosynthetic pathway genes for a cyanogenic glucoside were first identified and cloned from sorghum (Sorghum bicolor), which accumulates the Tyr-derived dhurrin (Koch et al., 1995; Bak et al., 1998; Jones et al., 1999). This provided a starting point for the identification of cyanogenic glucoside pathway genes in other plant species. The biosynthetic pathway for the Val-derived linamarin and Ile-derived lotaustralin has been elucidated from cassava (Manihot esculenta; Andersen et al., 2000; Jørgensen et al., 2011; Kannangara et al., 2011) and Lotus japonicus (Forslund et al., 2004; Takos et al., 2011), and recently, the full pathway for the Phe-derived diglucoside amygdalin from almond (Prunus dulcis) was identified (Thodberg et al., 2018). In species with characterized cyanogenic glucoside biosynthetic pathways, the production of monoglucosides involves two cytochrome P450s (CYPs) and a soluble UDP-glucosyltransferase (UGT). The first CYP is a member of the CYP79 family and catalyzes the conversion of an amino acid to the corresponding aldoxime. A CYP71 or CYP736 family member subsequently converts the aldoxime to a cyanohydrin. The labile cyanohydrin is stabilized by glucosylation to form the final cyanogenic glucosides in a reaction catalyzed by a UGT from the UGT85 family. Work on dhurrin biosynthesis in sorghum shows that the two CYPs and UGT of the pathway form a metabolon enabling efficient channeling between enzymes that avoids the accumulation of toxic intermediates (Laursen et al., 2016; Bassard et al., 2017).

Eucalyptus is a large genus with over 800 species (Coppen, 2002). They are the world’s most widely planted forest hardwood trees due to their fast growth, environmental plasticity, and exceptional wood properties (Myburg et al., 2014). The trees are grown for industrial purposes such as the production of pulp for paper, firewood, charcoal, and essential oils. Eucalyptus plants synthesize a broad spectrum of specialized metabolites, including terpenes, phenolics, formylated phloroglucinols, and cyanogenic glucosides (Eschler et al., 2000; Gleadow et al., 2008; Padovan et al., 2014; Marsh et al., 2017). Very little is known about the biosynthesis of specialized metabolites in Eucalyptus trees. The genome of Eucalyptus grandis, published in 2014 (Myburg et al., 2014), will potentially facilitate the identification of enzymes involved in these pathways. E. grandis has many tandem duplicated genes and tandem expanded clusters, as exemplified by the large number of terpene synthase genes (113) and the expansion of some phenylpropanoid genes (Myburg et al., 2014; Külheim et al., 2015). The significant expansion of some gene families in Eucalyptus species provides challenging obstacles toward the identification of pathway genes due to the large number of possible candidates.

More than 400 Eucalyptus species have been screened for cyanogenesis and 23 cyanogenic species have been identified, all containing prunasin as the major cyanogen (Fig. 1B; Gleadow et al., 2008). The South Australian species Eucalyptus cladocalyx (sugar gum) was first reported to be cyanogenic in 1928 (Finnemore and Cox, 1928), and a few years later, the foliar hydrogen cyanide source was identified as prunasin (Finnemore et al., 1935). In particular, the apical tips and young leaf tissue of E. cladocalyx can be highly cyanogenic, with up to 20% of leaf nitrogen allocated to prunasin biosynthesis (Gleadow et al., 1998; Gleadow and Woodrow, 2000b). Cyanogenic Eucalyptus species display unique ontogenetic variation. For example, E. cladocalyx has high levels of prunasin as a seedling compared with the adult, while the opposite trend is observed for Eucalyptus yarraensis and Eucalyptus camphora (Goodger et al., 2006; Neilson et al., 2011). As such, cyanogenic Eucalyptus species are an excellent system in which to investigate the regulation of chemical defense (Møller, 2010).

Item Type:	Journal Article
Publisher:	American Society of Plant Biologists
Copyright:	© 2018 American Society of Plant Biologists
URI:	http://researchrepository.murdoch.edu.au/id/eprint/56298

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