Reconfigured Cyanogenic Glucoside Biosynthesis in Eucalyptus cladocalyx Involves a Cytochrome P450 CYP706C55

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.

Figure 1.

Prunasin and amygdalin accumulation in E. cladocalyx. A, Prunasin (dark gray) and amygdalin (light gray) content in expanded leaves from seedling and adult trees, immature and mature flower buds, flowers, and fruits. Bars represent means with se. Magnified amygdalin concentrations are shown in the insert. Letters denote significance at P < 0.05 by one-way ANOVA. DW, Dry weight. B, Structures of prunasin and amygdalin.

Figure 2.

Localization of cyanogenic glucosides in E. cladocalyx tissue. A and B, Cross sections of seedling dorsoventral (A) and adult isobilateral (B) leaves prepared for MALDI-MSI, with corresponding ion maps of prunasin (yellow; mass-to-charge ratio [m/z] 334.0687; [M+K]⁺) localizing to the mesophyll and vascular tissue. C, Longitudinal section of a flower bud prepared for MALDI-MSI, with corresponding ion maps of prunasin and amygdalin (pink; m/z 496.1216; [M+K]⁺). D, Enlarged portions of the flower bud, with the section overlaid shown in the corresponding boxes 1 and 2. Prunasin ions were distributed in the ground tissue within the hypanthium, stamens, and operculum. Colocalization with amygdalin occurs in the filaments and around the staminal ring (indicated by white arrows). All images are root mean square normalized, with internal scaling of 100% for prunasin and 25% for amygdalin. EG, Embedded gland; PM, palisade; SM, spongy mesophyll; VB, vascular bundle. Bars = 500 μm.

Figure 3.

E. cladocalyx leaves of different developmental stages can synthesize prunasin from Phe. A, E. cladocalyx seedling withthe tissues used for administration with l-[¹⁴C]Phe are indicated. B, Thin-layer chromatography (TLC) plate showing the production of prunasin from l-[¹⁴C]Phe in apical tips, first and second expanding leaves, and fully expanded leaves.

Figure 4.

Prunasin biosynthesis is catalyzed by three CYPs and a UGT. A, LC-MS extracted ion chromatograms (EIC) for prunasin ([M+Na]⁺ m/z 318) of N. benthamiana leaf extracts transiently expressing candidate genes. B, Microsomes prepared from N. benthamiana leaves transiently expressing candidate CYP genes were assayed with [¹⁴C]Phe, and products were separated by TLC. C, Proposed pathway for prunasin synthesis in E. cladocalyx.

Figure 5.

Phylogenetic analysis of functionally characterized CYPs belonging to the CYP79, CYP71, CYP706, and CYP736 families, including members involved in cyanogenic glucoside biosynthesis (indicated by asterisks). Phylogeny was inferred using the neighbor-joining method with n = 1,000 bootstrap replicates, with bootstrap values above 70 shown. The E. cladocalyx CYPs are marked in bold. Full species names, GenBank accession numbers, and references are listed in Supplemental Table S1.

Figure 6.

Prunasin accumulation and expression of the biosynthetic genes in 4-, 7-, and 10-month-old E. cladocalyx plants (n = 6–8). A, Prunasin accumulation in apical tips, expanding leaves, and expanded leaves. Bars represent means ± se. Letters denote significance at P < 0.05 by one-way ANOVA. dw, Dry weight. B, Normalized gene expression of CYP79A125, CYP706C55, CYP71B103, and UGT85A59 in apical tips, expanding leaves, and expanded leaves. Symbols represent biological replicates (n = 3). C, Spearman correlation analysis of gene expression showing the coregulation of some genes.