Convergent evolution in biosynthesis of cyanogenic defence compounds in plants and insects

Abstract

For more than 420 million years, plants, insects and their predators have co-evolved based on a chemical arms race including deployment of refined chemical defence systems by each player. Cyanogenic glucosides are produced by numerous plants and by some specialized insects and serve an important role as defence compounds in these intimate interactions. Burnet moth larvae are able to sequester cyanogenic glucosides from their food plant as well as to carry out de novo biosynthesis. Here we show that three genes (CYP405A2, CYP332A3 and UGT33A1) encode the entire biosynthetic pathway of cyanogenic glucosides in the Burnet moth Zygaena filipendulae. In both plants and insects, convergent evolution has led to two multifunctional P450 enzymes each catalysing unusual reactions and a glucosyl-transferase acting in sequence to catalyse cyanogenic glucoside formation. Thus, plants and insects have independently found a way to package a cyanide time bomb to fend off herbivores and predators.

Figure 1. Zygaena larva feeding on its cyanogenic host plant Lotus corniculatus.

The larva was stimulated to secrete defence droplets (marked by white circles) containing the two cyanogenic glucosides linamarin and lotaustralin. Scale bar, ~2 cm.

Figure 2. Biosynthetic pathway of cyanogenic glucoside synthesis in plants and Zygaena.

Plant (green) and Zygaena enzymes (maroon) are shown. Glc, glucose.

Figure 3. Expression and screening results of insect cytochrome P450s and UGTs.

(a) CO-difference spectra from yeast microsomes harbouring A, CYP405A2; B, CYP304F2; C, CYP405A3; D, CYP332A3; E, CYP9A36; F, CYP9A37; G, CYP6CT1; H, CYP6AE27; I, CYP4G47 and J, CYP4L17. (b) Screening of yeast microsome preparations for the ability of the P450s to convert Val/Ile into their corresponding oximes, 2-methylpropanal oxime (ValOx) and 2-methylbutanal oxime (IleOx) as monitored by SPME-GC-MS. Extracted ion chromatograms corresponding to the most abundant ion of the mass spectra for the two oximes (ValOx: m/z 70; IleOx: m/z 59) are shown in identical scale for all samples belonging to the respective assays. The double peaks represent the E and Z isomers of the oximes. (c) Screening of yeast microsome preparations for the ability of P450s to convert ValOx or IleOx to the corresponding cyanohydrins. Cyanohydrin formation was measured as the dissociation products acetone or 2-butanone following DNPH derivatization giving rise to acetone-DNPH and 2-butanone-DNPH monitored by LC-MS analysis. Extracted-ion chromatograms corresponding to the two adducts are shown (acetone-DNPH: m/z 239; 2-butanone-DNPH: m/z 253). CYP332A3 chromatograms have been highlighted in red. The background levels of acetone-DNPH and 2-butanone-DNPH originated from the omnipresence of acetone and 2-butanone in laboratory air and plastic ware. The capital letters in b and c represent the same P450s as in a with K being the empty vector control. (d) Screening of yeast microsomes for the ability of UGTs to convert acetone cyanohydrin (Ach) or 2-butanone cyanohydrin (Bch) into radiolabelled linamarin and lotaustralin following administration of ¹⁴C-labelled UDP-glucose, and analysis of the radiolabelled products formed by thin-layer chromatography.

Figure 4. Phylogenetic analysis of P450 genes and models of metabolon organization.

(a) Neighbour joining phylogenetic tree with representatives from all A. thaliana (green) and B. mori (blue) P450 groups. Ten plant genes known from the cyanogenic glucoside pathway from L. japonicus (purple), S. bicolor (black), M. esculenta (olive), T. repens (silver) and T. maritima (lime), as well as ten genes from Z. filipendulae (maroon) are included. CYP405A1 from Epiphyas postvittana (fuchsia) and CYP332s from Helicoverpa armigera (navy), Heliothis virescens (grey), Spodoptera frugiperda (teal) and Manduca sexta (aqua) are also included. • Denotes P450s known to catalyse conversion of amino acids into corresponding oximes and * denotes P450s known to catalyse metabolism of oximes. • and * represent enzymes involved in cyanogenic glucoside synthesis. •• and ** represent enzymes involved in glucosinolate production. Alignment (plant–insectP450tree.mas) used for the tree can be accessed at http://genome.ku.dk/resources/zygaena/. (b) Sequence logos of conserved regions in P450s created at http://weblogo.berkeley.edu/logo.cgi. Plant/insect P450 sequence logos were generated from 173 insect sequences from B. mori, Drosophila melanogaster, Anopheles gambiae and Musca domestica, and all A. thaliana sequences (excluding CYP79s). CYP79 sequence logos were generated from 12 CYP79s from plants (A. thaliana, L. japonicus, M. esculenta, S. bicolor, T. maritima) known to catalyse conversion of amino acids into corresponding oximes. CYP71E logos were generated from two CYP71Es (S. bicolor, M. esculenta) known to catalyse conversion of oximes into cyanohydrins. (c) The proposed plant metabolon, and the putative insect metabolon including the two P450s, the UGT and the CPR. In plants, the non-covalently bound UGT is thought to be anchored on the ER membrane via the P450s and with the catalytic domain facing the cytosol. In insects, the catalytic domain of the membrane bound UGT is possibly situated inside the ER lumen. If these models are correct, the cyanogenic glucoside would be liberated from the metabolon into the cytosol in plants and inside the ER lumen in insects.