As plants are fixed to their habitat they produce specialized metabolites as chemical defenses to fight off herbivores. As an example, many plants produce cyanogenic glucosides and release toxic cyanide upon tissue damage (“cyanide bomb”). As a prerequisite for exploring cyanogenic plants as hosts, herbivores have evolved mechanisms to overcome cyanogenic defenses. Mammals metabolize cyanide to thiocyanate by rhodaneses. In arthropods, both rhodaneses and β-cyanoalanine synthases which transfer cyanide to cysteine contribute to cyanide detoxification. However, based on enzyme activity tests some arthropod species possess only one of these activities, and some possess both. Recently, cloning and characterization of first arthropod β-cyanoalanine synthases provided evidence for their involvement in cyanide detoxification. Phylogenetic analyses suggest that they have been recruited from microbial symbionts. Investigations with Zygaena filipendulae revealed that the avoidance of cyanide release is the primary mode of overcoming cyanide in this specialist. Some herbivores are able to sequester, de novo synthesize, and store cyanogenic glucosides for their defense and as nitrogen source. Thus, herbivores have evolved various mechanisms to counteract host plant cyanide defenses. These mechanisms are likely to have played a key role in the evolution of plant-herbivore interactions as well as in speciation and diversification of arthropods.
- cyanide detoxification
- plant secondary/specialized metabolism
- cyanogenic glucosides
- β-cyanoalanine synthase
Herbivores are a main threat for plants as their feeding destroys vegetative and generative parts of the plant, that is, organs needed for assimilation, nutrient storage and reproduction. In order to cut their losses, plants have developed physical and chemical defenses to fight off herbivores and to survive in their ecosystem. Very effective means to defend against predators are provided by the so-called specialized (or “secondary”) metabolism, which is not required for growth and development, but for the plant’s interaction with its environment [1, 2]. Specialized metabolism is the source of diverse low molecular weight compounds such as alkaloids, terpenes, glucosinolates or cyanogenic glucosides, which are often specific to certain families or species. These compounds may repel the potential predator before contact or harm the herbivore upon ingestion. Defensive metabolites may have herbivore-specific effects or be universally toxic. In the latter case, they need to be stored in an inactive or nontoxic form in the plant to avoid self-intoxication.
Chemical defense through cyanide is widespread in the plant kingdom. As a universal respiration toxin, cyanide is not accumulated in free form in plants but released from cyanogenic precursors upon tissue damage (“cyanide bomb”), in the course of metabolic reactions in intact plant tissue or upon ingestion by herbivores (Figure 1). The acute and universal toxicity of cyanide in combination with its frequent occurrence in the plant kingdom calls for efficient cyanide detoxification mechanisms in herbivores. As soon as cyanide is liberated upon ingestion of cyanogenic plant material, an enzymatic detoxification is vital for the protection of the herbivore’s cellular metabolism. Although diverse enzymatically catalyzed reactions for the detoxification of cyanide have been described in microorganisms , only two main pathways of cyanide detoxification are present in higher animals. These are on the one hand the rhodanese or 3-mercaptopyruvate sulfurtransferase-catalyzed transfer of sulfur from a donor substrate to cyanide, leading to the formation of thiocyanate, and, on the other hand, the β-cyanoalanine synthase-catalyzed substitution of cysteine’s sulfhydryl group by cyanide, leading to the formation of β-cyanoalanine.
An efficient way to minimize the risk of cyanide poisoning is to prevent its formation. Therefore, herbivorous arthropods that colonize plants with high cyanide potential often possess specialized adaptations, which allow them to avoid cyanide release upon ingestion of plant material. This chapter introduces cyanide as a ubiquitous plant-produced compound and summarizes the present understanding of cyanide detoxification pathways and the involved enzymes as well as the current knowledge on cyanide avoidance mechanisms in herbivores with a special focus on arthropods. As certain arthropod species are able to synthesize cyanogenic compounds themselves and/or to sequester cyanogenic compounds from their food plants, we also discuss cyanogenesis in herbivores from an ecological and evolutionary perspective.
2. Sources of cyanide exposure
The most common storage form of cyanide in plants is cyanogenic glucosides, which are potent antiherbivore defenses with an additional function as nitrogen storage compounds [4–8]. The intact glucosides are water-soluble and nontoxic compounds, but hydrolysis catalyzed by β-glucosidases liberates the cyanohydrins (α-hydroxynitriles), which, spontaneously or under catalysis by α-hydroxynitrilases, release the hydrogen cyanide next to aldehydes or ketones (Figure 1A) . Their broad distribution among more than 2650 plant species from the pteridophytes, gymnosperms and angiosperms [9, 10] may be explained by their biosynthetic origin. Cyanogenic glucosides are biosynthesized through oxidation of common aliphatic and aromatic amino acids by members of the wide-ranging cytochrome P450 family with oximes and cyanohydrins as intermediates and subsequent
Some cyanogenic glucosides have not only been found in plants, but are sequestered and even biosynthesized
A further class of plant secondary compounds carrying a cyanide group is the cyanolipids, a group of lipids possessing a branched five carbon skeleton with a nitrile group . Cyanolipids occur in the seed oil of diverse species of the Sapindaceae . They are cyanohydrin esters, that is, they possess an esterified hydroxyl group in α-position to the nitrile moiety and will form unstable α-hydroxynitriles upon spontaneous or lipase-catalyzed ester hydrolysis (Figure 1D) . As described above, α-hydroxynitriles are a source of cyanide as they readily decompose either spontaneously or enzymatically catalyzed.
Cyanide may also be liberated upon metabolism of another group of specialized metabolites, the glucosinolates, inside the herbivore by the consecutive action of plant- and herbivore-expressed enzymes. Glucosinolates are amino acid-derived thioglucosides with a sulfated aldoxime core and a variable side chain  (Figure 1B). They are part of the glucosinolate-myrosinase system or “mustard oil bomb,” a constitutive defense mechanism common to all families of the Brassicales. The products arising from glucosinolate hydrolysis have manifold effects on herbivores feeding on Brassicales plants, including general deterrence and toxicity, but may also be perceived by specialist herbivores and their parasitoids as host identification cues . The primary defense compounds derived from this system are the isothiocyanates which result from rearrangement of the aglucone formed upon hydrolysis by co-occurring thioglucosidases (myrosinases) when tissue is disrupted [23, 25]. Besides isothiocyanates, other products such as nitriles, epithionitriles and organic thiocyanate can also be formed depending on the structure of the glucosinolate side chain and the presence of additional plant-expressed proteins, the so-called specifier proteins [26–29]. Cyanide release from a glucosinolate-derived nitrile has been demonstrated to occur in larvae of
Apart from the accumulation of cyanogenic precursors as part of specialized metabolism for defense against herbivores, plants from all families generate cyanide as a by-product during the formation of ethylene, a ubiquitous plant hormone. In the last step of ethylene biosynthesis, the oxidation of 1-aminocyclopropane-1-carboxylic acid to ethylene, one mole of cyanide is liberated per mole of ethylene formed (Figure 1C) . Although the steady-state concentration of cyanide from this pathway is normally kept at a low level of 0.2 μM by action of cyanide detoxifying enzymes , this demonstrates the ubiquitous occurrence of cyanide in the feed of herbivores.
Taken together, cyanide is universally present in plants and herbivores are frequently confronted with this toxin through their diet. Thus, safe handling of cyanide is a necessary prerequisite for herbivory by both specialists feeding exclusively on cyanide-defended plants and generalists with occasional cyanide ingestion. Nevertheless, a varying cyanide content in the host plant seems to influence generalist herbivory more severely than specialist feeding indicating the existence of efficient adaptations to deal with this toxin .
3. Cyanide toxicity
Uptake of the small and simple ion cyanide has tremendous effects on the metabolism of all aerobic cells, resulting from its high reactivity and efficient binding to various proteins of cellular respiration and regulation. The main reason for its acute and universal toxicity is the formation of stable complexes between cyanide and the Fe3+-ion of heme a3 of cytochrome c oxidase, one of the electron carriers in the respiratory chain. Cyanide binds to cytochrome c oxidase and acts as a noncompetitive inhibitor of cytochrome c. This stops electron transfer, leading to termination of the respiratory chain and the citric acid cycle due to a shortage of the electron acceptor NAD+ . The resulting lack of ATP is detrimental to the cell. As a consequence, glycolysis, the alternative, but inefficient pathway of ATP generation, is accelerated in combination with lactic acid fermentation for regeneration of NAD+. In humans, metabolic acidosis resulting from high lactic acid levels is responsible for most of the symptoms of cyanide intoxication . Besides the Fe3+ of cytochrome c oxidase cyanide binds metal ions of various metalloenzymes, in particular molybdoenzymes, and forms Schiff base intermediates with pyridoxal phosphate-dependent enzymes causing an efficient inhibition of a wide range of metabolic reactions and regulatory processes in the cell .
In vertebrates, cyanide does not only influence cellular metabolism but also diverse physiological processes. By binding to chemoreceptors, cyanide causes vasoconstriction of main arteries which may lead to cardiac shock or pulmonary edema [43, 45]. In addition, cyanide may increase neurotransmitter release by influencing calcium channels in neural cell membranes . Even sublethal doses of cyanide may harm the brain of mammals by altering the membrane lipid peroxidation and the response of antioxidant enzymes .
Several studies performed by Edwin J. Bond in the 1960s on the beetle
Thus, the mode of action of the poison cyanide is complex, and the lethal effects differ between species. Nevertheless, cyanide is one of the most potent toxins and an efficient and universal weapon of plants against herbivore foraging.
4. Cyanide detoxification enzymes
4.1. Sulfur transferases
Sulfur transferases such as the rhodanese (thiosulfate:cyanidesulfurtransferase, EC 22.214.171.124; see Figure 2A), and its close relative, the 3-mercaptopyruvate sulfurtransferase (EC 126.96.36.199), are enzymes described in plants, fungi, bacteria and a wide range of animals including snails, insects, fish and mammals (see Figure 3) [52–58]. Enzymatic formation of thiocyanate, the so-called rhodanide, was first described in 1933 using vertebrate tissues as discussed by Lewis . Rhodaneses from mammals have been investigated most thoroughly and most insight has been gained from the examination of human and bovine liver rhodaneses [60, 61]. These two enzymes served to uncover the first protein structure of a rhodanese which revealed two similarly folded “rhodanese domains” . In contrast to the highly similar tertiary structure, the two domains differ strongly in their amino acid sequences in agreement with their divergent functions as C-terminal catalytical and N-terminal regulatory domains . At the level of primary structure, rhodaneses from different organisms show little similarity apart from two conserved, 11–13 amino acids long “signature” regions at the N- and C-termini which are also present in distantly related proteins of the rhodanese superfamily such as cdc25 phosphatase and heat shock proteins . This low identity at the amino acid level and the involvement of single rhodanese domains in aberrant proteins make homology-based identification of rhodaneses from further species difficult. To specify the sulfur transferases involved in cyanide detoxification among the members of the diverse superfamily, the tertiary structure has to be taken into account, classifying the true rhodaneses as tandem domain thiosulfate:cyanide sulfurtransferases . This group also comprises the 3-mercaptopyruvate sulfurtransferases. Both types of enzymes have distinct substrate and product spectra, but are yet interconvertible by few amino acid substitutions .
Rhodaneses do not only accept thiosulfate as sulfur donor, but all sulfane anions such as organic sulfanes and persulfides [55, 65]. Next to cyanide, the sulfur atom may be accepted by other thiophilic substrates such as the amino acids cysteine and glutathione . The kinetic mechanism of rhodanese was uncovered with its classical substrates cyanide and thiosulfate by Westley and coworkers [60, 66]. In a ping-pong reaction, the sulfane sulfur atom is abstracted from the donor substrate thiosulfate and bound to a cysteine residue in the active site of rhodanese . This is followed by entrance of the acceptor substrate cyanide into the active site and transfer of the sulfur atom [60, 66]. In contrast, the reaction of 3-mercaptopyruvate sulfurtransferase (which can also convert cyanide to thiocyanate ) follows a sequential mechanism with formation of a ternary complex composed of the enzyme and both substrates (3-mercaptopyruvate and cyanide) [68, 69].
A main function of the rhodaneses in cyanide detoxification is in agreement with their subcellular localization. Rhodanese activity is predominantly detected in the mitochondria, the site of cellular respiration with the cyanide-susceptible cytochrome c oxidase [70, 71]. Nevertheless, for 3-mercaptopyruvate sulfurtransferase and rhodanese of some species, an additional localization in the cytosolic fraction has been described [55, 71, 72]. The cytosolic enzymes may serve to reduce cyanide levels in this compartment whose components (glycolysis intermediates, proteins) may also be affected by cyanide poisoning (see above) . The reaction product of rhodanese-catalyzed cyanide detoxification, thiocyanate, possesses a toxic potential toward mitochondria which could be an additional reason why rhodanese isoforms are also localized in the cytosol in several species.
Although a major role of rhodaneses in cyanide detoxification seems likely based on the present knowledge, cyanide detoxification might not be their exclusive physiological function. The ubiquitous occurrence of rhodaneses in organisms and tissues with no obvious cyanide exposure as well as the low physiological concentration of their substrate thiosulfate in the mitochondria has fueled doubts about their main role in cyanide detoxification . In support of a major role in cyanide detoxification in mammals, rhodanese activity is inducible in rats by exposition to cyanide or supplementation with thiosulfate [74, 75].
4.2. β-Cyanoalanine synthase
The main enzyme of cyanide detoxification in plants and many bacteria, β-cyanoalanine synthase (EC 188.8.131.52, Figures 2B and 3), belongs to the family of β-substituted alanine synthases sharing the cofactor pyridoxal-5′-phosphate and a uniform fold [80, 81]. This family also comprises
Next to amino acid biosynthesis, β-substituted alanine synthases are involved in cellular sulfur and redox homeostasis [84, 91–93]. Cyanide detoxification by these enzymes is mainly catalyzed by β-cyanoalanine synthases, but
β-Cyanoalanine itself may also exert harmful effects. It has been identified as a neurotoxin and can also be lethal to plants [96, 97]. In order to protect themselves from poisoning with β-cyanoalanine and to minimize costs, plants and microorganisms are able to turn over β-cyanoalanine by nitrile hydratases and nitrilases . Nitrile hydratases catalyze the addition of a water molecule to β-cyanoalanine leading to the formation of the proteinogenic amino acid asparagine. Nitrilases convert β-cyanoalanine to the proteinogenic amino acid aspartate by addition of two water molecules . In addition, the conversion of β-cyanoalanine to asparagine with γ-glutamyl-β-cyanoalanine as an intermediate has been described for some plants . Recycling of β-cyanoalanine has also been shown in some arthropod species (see below). Due to its neurotoxic effect, β-cyanoalanine stored in the defensive droplets of the cyanogenic lepidopteran species
5. Cyanide detoxification strategies in herbivores
5.1. Cyanide detoxification in mammalian herbi- and omnivores
In mammals, rhodanese is generally believed to be the major enzyme for cyanide detoxification, while β-cyanoalanine synthase activity has not been detected in a mammal so far. A comparison of rhodanese activity between mammalian herbi-, omni- and carnivores shows highest activities in herbivores, especially in ruminants which feed on a broad range of plant material including plants with high cyanide potential . In several mammalian species such as plant-feeding rabbits, rhodanese activity is ubiquitously distributed in the body with the highest activity in hepatocytes, the main detoxification site of e.g. xenobiotics [101, 102]. Rhodanese activity is also localized in the mammalian brain, where cyanide acts as a neuromodulator .
5.2. Cyanide detoxification in invertebrates
Intensive research on mammalian rhodaneses also raised the question whether these enzymes are involved in cyanide detoxification in other animals. Rhodanese activity is widely distributed in insects and occurs in snails (see Figure 3) [55, 103]. The level of activity is comparable to that of mammalian gut tissue . However, activity levels are largely in the same range among herbivores which frequently or rarely encounter high cyanide levels. The basal rhodanese activity might be sufficient to capture dietary cyanide in herbivores regardless of the cyanide level in the diet. Alternatively, the uniform distribution of rhodanese activity among herbivores could indicate that arthropod rhodaneses possess an additional function unrelated to cyanide detoxification. This would likely require other mechanisms of cyanide detoxification such as β-cyanoalanine synthase activity . In agreement with this, β-cyanoalanine activity has been found to be broadly distributed in arthropod herbivores (see Figure 3) [86, 95, 104]. In support of the activity data, labeled β-cyanoalanine can be detected in arthropods after feeding of or exposition to isotopically labeled cyanide [30, 105]. Further support for the relevance of β-cyanoalanine synthases comes from experiments with several cyanide-forming lepidopteran species in whose defensive glands β-cyanoalanine and its hydration product asparagine were detected . In millipedes, β-cyanoalanine synthase-catalyzed detoxification of cyanide and further metabolism of β-cyanoalanine to asparagine was demonstrated by studies with radiolabeled precursors [14, 107]. For insects, a similar utilization of cyanide for the formation of proteinogenic amino acids using β-cyanoalanine as an intermediate has been discussed , but has only be proven for one beetle by radioactive feeding experiments so far . In the beetle, the radioactive label was recovered from a polypeptide rich in aspartate, the product of nitrilase-catalyzed conversion of β-cyanoalanine .
In order to estimate the relevance of rhodanese
Most β-cyanoalanine synthase activity data for animals were generated with intact, partly dried and stored animal tissues, but some enzymes have been purified and characterized [84, 104]. In the nematode
In general, proteins involved in the adaptation of herbivores to their host plants and, in particular, those catalyzing the detoxification and transport of host plant xenobiotics are thought to be under narrow transcriptional regulation . For the mite β-cyanoalanine synthase, an induction by cyanide exposure over 30 generations led to a transcriptional response allowing for the identification of the detoxification enzyme . Thus, these enzymes are among the most variable ones and play a key role in the adaptation to and population of new host plants . As the relationship between herbivore and host plant is close and evolution favors adapted defense of the plant in order to diminish resource and tissue loss through predation , transcriptional responses could be discovered also in the host plant . This close coevolution between herbivores and their host plants has therefore shaped both partners and is likely to underlie the higher cyanide tolerance of specialist herbivores on cyanogenic plants [30, 115].
6. Alternative herbivore strategies to cope with cyanogens
Next to efficient means of cyanide detoxification, herbivores have developed alternative ways to avoid intoxications when feeding on cyanide-defended plants. Often, the cyanide potential of food plants is below a toxic threshold . As generalist herbivores usually change their food plants frequently, this allows them to mix a cyanide-rich diet with a diet low in cyanide to keep the overall cyanide intake below a toxic threshold . Moreover, cyanogenic glucoside occurrence is often accompanied by a bitter taste of the potential food plant and many herbivores therefore avoid feeding on these plants if other host plants are available . Nevertheless, in no-choice feeding experiments or if no other food plant is available in the habitat, herbivores may consume high amounts of cyanide-defended plants leading to intoxication or even death [116, 117]. Adaptations to reduce this risk include morphological, behavioral, physiological and biochemical mechanisms as outlined in the following paragraphs.
The mouthparts of herbivores from the Aphididae have evolved to specialized sucking styli which they insert through the apoplast into the sieve elements to suck phloem sap. This feeding mode avoids tissue disruption and therefore the mixing of plant cyanogenic glucosides and their spatially separated hydrolysis enzymes [41, 116, 118, 119]. In the lepidopteran specialist
As a physiological adaptation, the strongly alkaline midgut pH found in some generalist and specialist herbivores allows for the inhibition of the ingested plant β-glucosidases and avoidance of cyanide liberation [118, 120] in contrast to other species with a slightly acidic midgut pH that are prone to cyanide intoxication [121, 122]. Further, properties and expression of the herbivore’s endogenous β-glucosidases have undergone adaptational adjustments to reduce cyanide release from ingested plant material. As an example, the β-glucosidases localized in the saliva and midgut lumen of the cyanogenic glucoside-feeding specialist
Yet another mechanism protects millipede species (Diplopoda) from cyanide poisoning. These animals possess a highly tolerant cytochrome c oxidase, making cyanide poisoning less effective . Instead of or in combination with a cyanide-resistant terminal oxidase, a complete cyanide-insensitive oxidative pathway has also been proposed . Studies on the respiratory rate of larvae of the lepidopteran generalist herbivore,
7. Cyanogenic compounds in arthropod herbivores
7.1. Occurrence of cyanogenic compounds in arthropods
Arthropods are the only phylum of animals in which biosynthesis or sequestration of cyanogenic compounds has been shown . Within arthropods, the presence of cyanogenic glucosides appears to be restricted to millipedes (Diplopoda), centipedes (Chilopoda) and three orders within the Insecta (Lepidoptera, Coleoptera and Hemiptera) . The Lepidoptera and Hemiptera are the only groups containing cyanogenic compounds with aliphatic side chains, while in the others groups of arthropods, cyanogenic compounds possess aromatic side chains [4, 129]. Among the most intensely studied species, larvae of
biosynthesis of cyanogenic compounds in arthropods De novo
First indications for
Among arthropods, millipedes also contain cyanogenic compounds, namely cyanohydrins such as mandelonitrile, and use them as defense against predators as discussed by Shear . Synthesis of cyanide and cyanohydrins such as mandelonitrile was demonstrated for different species of millipedes using feeding tests with 14C-phenylalanine and further radioactively labeled precursors [14, 107, 138]. As this resulted in labeling of phenylacetaldoxime and phenylacetonitrile as potential pathway intermediates, a biosynthesis pathway very alike the one described in plants and later in insects was proposed [14, 107, 138] (Figure 4). The unwanted release of cyanide is prevented by specifically shaped, two chamber glands where cyanide precursor and the hydrolyzing enzyme α-hydroxynitrile lyase are stored separately [107, 133]. In the α-hydroxynitrile chamber, organic acids generate low pH values to stabilize the α-hydroxynitrile . Upon attack by a predator, gland secretions are mixed to generate cyanide. This mechanism allows the millipede to liberate cyanide in a controlled way, thereby economizing its chemical defense and protecting its own tissue from poisoning.
Although the presence of cyanogenic glucosides and, in part, their
7.3. Sequestration of cyanogenic compounds from the food plants
Most data known on sequestration of cyanogenic compounds in insects were generated using larvae of
Sequestration of cyanogens has also been shown for gynocardin, a cyclic α-hydroxynitrile glucoside, in
Larvae of the bug
7.4. Benefit of cyanogenesis for herbivores
Cyanogenic glucosides derived from
Nevertheless, cyanide alone is not always efficient for the animal’s defense. It has been shown that in millipedes, not cyanide itself but the second product of mandelonitrile hydrolysis, benzaldehyde, is repellent to ants . In contrast, for the intact cyanogenic glucoside cardiospermin a deterrent effect on ants has been shown which could not be observed for any cyanogenic glucoside before . Thus, predating insects facing cyanide in their prey may have evolved a sensitive perception of substances with stronger odor or taste usually occurring alongside cyanide . Alternatively, it was proposed that cyanide is the main means of defense against vertebrate predators, while benzaldehyde is used to repel arthropod enemies . Phylogenetic and physiological data indicate that cyanogenesis as defense strategy has been lost and replaced by ancient phenolic defense compounds in some groups among the Polydesmida, mainly those unlikely to be targeted by vertebrate predators .
Next to their defensive roles, linamarin and lotaustralin are also used as nitrogen sources for chitin biosynthesis based on their turnover during metamorphosis . During the formation of the pupal cuticle, cyanogenic glucosides are a key nitrogen source [146, 147]. However, mobilization through β-cyanoalanine synthase and nitrilase/nitrile hydratase leading to the formation of asparagine and aspartate similar to plants  has not been demonstrated in insects so far. The efficient transport of cyanogenic glucosides in
Based on transcriptional and metabolite analyses , it has been hypothesized that the biosynthesis of cyanogenic glucosides in arthropods is older than their sequestration . The biosynthesis is thought to have been constitutive in the ancestors of Zygaena which did not live on cyanogenic plants (but on Celastraceae) and were, therefore, not able to receive cyanogenic glucosides from their host plants. Upon exploration of cyanogenic plants, the insects’ endogenous biosynthesis became inducible as sequestration helped to reduce metabolic costs for
8. Conclusions and perspectives
The past 15 years have witnessed an enormous progress in our understanding of herbivore adaptational mechanisms to plant cyanide defenses and their evolution. A lot of the present knowledge has been acquired through the application of state-of-the-art analytical and molecular tools as well as imaging techniques to the model species
Future research will have to extend the present insights by studying a broader range of species with respect to their behavioral, physiological and biochemical adaptations to cyanogens. Besides the identification and detailed characterization of cyanide detoxification enzymes from additional species, transporters involved in cyanogen sequestration will be an interesting target of future investigations. In addition, experimental proof of essential roles of herbivore proteins involved in overcoming plant cyanide defenses might become possible
Financial support of our research on cyanide detoxification in arthropods by the German Research Foundation is gratefully acknowledged.