Cyanogenic glycosides of the Turneraceae

Eighty species of Turneraceae were tested and found to be cyanogenic. Analysis by HPLC and NMR and comparisons of R_t values from paper chromatograms showed all to possess tetraphyllin A and B and epitetraphyllin B and deidaclin as their cyanogens. These data confirm the close relationship of this family with other families which produce structurally related cyanogens: the Passifloraceae, Malesherbiaceae and Flacourtiaceae.     

THE RELATIVE IMPORTANCE OF MOLLUSCS AND INSECTS AS SELECTIVE GRAZERS OF ACYANOGENIC WHITE CLOVER (TRIFOLIUM REPENS)

Selective grazing of acyanogenic and cyanogenic white cloverwas investigated by a field experiment using biocides to maintain(a) mollusc only, (b) insect only, (c) both mollusc and insect,and (d) neither mollusc nor insect, grazing regimes. Molluscsfed more on acyanogenic clover throughout the year, whereasinsects damaged more acyanogenic plants in late summer, butmore cyanogenic plants in spring. When both molluscs and insectswere present, the net effect was greater damage to acyanogenicplants. However, in situations where insects are more abundantthan molluscs there might be a net selection against cyanogenicclover. (Received 16 December 1988; accepted 22 February 1989)     

Endogenous evolution of HCN during pre-germination periods in many seed species

Both cyanogenic ( Malus pumila Mill) and acyanogenic ( Oryza sativa L., Hordeum vulgare L., Zea mays L., Glycine max Merr., Lactuca sativa L., and Xanthium pennsylvanicum Wallr. etc.) seeds evolve HCN gas during the early periods of water imbibition. All tested seeds contained reserve cyanogens which liberated HCN upon hydrolysis with H₂SO₄ and with β-glucosidase and/or lipase. The amounts of liberated HCN were roughly comparable to those of unidentified cyanogens. It is thus conceivable that the cyanogens within seeds are available as precursors for free HCN evolved in the pre-germination period. The amounts of HCN evolved in the acyanogenic seeds were only 0.002 to 1% of that in apple, but the contents of the cyanogenic compounds in rice and cocklebur increased temporarily during the pre-germination period, then decreased and, finally, disappeared completely with the start of germination.     

Effects of cyanogenesis in bracken fern (Pteridium aquilinum) on associated insects

Abstract. 1. The relationship between cyanogenesis in bracken fern and the insect fauna feeding on the plant was investigated over a 3 year period. The most common insects between May and July, while cyanide levels were high, were the sawflies Strongylogaster impressata Provancher, S.multicincta Norton, Aneug-menus fzavipes (Norton), the aphid Macrosiphum euphorbiae (Thomas) and a microlepidopteran species of Monochroa .
2. Collections of insects from cyanogenic and acyanogenic fronds showed significantly fewer sawflies on the cyanogenic fronds. The aphid and the microlepidopteran were randomly distributed with respect to cyanogenicity.
3. Feeding tests for two of the sawfly species ( Simpressata and Smulticincta ) showed that larvae grew more slowly and had a higher mortality when raised on cyanogenic fronds than on acyanogenic ones.
4. Field collected cyanogenic bracken fronds were found to have sustained less damage from chewing herbivores compared with acyanogenic fronds.     

An investigation of the responses of herbivores to cyanogenesis in Lotus corniculatus L.

In laboratory feeding choice experiments several species of insects and molluscs eat acyanogenic Lotus corniculatus leaves or petals in preference to cyanogenic alternatives. These species can be characterized as generalist feeders for which L. corniculatus forms only an occasional dietary item for any particular individual. Some of the insects that were found to display selective eating were maintained on exclusive diets of either cyanogenic or acyanogenic L. corniculatus . Survival times varied considerably between species, but there was no evidence that mortalities were higher amongst the groups exposed to cyanide. These results suggest that the major role of cyanogenesis may be as a feeding inhibitor, not as a toxin.     

Positive effects of cyanogenic glycosides in food plants on larval development of the common blue butterfly

Cyanogenesis is a widespread chemical defence mechanism in plants against herbivory. However, some specialised herbivores overcome this protection by different behavioural or metabolic mechanisms. In the present study, we investigated the effect of presence or absence of cyanogenic glycosides in birdsfoot trefoil (Lotus corniculatus, Fabaceae) on oviposition behaviour, larval preference, larval development, adult weight and nectar preference of the common blue butterfly (Polyommatus icarus, Lycaenidae). For oviposition behaviour there was a female-specific reaction to cyanogenic glycoside content; i.e. some females preferred to oviposit on cyanogenic over acyanogenic plants, while other females behaved in the opposite way. Freshly hatched larvae did not discriminate between the two plant morphs. Since the two plant morphs differed not only in their content of cyanogenic glycoside, but also in N and water content, we expected these differences to affect larval growth. Contrary to our expectations, larvae feeding on cyanogenic plants showed a faster development and stronger weight gain than larvae feeding on acyanogenic plants. Furthermore, female genotype affected development time, larval and pupal weight of the common blue butterfly. However, most effects detected in the larval phase disappeared for adult weight, indicating compensatory feeding of larvae. Adult butterflies reared on the two cyanogenic glycoside plant morphs did not differ in their nectar preference. But a gender-specific effect was found, where females preferred amino acid-rich nectar while males did not discriminate between the two nectar mimics. The presented results indicate that larvae of the common blue butterfly can metabolise the surplus of N in cyanogenic plants for growth. Additionally, the female-specific behaviour to oviposit preferably on cyanogenic or acyanogenic plant morphs and the female-genotype-specific responses in life history traits indicate the genetic flexibility of this butterfly species and its potential for local adaptation.     

Cyanogenic glycosides of Malesherbia

Twenty-eight species of Malesherbia were tested and found to be cyanogenic. Analysis of HPLC, GC, NMR and comparisons of R_f values on paper chromatograms showed all to possess tetraphyllin A and B as major cyanogens, with epitetraphyllin B and deidaclin being present occasionally. These data confirm the close relationship of this family with other families which produce structurally related cyanogens, the Passifloraceae, Turneraceae and Flacourtiaceae. In none of these families is tetraphyllin A the major cyanogen, however. The genus Gynopleura, sometimes segregated from Malesherbia, does not differ in this character.     

Cyanogenic glycosides in Lotus corniculatus

Summary Two genotypes (one cyanogenic and the other acyanogenic) of birds-foot trefoil, Lotus corniculatus L., were used to study the effects of cyanogenic glycosides in leaf tissues upon a polyphagous herbivore, the southern armyworm, Spodoptera eridania Cram. (Lepidoptera). No differences were observed in consumption rate, assimilation efficiency, utilization of plant biomass, or metabolic costs in terms of expended calories between larvae fed acyanogenic or cyanogenic leaves. Similarly no differences were seen in the nitrogen or caloric utilization efficiencies, or in the nitrogen accumulation rate or growth rate of larvae on cyanogenic versus acyanogenic plants. Larval performance and growth on 20-week old plants was generally poorer than on 4 week old plants, however. This was reflected in slower growth, smaller pupal weights, lower nitrogen utilization efficiencies (N.U.E.) and biomass assimilation efficiencies (A.D.) on both the cyanogenic and acyanogenic plants.Although useful as a deterrent to some herbivores, cyanogenesis does not seem to provide an effective defense against this adapted herbivore. This study supports current hypotheses of insect/plant coevolution, and suggests that the metabolic costs of processing cyanogenic plant biomass are small in comparison to those imposed by the nutritional status of the plant leaves.     

Selective grazing of acyanogenic white clover: Variation in behaviour among populations of the slug Deroceras reticulatum

Summary Collections of the slug Deroceras reticulatum were made from grassland sites containing contrasting frequencies of the cyanogenic morph of white clover, Trifolium repens. In choice chamber experiments, slugs obtained from sites with a low frequency of cyanogenic clover showed a significantly greater degree of selective eating of acyanogenic morphs than slugs taken from a site containing a high frequency of cyanogenic clover. Differences in selectivity between populations were caused both by differences in the rate of initiation of feeding on cyanogenic morphs, and by differences in the extent of damage once feeding had been initiated. The implications of these results for the cyanogenic polymorphism of T. repens are discussed.     

Phylogenetic analysis of Zygaenoidea small-subunit rRNA structural variation implies initial oligophagy on cyanogenic host plants in larvae of the moth genus Zygaena (Insecta: Lepidoptera)

Cyanogenesis, the release of toxic cyanide from living cells, plays an important role in the defence system of certain plant (e.g. Fabaceae) and animal (e.g. Zygaenidae) taxa. The larvae of a significant number of Zygaena moth species (Zygaenidae) preferentially feed on cyanogenic Fabaceae and some of them are able to sequester cyanogenic compounds of their host plants. Using secondary structure variation of the small-subunit rRNA, we tested the currently accepted evolutionary hypothesis explaining species diversification in the genus Zygaena . We derived secondary structures considering evidence from covariation patterns and thermodynamic folding and applied structural information in a phylogenetic analysis. Contrary to previous assumptions, our results suggest that the use of cyanogenic larval host plants is an ancient trait and that the ability to feed on cyanogenic plants was probably already present in the most recent common ancestor of Zygaena . The utilization of acyanogenic plants in Zygaena species appears to be the result of a single secondary, reverse, larval host-plant shift. © 2006 The Linnean Society of London, Zoological Journal of the Linnean Society , 2006, 147 , 367–381.     

Cyanogenic glycosides: a case study for evolution and application of cytochromes P450

Cyanogenic glycosides are ancient biomolecules found in more than 2,650 higher plant species as well as in a few arthropod species. Cyanogenic glycosides are amino acid-derived β-glycosides of α-hydroxynitriles. In analogy to cyanogenic plants, cyanogenic arthropods may use cyanogenic glycosides as defence compounds. Many of these arthropod species have been shown to de novo synthesize cyanogenic glycosides by biochemical pathways that involve identical intermediates to those known from plants, while the ability to sequester cyanogenic glycosides appears to be restricted to Lepidopteran species. In plants, two atypical multifunctional cytochromes P450 and a soluble family 1 glycosyltransferase form a metabolon to facilitate channelling of the otherwise toxic and reactive intermediates to the end product in the pathway, the cyanogenic glycoside. The glucosinolate pathway present in Brassicales and the pathway for cyanoalk(en)yl glucoside synthesis such as rhodiocyanosides A and D in Lotus japonicus exemplify how cytochromes P450 in the course of evolution may be recruited for novel pathways. The use of metabolic engineering using cytochromes P450 involved in biosynthesis of cyanogenic glycosides allows for the generation of acyanogenic cassava plants or cyanogenic Arabidopsis thaliana plants as well as L. japonicus and A. thaliana plants with altered cyanogenic, cyanoalkenyl or glucosinolate profiles.     

Cassava plants with a depleted cyanogenic glucoside content in leaves and tubers. Distribution of cyanogenic glucosides, their site of synthesis and transport, and blockage of the biosynthesis by RNA interference technology

Transgenic cassava (Manihot esculenta Crantz, cv MCol22) plants with a 92% reduction in cyanogenic glucoside content in tubers and acyanogenic (<1% of wild type) leaves were obtained by RNA interference to block expression of CYP79D1 and CYP79D2, the two paralogous genes encoding the first committed enzymes in linamarin and lotaustralin synthesis. About 180 independent lines with acyanogenic (<1% of wild type) leaves were obtained. Only a few of these were depleted with respect to cyanogenic glucoside content in tubers. In agreement with this observation, girdling experiments demonstrated that cyanogenic glucosides are synthesized in the shoot apex and transported to the root, resulting in a negative concentration gradient basipetal in the plant with the concentration of cyanogenic glucosides being highest in the shoot apex and the petiole of the first unfolded leaf. Supply of nitrogen increased the cyanogenic glucoside concentration in the shoot apex. In situ polymerase chain reaction studies demonstrated that CYP79D1 and CYP79D2 were preferentially expressed in leaf mesophyll cells positioned adjacent to the epidermis. In young petioles, preferential expression was observed in the epidermis, in the two first cortex cell layers, and in the endodermis together with pericycle cells and specific parenchymatic cells around the laticifers. These data demonstrate that it is possible to drastically reduce the linamarin and lotaustralin content in cassava tubers by blockage of cyanogenic glucoside synthesis in leaves and petioles. The reduced flux to the roots of reduced nitrogen in the form of cyanogenic glucosides did not prevent tuber formation.     

Influence of two selective factors on cyanogenesis polymorphism ofTrifolium repens L. in Darjeeling Himalaya

Cyanogenesis-the production of toxic hydrogen cyanide (HCN) by damaged tissue-inTrifolium repens L. (white clover), a type of most important pasture legume, has been studied at different elevations of Darjeeling Himalaya (latitude-27° 2′ 57″ N, longitude-88° 15′ 45″ E). Release of HCN takes place due to reaction between cyanogenic glucosides stored in vacuoles of the leaf cell and the corresponding enzyme β-glucosidase present in another compartment, often cell wall. Cyanogenesis, a defense system in plant, protects the clover from herbivore and inhibits grazing. Biochemical analysis showed the presence and absence of the cyanogenesis trait within the population in different proportions at different elevations. Acyanogenic individuals also showed variations with respect to presence or absence of either cyanogenic glucosides or β-glucosidase enzyme or both. The distribution of cyanogenic and acyanogenic plants was found in all places, but at lower altitudes (2084–2094 m) the dominating plants were cyanogenic whereas in higher altitude (2560 m) the dominating plants were acyanogenic. It was observed that blister beetle (Mylabris pustalata Thunb.) and the mollusc (Macrochlamys tusgurium Benson.) were the most common consumer of leaflets ofT. repens. Six categories of damage on white clover leaf by these animals were recorded. Our results suggest that the two selective factors or forces i.e. very cold temperature (harmful to cyanogenic plants) at higher altitude as well as indiscriminate but preferential predation (harmful to acyanogenic plants) interact to affect the system of cyanogenesis and also to cause the stable and protective polymorphism inT. repens rather than genotypic differences present among the plants.     

The cyanogenic glucoside composition of Zygaena filipendulae (Lepidoptera: Zygaenidae) as effected by feeding on wild-type and transgenic lotus populations with variable cyanogenic glucoside profiles

Zygaena larvae sequester the cyanogenic glucosides linamarin and lotaustralin from their food plants (Fabaceae) as well as carry out de novo biosynthesis of these compounds. In this study, Zygaena filipendulae were reared on wild-type Lotus corniculatus and wild-type and transgenic L. japonicus plants with differing content and ratios of the cyanogenic glucosides linamarin and lotaustralin and of the cyanoalkenyl glucosides rhodiocyanoside A and D. LC-MS analyses, free choice feeding experiments and developmental studies were used to examine the effect of varying content and ratios of these secondary metabolites on the feeding preferences, growth and development of Z. filipendulae. Larvae reared on cyanogenic L. corniculatus developed faster compared to larvae reared on L. japonicus although free choice feeding trials demonstrated that the latter plant source was the preferred food plant. Larvae reared on acyanogenic L. corniculatus showed decelerated development. Analysis of different life stages and tissues demonstrate that Z. filipendulae strive to maintain certain threshold content and ratios of cyanogenic glucosides regardless of the composition of the food plants. Despite this, the ratios of cyanogenic glucosides in Z. filipendulae remain partly affected by the ratio of the food plant due to the high proportion of sequestering that takes place.     

Symbiotic soil microorganisms as players in aboveground plant–herbivore interactions – the role of rhizobia

Rhizobia play a key role for performance of leguminous plants and ecosystem productivity. However, no studies to date have investigated the importance of the rhizobial symbiosis for legume–herbivore interactions. The additional nitrogen provided by the rhizobia improves the nutritional quality of plants, but may also be used for the synthesis of defence compounds. We performed greenhouse experiments with nodulating and non-nodulating, as well as cyanogenic and acyanogenic strains of Trifolium repen s to study the effects of rhizobia Rhizobium leguminosarum on plant growth and the performance of the chewing herbivore Spodoptera littoralis and the phloem-sucking aphid Myzus persicae . We demonstrate that for nodulating strains of T. repens rhizobia increased plant growth and the performance of Spodoptera littoralis . However, this positive effect of rhizobia on the caterpillars did not occur in a cyanogenic clover strain. Reproduction of the phloem-sucking aphid Myzus persicae was inconsistently affected by rhizobia. Our study provides evidence that the additional nitrogen provided by the rhizobia may be used for the production of nitrogen-based defence compounds, thereby counteracting positive effects on the performance of chewing herbivores. The symbiosis with rhizobia is therefore an important driver of legume–herbivore interactions.     

Uptake of linamarin and lotaustralin from their foodplant by larvae of Zygaena trifolii

Experiments in which unlabelled and [aglycone ¹⁴C-labelled cyanogenic glycosides, linamarin and lotaustralin, were fed to larvae of the moth Zygaena trifolii on leaves of an acyanogenic strain of their food plant, Lotus corniculatus, showed that the larvae retained about 20–45% of the glucosides consumed. The larvae in nature usually feed on plants of L. corniculatus which themselves contain linamarin and lotaustralin. Earlier experiments had shown that the larvae of Zygaena spp. are able to synthesize these glucosides from valine and isoleucine and so both sequestration and biosynthesis of the same compounds can occur. This is the only such occurrence yet known in the relationships between plants and insects.     

Herbivores and plant defences affect selection on plant reproductive traits more strongly than pollinators

Pollinators and herbivores can both affect the evolutionary diversification of plant reproductive traits. However, plant defences frequently alter antagonistic and mutualistic interactions, and therefore, variation in plant defences may alter patterns of herbivore‐ and pollinator‐mediated selection on plant traits. We tested this hypothesis by conducting a common garden field experiment using 50 clonal genotypes of white clover (Trifolium repens) that varied in a Mendelian‐inherited chemical antiherbivore defence—the production of hydrogen cyanide (HCN). To evaluate whether plant defences alter herbivore‐ and/or pollinator‐mediated selection, we factorially crossed chemical defence (25 cyanogenic and 25 acyanogenic genotypes), herbivore damage (herbivore suppression) and pollination (hand pollination). We found that herbivores weakened selection for increased inflorescence production, suggesting that large displays are costly in the presence of herbivores. In addition, herbivores weakened selection on flower size but only among acyanogenic plants, suggesting that plant defences reduce the strength of herbivore‐mediated selection. Pollinators did not independently affect selection on any trait, although pollinators weakened selection for later flowering among cyanogenic plants. Overall, cyanogenic plant defences consistently increased the strength of positive directional selection on reproductive traits. Herbivores and pollinators both strengthened and weakened the strength of selection on reproductive traits, although herbivores imposed ~2.7× stronger selection than pollinators across all traits. Contrary to the view that pollinators are the most important agents of selection on reproductive traits, our data show that selection on reproductive traits is driven primarily by variation in herbivory and plant defences in this system.     

Spectrophotometer-aided evaluation of cyanogenic potential in white clover (Trifolium repens L.)

In comparison with ordinary methods of colorimetric evaluation of cyanogenic potential based on visual evaluation of the alkaline picrate reaction, a spectrophotometer-aided method could be more accurate (since it determines the exact amount of hydrogen cyanide released by the plant material), and less time-consuming as it can be performed on bulk material rather than on a number of individual plants. Ten white clover populations were evaluated by a spectrophotometer-aided method and by two visual evaluation criteria. All methods indicated the presence of large variation between populations. Visual methods gave almost identical results and allowed only for the distinction between cyanogenic and substantially acyanogenic populations. The results were only moderately consistent with those obtained by the spectrophotometer-aided method, which could detect the presence of variation also between cyanogenic populations. The effects of various incubation times (from 4 to 48 h) and of the addition of β-glucosidase on hydrogen cyanide release were also investigated. Comparable results for ranking of populations could be obtained over a range of incubation times, but at least 24 h were needed for a reliable estimation of the hydrogen cyanide produced by plants. The addition of enzyme did not increase the released cyanide. The effect of season and/or conditions of evaluation was marked on mean cyanogenic potential but limited on ranking of populations. Copyright © 2000 John Wiley & Sons, Ltd.     

Evidence on the molecular basis of the Ac/ac adaptive cyanogenesis polymorphism in white clover (Trifolium repens L)

White clover is polymorphic for cyanogenesis, with both cyanogenic and acyanogenic plants occurring in nature. This chemical defense polymorphism is one of the longest-studied and best-documented examples of an adaptive polymorphism in plants. It is controlled by two independently segregating genes: Ac/ac controls the presence/absence of cyanogenic glucosides; and Li/li controls the presence/absence of their hydrolyzing enzyme, linamarase. Whereas Li is well characterized at the molecular level, Ac has remained unidentified. Here we report evidence that Ac corresponds to a gene encoding a cytochrome P450 of the CYP79D protein subfamily (CYP79D15), and we describe the apparent molecular basis of the Ac/ac polymorphism. CYP79D orthologs catalyze the first step in cyanogenic glucoside biosynthesis in other cyanogenic plant species. In white clover, Southern hybridizations indicate that CYP79D15 occurs as a single-copy gene in cyanogenic plants but is absent from the genomes of ac plants. Gene-expression analyses by RT-PCR corroborate this finding. This apparent molecular basis of the Ac/ac polymorphism parallels our previous findings for the Li/li polymorphism, which also arises through the presence/absence of a single-copy gene. The nature of these polymorphisms may reflect white clover's evolutionary origin as an allotetraploid derived from cyanogenic and acyanogenic diploid progenitors.     

Cyanogenic Eucalyptus nobilis is polymorphic for both prunasin and specific beta-glucosidases

Cyanogenesis (i.e. the evolution of HCN from damaged plant tissue) requires the presence of two biochemical pathways, one controlling synthesis of the cyanogenic glycoside and the other controlling the production of a specific degradative beta-glucosidase. The sole cyanogenic glycoside in Eucalyptus nobilis was identified as prunasin (D-mandelonitrile beta-D-glucoside) using HPLC and GC-MS. Seedlings from three populations of E. nobilis were grown under controlled conditions and 38% were found to be acyanogenic, a proportion far greater than reported for any other cyanogenic eucalypt. A detailed study of the acyanogenic progeny from a single open-pollinated parent found that 23% lacked a cyanogenic beta-glucosidase, 32% lacked prunasin and 9% lacked both. Of the remaining seedlings initially identified as acyanogenics, 27% contained either trace amounts of beta-glucosidase or prunasin, while 9% contained trace amounts of both. Results support the hypothesis that the two components necessary for cyanogenesis are inherited independently. Trace amounts are likely to result from the presence of non-specific beta-glucosidases or the glycosylation of the cyanohydrin intermediate by non-specific UDP glycosyl transferases.