Cyanide production by Pseudomonas fluorescens and Pseudomonas aeruginosa.

Of 200 water isolates screened, five strains of Pseudomonas fluorescens and one strain of Pseudomonas aeruginosa were cyanogenic. Maximum cyanogenesis by two strains of P. fluorescens in a defined growth medium occurred at 25 to 30 degrees C over a pH range of 6.6 to 8.9. Cyanide production per cell was optimum at 300 mM phosphate. A linear relationship was observed between cyanogenesis and the log of iron concentration over a range of 3 to 300 microM. The maximum rate of cyanide production occurred during the transition from exponential to stationary growth phase. Radioactive tracer experiments with [1-14C]glycine and [2-14C]glycine demonstrated that the cyanide carbon originates from the number 2 carbon of glycine for both P. fluorescens and P. aeruginosa. Cyanide production was not observed in raw industrial wastewater or in sterile wastewater inoculated with pure cultures of cyanogenic Pseudomonas strains. Cyanide was produced when wastewater was amended by the addition of components of the defined growth medium.     

Cyanide production by Pseudomonas fluorescens and Pseudomonas aeruginosa

Of 200 water isolates screened, five strains of Pseudomonas fluorescens and one strain of Pseudomonas aeruginosa were cyanogenic. Maximum cyanogenesis by two strains of P. fluorescens in a defined growth medium occurred at 25 to 30 degrees C over a pH range of 6.6 to 8.9. Cyanide production per cell was optimum at 300 mM phosphate. A linear relationship was observed between cyanogenesis and the log of iron concentration over a range of 3 to 300 microM. The maximum rate of cyanide production occurred during the transition from exponential to stationary growth phase. Radioactive tracer experiments with [1-14C]glycine and [2-14C]glycine demonstrated that the cyanide carbon originates from the number 2 carbon of glycine for both P. fluorescens and P. aeruginosa. Cyanide production was not observed in raw industrial wastewater or in sterile wastewater inoculated with pure cultures of cyanogenic Pseudomonas strains. Cyanide was produced when wastewater was amended by the addition of components of the defined growth medium.     

The relationship between growth phase and cyanogenesis inPseudomonas aeruginosa

In batch cultures ofPseudomonas aeruginosa, hydrogen cyanide is produced primarily during the transition between logarithmic and stationary phases. This transient response is due to the synthesis of the enzyme system of cyanogenesis during mid to late logorithmic and the inactivation of this system in early stationary phase. Although glycine, the metabolic precursor of cyanide, stimulates cyanogenesis, it is not necessary to incorporate this amino acid in the growth medium to produce elevated enzyme levels. Under conditions of iron limitation (1×10⁻⁶ M), phosphate limitation (0.1 mM), and excess phosphate (250 mM), the culture produces low levels of the cyanogenic enzyme system. Increasing the carbon and energy source,l-glutamate, prolongs cyanogenesis and postpones the inactivation of the cyanogenic enzyme system.     

Aridity shapes cyanogenesis cline evolution in white clover (Trifolium repens L.)

Adaptive differentiation between populations is often proposed to be the product of multiple interacting selective pressures, although empirical support for this is scarce. In white clover, populations show adaptive differentiation in frequencies of cyanogenesis, the ability to produce hydrogen cyanide after tissue damage. This polymorphism arises through independently segregating polymorphisms for the presence/absence of two required cyanogenic components, cyanogenic glucosides and their hydrolysing enzyme. White clover populations worldwide have evolved a series of recurrent, climate‐associated clines, with higher frequencies of cyanogenic plants in warmer locations. These clines have traditionally been hypothesized to reflect a fitness trade‐off between chemical defence in herbivore‐rich areas (warmer climates) and energetic costs of producing cyanogenic components in areas of low herbivore pressure (cooler climates). Recent observational studies suggest that cyanogenic components may also be beneficial in water‐stressed environments. We investigated fitness trade‐offs associated with temperature‐induced water stress in the cyanogenesis system using manipulative experiments in growth chambers and population surveys across a longitudinal precipitation gradient in the central United States. We find that plants producing cyanogenic glucosides have higher relative fitness in treatments simulating a moderate, persistent drought stress. In water‐neutral treatments, there are energetic costs to producing cyanogenic components, but only in treatments with nutrient stress. These fitness trade‐offs are consistent with cyanogenesis frequencies in natural populations, where we find clinal variation in the proportion of plants producing cyanogenic glucosides along the precipitation gradient. These results suggest that multiple selective pressures interact to maintain this adaptive polymorphism and that modelling adaptation will require knowledge of environment‐specific fitness effects.     

Influence of aeration on hydrogen cyanide biosynthesis byPseudomonas aeruginosa

Batch cultures ofPseudomonas aeruginosa were able to produce only low levels of cyanide during logarithmic growth with adequate aeration. The reduction of aeration caused a rapid increase in the ability of such cultures to produce hydrogen cyanide. The immediacy and the magnitude of this response depended on the oxygen level, with a concentration of 4% in the aeration gas giving optimal results. The reestablishment of normal aeration resulted in a cessation of the increase of the culture's cyanogenic capacity. This effect appeared to be a combination of inactivation of the hydrogen cyanide synthase and repression of synthesis of this enzyme.     

Influence of oxygen on thePseudomonas aeruginosa hydrogen cyanide synthase

Partially purified HCN Synthase (HCS) required exogenous electron acceptors for activity. Phenazine methosulfate (PMS) provided the greatest activity, whereas oxygen allowed only a limited response. TheP. aeruginosa secondary metabolite pyocyanin supported HCS-mediated cyanide production. HCN production by whole cells operated maximally at low oxygen levels, whereas moderate oxygen levels limited HCS activity. Respiration and cyanogenesis by whole cells were equally sensitive to azide; HCS was completely resistant.     

Cyanogenesis of Wild Lima Bean (Phaseolus lunatus L.) Is an Efficient Direct Defence in Nature

In natural systems plants face a plethora of antagonists and thus have evolved multiple defence strategies. Lima bean (Phaseolus lunatus L.) is a model plant for studies of inducible indirect anti-herbivore defences including the production of volatile organic compounds (VOCs) and extrafloral nectar (EFN). In contrast, studies on direct chemical defence mechanisms as crucial components of lima beans'' defence syndrome under natural conditions are nonexistent. In this study, we focus on the cyanogenic potential (HCNp; concentration of cyanogenic glycosides) as a crucial parameter determining lima beans'' cyanogenesis, i.e. the release of toxic hydrogen cyanide from preformed precursors. Quantitative variability of cyanogenesis in a natural population of wild lima bean in Mexico was significantly correlated with missing leaf area. Since existing correlations do not by necessity mean causal associations, the function of cyanogenesis as efficient plant defence was subsequently analysed in feeding trials. We used natural chrysomelid herbivores and clonal lima beans with known cyanogenic features produced from field-grown mother plants. We show that in addition to extensively investigated indirect defences, cyanogenesis has to be considered as an important direct defensive trait affecting lima beans'' overall defence in nature. Our results indicate the general importance of analysing ‘multiple defence syndromes’ rather than single defence mechanisms in future functional analyses of plant defences.     

Cyanogenic potential in cassava and its influence on a generalist insect herbivore Cyrtomenus bergi (Hemiptera: Cydnidae)

The hypothesis that cyanogenic potential in cassava is a defense mechanism against arthropod pests is one of the crucial questions relevant to current efforts to reduce or eliminate cyanogenic potential (CNP) in cassava. The generalist arthropod Cyrtomenus bergi, which attacks cassava roots, was used in a bioassay relating oviposition and survival to CNP, concentration of nonglycosidic cyanogens, and linamarase (beta-glycosidase) activity in twelve selfed cassava siblings and their parental clone, which has segregated for different levels of cyanogenesis. Electron microscopic evaluation revealed an intracellular pathway of the stylet of C. bergi in the cassava root tissue to rupture cell walls. This feeding behavior causes cyanogenesis and increased linamarin content in the hemolymph of C. bergi while feeding on a cyanogenic diet. This diet resulted in a significant reduction in oviposition, especially at levels of CNP above 150 ppm (expressed as hydrogen cyanide) on fresh weight basis (or 400 ppm on dry weight basis) in cassava roots. An exponential decline in oviposition was observed with increasing levels of CNP, beginning 12 d after exposure to the cyanogenic diet. Cyanogenic potential and dry matter content showed a positive effect on survival. No relationship was found between concentrations of nonglycosidic cyanogens or linamarase activity in the cassava root and either oviposition or survival. According to our results, there is a significant difference between potentially noncyanogen and high cyanogen clones, but there may not be a significant difference between potentially noncyanogen and low cyanogen clones. Consequently, more frequent outbreaks or higher levels of damage might not be anticipated in potentially noncyanogen cassava clones than that anticipated in low cyanogenic clones. The negative effect of cyanogenesis on oviposition concurrent with a positive effect on survival of this pest is most likely the result of a physiological trade-off between survival and oviposition. The question of whether ovipositional rates could be recovered after a long-term exposure to cyanide remains unanswered.     

Bacterial cyanogenesis: impact on biotic interactions

The ability of bacteria to influence organisms that they associate with via metabolite production is one of the hallmarks of microbial interactions. One metabolite of interest is the metabolic poison cyanide. Production of this metabolite is an unique characteristic of certain bacteria that inhabit a wide array of habitats ranging from the human body to the rhizosphere. This review focuses on four targets of cyanogenic bacteria: the human lung, plant pathogens, plants and invertebrates. For a number of cyanogenic bacteria, the contribution of cyanide to the interaction has been rigorously tested using mutants altered in cyanide production. Both deleterious and stimulatory effects of cyanogenic bacteria on other organisms have been documented. In addition, the HCN synthase‐encoding gene cluster hcnABC has served as a marker of cyanogenic capability in the soil environment revealing both genetic diversity at this locus and regulatory influences by other organisms. The pervasive nature of cyanogenesis in a number of different ecological contexts encourages exploration of this bacterial ability and its possible optimization for improving human health, crop production and pest control.     

Relationship between cyanogenesis and latex stability on tapping panel dryness in rubber trunk girth

The presence of high cyanogenic glycoside concentrations may predispose plant to the tapping panel dryness (TPD). This study aimed to verify the involvement of cyanogenesis in the reduction of latex stability and in the establishment of TPD. The following parameters were evaluated in rubber tree trunk bark: concentration of cyanogenic glycosides with determination of cyanogenic potential (HCNp) and latex stability with lutoid bursting index (LBI). The study of the relationship between cyanogenesis and TPD was performed by semiquantitative comparison of hydrogen cyanide (HCN) gas released from the trunk bark under the following conditions: without (0%) and with (100%) TPD. The positive correlations between HCNp values and LBI indicate that cyanogenic glycosides present in the bark reduce latex stability, resulting in low yield due to the short duration of flow during tapping. The largest amount of HCN released by trunk bark tissues when the plant exhibits TPD symptoms strengthens the evidence of the involvement of this compound in the establishment of this condition.     

Cyanogenesis in plants

Several thousand plant species, including many economically important food plants, synthesize cyanogenic glycosides and cyanolipids. Upon tissue disruption, these natural products are hydrolyzed liberating the respiratory poison hydrogen cyanide. This phenomenon of cyanogenesis accounts for numerous cases of acute and chronic cyanide poisoning of animals including man. This article reviews information gathered during the past decade about the enzymology and molecular biology of cyanogenesis in higher plants. How compartmentation normally prevents the large-scale, suicidal release of HCN within the intact plant is discussed. A renewed interest in the physiology of these cyanogenic compounds has revealed that, in addition to providing protection for some species against herbivory, they may also serve as storage forms for reduced nitrogen.     

Frequency of cyanogenesis in tropical rainforests of far north Queensland, Australia

BACKGROUND AND AIMS: Plant cyanogenesis is the release of toxic cyanide from endogenous cyanide-containing compounds, typically cyanogenic glycosides. Despite a large body of phytochemical, taxonomic and ecological work on cyanogenic species, little is known of their frequency in natural plant communities. This study aimed to investigate the frequency of cyanogenesis in Australian tropical rainforests. Secondary aims were to quantify the cyanogenic glycoside content of tissues, to investigate intra-plant and intra-population variation in cyanogenic glycoside concentration and to appraise the potential chemotaxonomic significance of any findings in relation to the distribution of cyanogenesis in related taxa. METHODS: All species in six 200 m(2) plots at each of five sites across lowland, upland and highland tropical rainforest were screened for cyanogenesis using Feigl-Anger indicator papers. The concentrations of cyanogenic glycosides were accurately determined for all cyanogenic individuals. KEY RESULTS: Over 400 species from 87 plant families were screened. Overall, 18 species (4.5 %) were cyanogenic, accounting for 7.3 % of total stem basal area. Cyanogenesis has not previously been reported for 17 of the 18 species, 13 of which are endemic to Australia. Several species belong to plant families or orders in which cyanogenesis has been little reported, if at all (e.g. Elaeocarpaceae, Myrsinaceae, Araliaceae and Lamiaceae). A number of species contained concentrations of cyanogenic glycosides among the highest ever reported for mature leaves-up to 5.2 mg CN g(-1) d. wt, for example, in leaves of Elaeocarpus sericopetalus. There was significant variation in cyanogenic glycoside concentration within individuals; young leaves and reproductive tissues typically had higher cyanogen content. In addition, there was substantial variation in cyanogenic glycoside content within populations of single species. CONCLUSIONS: This study expands the limited knowledge of the frequency of cyanogenesis in natural plant communities, includes novel reports of cyanogenesis among a range of taxa and characterizes patterns in intra-plant and intra-population variation of cyanogensis.     

Adaptive cyanogenesis clines evolve recurrently through geographical sorting of existing gene deletions

Identifying the genetic basis of parallel phenotypic evolution provides insight into the process of adaptation and evolutionary constraint. White clover (Trifolium repens) has evolved climate‐associated adaptive clines in cyanogenesis (the ability to produce hydrogen cyanide upon tissue damage) in several world regions where it has been introduced. Gene‐deletion polymorphisms at the CYP79D15 and Li loci underlie the presence/absence of the cyanogenic phenotype. Both loci have undergone multiple independent gene‐deletion events, which are identifiable through molecular signatures in flanking regions. To investigate whether cyanogenesis clines in introduced populations have evolved through the sorting of standing genetic variation or de novo gene deletions, we examined cyanogenesis gene‐flanking regions in three world regions. In comparison with native Eurasian populations, we find no evidence for novel gene deletion events in any introduced region, which suggests that these adaptive clines have evolved through the geographical sorting of pre‐existing genetic variation.     

Variation of cyanogenesis in some plant species of the midwestern United States

The frequency of cyanogenesis of 48 species of vascular plants was examined by testing 30 individuals from five populations of each species for release of cyanide. The rate at which cyanide was released and the amount of cyanide released varied widely among individuals of a population and among populations of a species. For many taxa, the frequency of cyanogenesis was highly variable among populations. Of the species examined, 20 have not been reported previously as being cyanogenic.     

Cyanide Production by Rhizobacteria and Potential for Suppression of Weed Seedling Growth

Rhizobacteria strains were characterized for ability to synthesize hydrogen cyanide and for effects on seedling root growth of various plants. Approximately 32% of bacteria from a collection of over 2000 isolates were cyanogenic, evolving HCN from trace concentrations to >30 nmoles/mg cellular protein. Cyanogenesis was predominantly associated with pseudomonads and was enhanced when glycine was provided in the culture medium. Concentrations of HCN produced by rhizobacteria were similar to exogenous concentrations inhibiting seedling growth in bioassays, suggesting that cyanogenesis by rhizobacteria in the rhizosphere can adversely affect plant growth. Growth inhibition of lettuce and barnyardgrass by volatile metabolites of the cyanogenic rhizobacteria confirmed that HCN was the major inhibitory compound produced. Our results suggest that HCN produced in the rhizospheres of seedlings by selected rhizobacteria is a potential and environmentally compatible mechanism for biological control of weeds. Received: 13 December 2000/Accepted: 6 February 2001     

Absence of cyanogenic glycosides in the tribe Miconieae (Melastomataceae)

In order to compare ant and non-ant defended species of Melastomataceae, production of hydrogen cyanide gas was tested in the field for 51 species of 10 genera of the tribe Miconieae. Using both the picric acid and the Feigl–Anger tests all populations surveyed tested negative for the presence of cyanogenic glycosides. These results confirm that cyanogenesis is rare in the family, although not completely absent. Cyanogenic glycosides are not responsible for the protection against herbivory in non-ant defended species, but this does not rule out that there are quantitative of qualitative differences in other secondary metabolites.     

Reconstitution of cyanogenesis in barley (Hordeum vulgare L.) and its implications for resistance against the barley powdery mildew fungus

Barley (Hordeum vulgare L.) produces a leucine-derived cyanogenic β-d-glucoside, epiheterodendrin that accumulates specifically in leaf epidermis. Barley leaves are not cyanogenic, i.e. they do not possess the ability to release hydrogen cyanide, because they lack a cyanide releasing β-d-glucosidase. Cyanogenesis was reconstituted in barley leaf epidermal cells through single cell expression of a cDNA encoding dhurrinase-2, a cyanogenic β-d-glucosidase from sorghum. This resulted in a 35–60% reduction in colonization rate by an obligate parasite Blumeria graminis f. sp. hordei, the causal agent of barley powdery mildew. A database search for barley homologues of dhurrinase-2 identified a (1,4)-β-d-glucan exohydrolase isozyme βII that is located in the starchy endosperm of barley grain. The purified barley (1,4)-β-d-glucan exohydrolase isozyme βII was found to hydrolyze the cyanogenic β-d-glucosides, epiheterodendrin and dhurrin. Molecular modelling of its active site based on the crystal structure of linamarase from white clover, demonstrated that the disposition of the catalytic active amino acid residues was structurally conserved. Epiheterodendrin stimulated appressoria and appressorial hook formation of B. graminis in vitro, suggesting that loss of cyanogenesis in barley leaves has enabled the fungus to utilize the presence of epiheterodendrin to facilitate host recognition and to establish infection.     

Constitutive polymorphic cyanogenesis in the Australian rainforest tree, Ryparosa kurrangii (Achariaceae)

Cyanogenesis, the liberation of volatile hydrogen cyanide from endogenous cyanide-containing compounds, is a proven plant defence mechanism and the particular cyanogens involved have taxonomic utility. The cyclopentenoncyanhydrin glycoside gynocardin was the only cyanogen isolated from foliar tissue of the rare Australian rainforest tree, Ryparosa kurrangii (Achariaceae). Mechanical damage simulating foliar herbivory did not induce a significant increase in the expression of cyanogenesis over a 24h period, indicating cyanogenic herbivore defence in R. kurrangii is constitutive. The cyanogenic potential of mature leaves was quantitatively polymorphic between trees in a natural population, ranging from 0.54 to 4.77 mg CN g(-1) dry wt leaf tissue.