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.     

Cyanogenesis variation in the Turnera sidoides L. polyploid complex (Turneraceae)

This study constitutes the first investigation of cyanogenesis variation within the Turnera sidoides autopolyploid complex. The qualitative and quantitative analysis carried out in 38 populations showed that the five subspecies of the complex are cyanogenic, although the levels of cyanide released varied both between and within subspecies. Between the subspecies, carnea , holosericea and integrifolia showed higher values of HCN released than subspecies pinnatifida and sidoides . Within subspecies, there is a tendency toward an increment of mean cyanide values with ploidy levels and different hypotheses are considered in order to explain this variation. The study of cyanogenesis in populations from different geographical areas cultivated under uniform greenhouse conditions showed that, in the widespread subspecies pinnatifida , HCN variation may also reflect the action of different selection pressures, since populations having dissimilar cyanide levels grow in different phytogeographical provinces with different climatic regimes. On the other hand, the fact that the variation of HCN released was greater between populations from different geographical areas than that observed between diploid and tetraploid populations of the same area provides additional evidence for the multiple origins of polyploids of the T. sidoides complex.  © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society , 2003, 141 , 85–94.     

Injury-induced Cyanogenesis in Vegetative and Reproductive Parts of Two Grevillea Species and their F1 Hybrid

When leaves of three Grevillea species were macerated, hydrogencyanide was only released from the soft, immature ones. Thebark but not wood of young stems was weakly cyanogenic. Thegreatest concentration of cyanoglucosides occurred in peduncles,flower buds, open flowers, immature seeds and germinants. Nocyanide was released from nectar, pedicels, dry fruit, elaiosomesand dry walls mature seeds. Addition of ß-glucosidase,and sometimes water only, increased cyanogenesis in young leaves,bark, some seed and fruit walls, and embryos of mature seeds.The endogenous levels of ß-glucosidase were sufficientto release all the cyanide present in at least 80% of cases. Young leaves and germinants of G. bipinnatifida (resprouter)released more cyanide than those of G. banksii, while flowerbuds, stigmatic region, styles, perianths and ovaries of G.banksii (non-sprouter) released far more cyanide than thoseof G. bipinnatifida. The interspecific and intraplant distributionof cyanoglucosides is consistent with the expected impact ofbeneficial and herbivorous animals on long term fitness of thesespecies. The F1 hybrid (G. 'Robyn Gordon') tended to have intermediatecyanoglucoside levels between the two putative parents.Copyright1993, 1999 Academic Press Grevillea bipinnatifida, G. banksii, G. 'Robyn Gordon', cyanide, herbivory, ß-glucosidase, cyanogenesis     

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.     

Cyanogenic polymorphism in Eucalyptus polyanthemos Schauer subsp. vestita L. Johnson and K. Hill (Myrtaceae)

Plant cyanogenesis, the release of cyanide from endogenous cyanide-containing compounds, is an effective herbivore deterrent. This paper characterises cyanogenesis in the Australian tree Eucalyptus polyanthemos Schauer subsp. vestita L. Johnson and K. Hill for the first time. The cyanogenic glucoside prunasin ((R)-mandelonitrile β-D-glucoside) was determined to be the only cyanogenic compound in E. polyanthemos foliage. Two natural populations of E. polyanthemos showed quantitative variation in foliar prunasin concentration, varying from zero (i.e. acyanogenic) to 2.07 mg CN g^-1 dry weight in one population and from 0.17 to 1.98 mg CN g^-1 dry weight in the other. No significant difference was detected between the populations with respect to the mean prunasin concentration or the degree of variation in foliar prunasin, despite significant differences in foliar nitrogen. Variation between individuals was also observed with respect to the capacity of foliage to catabolise prunasin to form cyanide. Moreover, variation in this capacity generally correlated with the amount of prunasin in the tissue, suggesting genetic linkage between prunasin and β-glucosidase.     

Cyanogenic pseudomonads influence multitrophic interactions in the rhizosphere

In the rhizosphere, plant roots cope with both pathogenic and beneficial bacterial interactions. The exometabolite production in certain bacterial species may regulate root growth and other root-microbe interactions in the rhizosphere. Here, we elucidated the role of cyanide production in pseudomonad virulence affecting plant root growth and other rhizospheric processes. Exposure of Arabidopsis thaliana Col-0 seedlings to both direct (with KCN) and indirect forms of cyanide from different pseudomonad strains caused significant inhibition of primary root growth. Further, we report that this growth inhibition was caused by the suppression of an auxin responsive gene, specifically at the root tip region by pseudomonad cyanogenesis. Additionally, pseudomonad cyanogenesis also affected other beneficial rhizospheric processes such as Bacillus subtilis colonization by biofilm formation on A. thaliana Col-0 roots. The effect of cyanogenesis on B. subtilis biofilm formation was further established by the down regulation of important B. subtilis biofilm operons epsA and yqxM. Our results show, the functional significance of pseudomonad cyanogenesis in regulating multitrophic rhizospheric interactions.     

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.     

Searching for the bull's eye: agents and targets of selection vary among geographically disparate cyanogenesis clines in white clover (Trifolium repens L.)

The recurrent evolution of adaptive clines within a species can be used to elucidate the selective factors and genetic responses that underlie adaptation. White clover is polymorphic for cyanogenesis (HCN release with tissue damage), and climate-associated cyanogenesis clines have evolved throughout the native and introduced species range. This polymorphism arises through two independently segregating Mendelian polymorphisms for the presence/absence of two required components: cyanogenic glucosides and their hydrolyzing enzyme linamarase. Cyanogenesis is commonly thought to function in herbivore defense; however, the individual cyanogenic components may also serve other physiological functions. To test whether cyanogenesis clines have evolved in response to the same selective pressures acting on the same genetic targets, we examined cyanogenesis cline shape and its environmental correlates in three world regions: southern New Zealand, the central United States and the US Pacific Northwest. For some regional comparisons, cline shapes are remarkably similar despite large differences in the spatial scales over which clines occur (40–1600 km). However, we also find evidence for major differences in both the agents and targets of selection among the sampled clines. Variation in cyanogenesis frequency is best predicted using a combination of minimum winter temperature and aridity variables. Together, our results provide evidence that recurrent adaptive clines do not necessarily reflect shared adaptive processes.     

Hydrogen cyanide production by Pseudomonas aeruginosa at reduced oxygen levels

Hydrogen cyanide production by Pseudomonas aeruginosa growing in a synthetic medium required aerobosis but operated efficiently at low dissolved oxygen concentration. Half maximum levels of cyanogenesis occurred at 0.015 microM oxygen; maximum cyanogenesis occurred over a wide range, 0.1-180 microM, of oxygen concentrations. These cells lost the ability to produce cyanide upon aerobic incubation in the absence of both the carbon energy source (L-glutamate) and the metabolic precursor of hydrogen cyanide (glycine). This loss of cyanogenesis was dependent on oxygen concentration; 1.0 microM oxygen produced no detectable loss, whereas 180 microM oxygen caused a rapid decline in cyanogenic ability. The endogenous cyanide production rate of cells in the presence of carbon energy source was not significantly influenced by oxygen concentration. During the batch culture cycle, the acquisition of the ability to produce HCN was preceded by oxygen reduction to growth-limiting levels. Cells which had lost the ability to produce hydrogen cyanide by oxygen treatment required protein synthesis before they could again become cyanogenic.     

Rapid evolution of an adaptive cyanogenesis cline in introduced North American white clover (Trifolium repens L.)

White clover is polymorphic for cyanogenesis (HCN production after tissue damage), and this herbivore defence polymorphism has served as a classic model for studying adaptive variation. The cyanogenic phenotype requires two interacting biochemical components; the presence/absence of each component is controlled by a simple Mendelian gene (Ac/ac and Li/li). Climate-associated cyanogenesis clines occur in both native (Eurasian) and introduced populations worldwide, with cyanogenic plants predominating in warmer locations. Moreover, previous studies have suggested that epistatic selection may act within populations to maintain cyanogenic (AcLi) plants and acyanogenic plants that lack both components (acli plants) at the expense of plants possessing a single component (Acli and acLi plants). Here, we examine the roles of selection, gene flow and demography in the evolution of a latitudinal cyanogenesis cline in introduced North American populations. Using 1145 plants sampled across a 1650 km transect, we determine the distribution of cyanogenesis variation across the central United States and investigate whether clinal variation is adaptive or an artefact of population introduction history. We also test for the evidence of epistatic selection. We detect a clear latitudinal cline, with cyanogenesis frequencies increasing from 11% to 86% across the transect. Population structure analysis using nine microsatellite loci indicates that the cline is adaptive and not a by-product of demographic history. However, we find no evidence for epistatic selection within populations. Our results provide strong evidence for rapid adaptive evolution in these introduced populations, and they further suggest that the mechanisms maintaining adaptive variation may vary among populations of a species.     

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.     

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.     

Human disturbance causes the formation of a hybrid swarm between two naturally sympatric fish species

Most evidence for hybrid swarm formation stemming from anthropogenic habitat disturbance comes from the breakdown of reproductive isolation between incipient species, or introgression between allopatric species following secondary contact. Human impacts on hybridization between divergent species that naturally occur in sympatry have received considerably less attention. Theory predicts that reinforcement should act to preserve reproductive isolation under such circumstances, potentially making reproductive barriers resistant to human habitat alteration. Using 15 microsatellites, we examined hybridization between sympatric populations of alewife (Alosa pseudoharengus) and blueback herring (A. aestivalis) to test whether the frequency of hybridization and pattern of introgression have been impacted by the construction of a dam that isolated formerly anadromous populations of both species in a landlocked freshwater reservoir. The frequency of hybridization and pattern of introgression differed markedly between anadromous and landlocked populations. The rangewide frequency of hybridization among anadromous populations was generally 0–8%, whereas all landlocked individuals were hybrids. Although neutral introgression was observed among anadromous hybrids, directional introgression leading to increased prevalence of alewife genotypes was detected among landlocked hybrids. We demonstrate that habitat alteration can lead to hybrid swarm formation between divergent species that naturally occur sympatrically, and provide empirical evidence that reinforcement does not always sustain reproductive isolation under such circumstances.     

Quantitative variability of cyanogenesis in Cathariostachys madagascariensis—the main food plant of bamboo lemurs in Southeastern Madagascar

Giant bamboo (Cathariostachys madagascariensis) is a major food plant for three sympatric species of bamboo‐eating lemurs (Hapalemur aureus, H. griseus, and Prolemur simus) in the rain forests of southeastern Madagascar. This plant species is strongly cyanogenic. However, quantitative data on cyanide concentration in C. madagascariensis are scarce. Previous studies reported 15 mg cyanide per 100 g fresh shoot material (corresponding to approx. 57 µmol cyanide per gram dry weight). However, we found mean concentrations (±SE) ranging from 139.3±19.32 in ground shoots to 217.7±16.80 µmol cyanide per gram dry weight in branch shoots. Thus, cyanogenesis of C. madagascariensis was up to four times higher than reported before. In contrast to the strongly cyanogenic shoots no cyanide could be detected in differently aged leaves of C. madagascariensis confirming earlier studies. Within individual shoots fine‐scaled analysis revealed a characteristic ontogenetic pattern of cyanide accumulation. Highest concentrations were found in youngest parts near the apical meristem, whereas concentrations decreased in older shoot parts. Beyond the general intra‐individual variability of cyanogenic features analyses indicated site‐specific variability of both, the ontogenetic pattern of cyanide concentration as well as the total amount of cyanide accumulated in shoots. Additionally, analyses of soluble proteins—one important nutritive measure affecting food plant quality—demonstrated a converse quantitative relation of protein concentrations in leaves to cyanide concentration in shoots at the site‐specific level. We, thus, suggest integrative analyses on quantitative variation of cyanogenesis together with nutritive plant parameters in future studies. This approach would allow obtaining more detailed insights into spatial variability of giant bamboo's overall browse quality and its impact on lemur herbivores. Am. J. Primatol. 71:305–315, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

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.     

Cyanogenic constituents in woody plants in natural lowland rain forest in Costa Rica

Screening of fresh leaves and other plant organs revealed the presence of cyanogenesis in 25 species in 16 families out of a total of 488 species in 79 families vouchered in natural lowland rain forest in Costa Rica. In a qualitative screening of a random sample based on seven one-hectare inventories of woody plants, we found cyanogenesis in 4.0% of all species; these represented 2.5% of all individuals present, and 3.0% of the total basal area of stems. The frequency of occurrence of cyanogenic compounds was higher in reproductive plant parts than in leaves, and highest in pericarps. Cyanogenesis was found in Annona amazonica, Annona pittieri, Cymbopetalum costaricense, Tabebuia chrysantha, Sloanea tuerckheimii, Sapium laurifolium, Lecointea amazonica, Carpotroche platyptera, Klayana odorata, Byrsonima crispa, Miconia splendens, Inga acuminata, Chaunochiton kappleri, Passiflora ambigua, P. pittieri, P. talamancensis, P. vitifolia, Panopsis costaricensis, Faramea pawibractea, Paullinia capreolata, Pouteria amygdalicarpa, P. campechiana, P. subrotata, P. torta and Rinorea guatemalensis. Some taxonomic implications are discussed. The identity of cyanogenic constituents found in dried material of these species is presented, as is the total cyanogenic potential (CNp) of 11 positive species. The CNp ranged from less than 5 to approx. 2000 mg kg^-1 f.w. Several species were cyanogenic only in some individuals. A total of 463 species gave no positive test for cyanogenic glycosides. Tests on leaves and seeds of Ampelocera macrocarpa indicated the content of a rare and unknown volatile constituent.     

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.