Purification and characterization of cyanide hydratase from the phytopathogenic fungus Gloeocercospora sorghi.

Previous studies have demonstrated that fungal pathogens of cyanogenic plants produce cyanide hydratase (CHT, EC 4.2.1.66), which converts HCN to formamide. Production of CHT in these fungi is thought to be a means of circumventing cyanide toxicity, and CHT is thus believed to be an important pathogenicity trait. In the present study, 13 species of fungi were assayed for CHT production, and all 7 species that were pathogens of sorghum, a cyanogenic plant, produced this enzyme. CHT was purified to apparent homogeneity from one of these sorghum pathogens, Gloeocercospora sorghi. The enzyme had a Km of 12 mM for KCN. Enzymatically functional CHT was obtained only as a large molecular entity of greater than 300 kDa. However, a polypeptide of approximately 45 kDa was identified as the only component of purified CHT detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 45-kDa polypeptide band could be resolved into three isozymes of pI 6.1, 6.3, and 6.5. Antibodies raised against the 45-kDa polypeptide inhibited the G. sorghi CHT activity and showed high specificity in Western blots to a polypeptide of approximately the same size. The evidence suggests that functional G. sorghi CHT is an aggregated protein that consists of 45-kDa polypeptides. A CHT with similar properties was also found in the fungus Colletotrichum graminicola, another pathogen of sorghum.     

Cloning and properties of a cyanide hydratase gene from the phytopathogenic fungus Gloeocercospora sorghi.

The Cht gene encoding cyanide hydratase (CHT, EC 4.2.1.66), which detoxifies HCN and is thought to be important in fungal infection of cyanogenic plants, has been cloned from the phytopathogenic fungus Gloeocercospora sorghi. The gene was isolated by screening an expression library of G. sorghi using a CHT-specific antibody and using one of the positive cDNA clones as a probe in Southern hybridization to identify a 3.1 kb PstI genomic fragment. This PstI fragment expressed CHT activity when transformed into Aspergillus nidulans, a fungus that normally lacks CHT activity. Sequence analysis identified a single open reading frame of 1,107 base pairs which encodes a polypeptide of 40,904 daltons. The deduced amino acid sequence of CHT shares 36.5% identity to a nitrilase from the bacterium Klebsiella pneumoniae subsp. ozaenae.     

Relationship of Cyanogenic Capacity (HCN-c) of the Rubber Tree Hevea brasiliensis to Susceptibility to Microcyclus ulei, the Agent Causing South American Leaf Blight

Liberation of HCN from cyanogenic plant tissue depends on cyanogen content (HCN-potential), cyanogen splitting enzymes, cyanohydrin cleaving activity (hydroxynitrile lyase) and nonenzymatic cyanide detoxifying compounds. The maximal amount of HCN potentially set free is governed by the total cyanogen content, whereas the velocity of HCN liberation depends on enzymatic activities (β-glucosidase [β-G], hydroxynitrile lyase [HN]). Plants revealing a high HCN-potential and a high β-glucosidase activity generally are susceptible to infection with Microcyclus ulei. Based on the data of HCN-p and β-G activity of different Hevea species a proposal for future screening work in Hevea resistance selection is given.     

Volatile-mediated killing of Arabidopsis thaliana by bacteria is mainly due to hydrogen cyanide

The volatile-mediated impact of bacteria on plant growth is well documented, and contrasting effects have been reported ranging from 6-fold plant promotion to plant killing. However, very little is known about the identity of the compounds responsible for these effects or the mechanisms involved in plant growth alteration. We hypothesized that hydrogen cyanide (HCN) is a major factor accounting for the observed volatile-mediated toxicity of some strains. Using a collection of environmental and clinical strains differing in cyanogenesis, as well as a defined HCN-negative mutant, we demonstrate that bacterial HCN accounts to a significant extent for the deleterious effects observed when growing Arabidopsis thaliana in the presence of certain bacterial volatiles. The environmental strain Pseudomonas aeruginosa PUPa3 was less cyanogenic and less plant growth inhibiting than the clinical strain P. aeruginosa PAO1. Quorum-sensing deficient mutants of C. violaceum CV0, P. aeruginosa PAO1, and P. aeruginosa PUPa3 showed not only diminished HCN production but also strongly reduced volatile-mediated phytotoxicity. The double treatment of providing plants with reactive oxygen species scavenging compounds and overexpressing the alternative oxidase AOX1a led to a significant reduction of volatile-mediated toxicity. This indicates that oxidative stress is a key process in the physiological changes leading to plant death upon exposure to toxic bacterial volatiles.     

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.     

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.     

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.     

编码腈水解酶的大豆疫霉基因PsNIA的克隆与转录分析

包括大豆在内的许多植物都可以产生氰化物,对侵染的病原菌产生毒害作用而阻碍其进一步扩展。采用抑制性差减杂交(suppression subtractive hybridization,SSH)的方法,筛选到一个在大豆疫霉侵染早期上调表达的、编码腈水解酶的cDNA片段;克隆了该基因的全长序列,命名为PsNIA。Southern杂交结果显示,PsNIA在大豆疫霉基因组中只有1个拷贝。系统发育分析表明,PsNIA与绿脓杆菌Pseudomonas aeruginosa的腈水解酶的序列同源性最高,且该基因编码的氨基酸序列具有腈水解酶的保守结构域。RT-PCR分析表明,该基因在大豆疫霉侵染大豆12h时可以检测到转录。     

Cyanide action in plants — from toxic to regulatory

Recent biochemical and genetic studies on hydrogen cyanide (HCN) metabolism and function in plants were reviewed. The potential sources of endogenous cyanide and the pathways of its detoxification are outlined and the possible signaling routes by which cyanide exerts its physiological effects are discussed. Cyanide is produced in plant tissues as the result of hydrolysis of cyanogenic compounds and is also released as a co-product of ethylene biosynthesis. Most cyanide produced in plants is detoxified primarily by the key enzyme β-cyanoalanine synthase. The remaining HCN at non-toxic concentration may play a role of signaling molecule involved in the control of some metabolic processes in plants. So, HCN may play a dual role in plants, depending on its concentration. It may be used in defense against herbivores at high toxic concentration and may have a regulatory function at lower concentration. Special attention is given to the action of HCN during biotic and abiotic stresses, nitrate assimilation and seed germination. Intracellular signaling responses to HCN involve enhancement of reactive oxygen species (ROS) generation and the expression of cyanide-insensitive alternative oxidase (AOX) and ACC synthase (ACS) genes. The biochemical and cellular mechanisms of these responses are, however, not completely understood.     

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     

Genomic clustering of cyanogenic glucoside biosynthetic genes aids their identification in Lotus japonicus and suggests the repeated evolution of this chemical defence pathway

Cyanogenic glucosides are amino acid-derived defence compounds found in a large number of vascular plants. Their hydrolysis by specific β-glucosidases following tissue damage results in the release of hydrogen cyanide. The cyanogenesis deficient1 (cyd1) mutant of Lotus japonicus carries a partial deletion of the CYP79D3 gene, which encodes a cytochrome P450 enzyme that is responsible for the first step in cyanogenic glucoside biosynthesis. The genomic region surrounding CYP79D3 contains genes encoding the CYP736A2 protein and the UDP-glycosyltransferase UGT85K3. In combination with CYP79D3, these genes encode the enzymes that constitute the entire pathway for cyanogenic glucoside biosynthesis. The biosynthetic genes for cyanogenic glucoside biosynthesis are also co-localized in cassava (Manihot esculenta) and sorghum (Sorghum bicolor), but the three gene clusters show no other similarities. Although the individual enzymes encoded by the biosynthetic genes in these three plant species are related, they are not necessarily orthologous. The independent evolution of cyanogenic glucoside biosynthesis in several higher plant lineages by the repeated recruitment of members from similar gene families, such as the CYP79s, is a likely scenario.     

Cyanogenesis by the entomopathogenic bacterium Pseudomonas entomophila

Aims:  To investigate whether the entomopathogenic bacterium Pseudomonas entomophila can synthesize hydrogen cyanide (HCN).
Methods and Results:  Cyanide production was assayed for during the growth of P. entomophila in liquid culture and during colonial growth. Pseudomonas entomophila produced HCN at a concentration of up to 40 μmol l⁻¹ during growth in liquid cultures and its production was found to be affected by oxygen availability, with levels increasing as the oxygen-transfer coefficient decreased. Pseudomonas entomophila made HCN during colonial growth at levels greater (approximately threefold) than those made by the well studied cyanogenic bacterium Pseudomonas aeruginosa .
Conclusions:  This study demonstrated unequivocally that P. entomophila can synthesize HCN, placing it among the small number of cyanogenic bacteria. Our data indicate that HCN production in P. entomophila is regulated by oxygen availability.
Significance and Impact of the Study:  Pseudomonas entomophila was recently identified to be the only pseudomonad that naturally infects and induces lethality of Drosophila melanogaster . The virulence factors which contribute to entomopathogenicity exerted by this species are largely unknown. In this study, we demonstrate that P. entomophila produces HCN, a secondary metabolite implicated in biocontrol properties and pathogenicity exerted by other bacteria.     

Changes in endogenous cyanide and 1-aminocyclopropane-1-carboxylic acid levels during the hypersensitive response of tobacco mosaic virus-infected tobacco leaves

Leaves of Nicotiana tabacum L. cv. Xanthi necroticum plants form local necrotic lesions at the site of infection by tobacco mosaic virus. During the first seven days post-inoculation, endogenous levels of 1-aminocyclopropane-1-carboxylic acid (ACC) and N-malonyl-ACC increased in the lesion area. The time course of ACC accumulation coincided with an increase in the endogenous cyanide level which began within two days after inoculation. Concomitantly, the activity of -cyanoalanine synthase, the main HCN detoxifying enzyme, decreased. Likewise, treatment of leaf discs of uninfected plants with ACC led to cyanide accumulation. Exogenously applied KCN caused necrotic spots on tobacco leaves very similar to the whitish centers of virus-induced local lesions. Possible implications of cyanide in cell death during TMV-induced lesion development are discussed.     

α-Hydroxynitrile lyase in Hevea brasiliensis and its significance for rapid cyanogenesis

The hydroxynitrile lyase (HNL, EC 4.2.1.-) of Hevea brasiliensis (Muell.-Arg.) catalyzes the dissociation of acetone cyanohydrin and mandelonitrile, but shows higher activity towards the natural substrate acetone cyanohydrin. The ratio between the activities of linamarase (β-glycosidase, EC 3.2.1.21) to HNL was screened for more than 30 Hevea plants. In mixed-enzyme incubations various ratios of HNL to β-glucosidase were analyzed for the rapidity of HCN liberation. Addition of HNL increased the rate of HCN liberation up to 20-fold, thus demonstrating the significance of the HNL for rapid cyanogenesis. Its physiological importance is shown by the fact that only plants possessing high HNL activity are able to liberate HCN efficiently. Cyanogenic plants have been described as being weakly or strongly cyanogenic depending on the total amount of HCN which is potentially liberated. The data presented in this paper suggest that cyanogenic plants should also be differentiated as fast or slow cyanogenic according to the observed velocity of HCN liberation. Thus, for evaluating the repellent action of cyanogenic plants not only the final level of the HCN liberated is important but rather the rate with which this level is reached.     

Hydrogen cyanide release during feeding of generalist and specialist lepidopteran larvae on a cyanogenic plant, Passiflora capsularis

Abstract.  The hydrogen cyanide-based interaction of a strongly cyanogenic plant, Passiflora capsularis , and larvae of two insect herbivores, a generalist ( Spodoptera frugiperda ) and a specialist ( Heliconius erato ), is examined in terms of the combined kinetics of the feeding process and the simultaneous hydrogen cyanide (HCN) liberation, as compared with the natural kinetics of hydrogen cyanide evolution by plant-leaf tissue. There are marked differences in acceptance of P. capsularis by third-instar larvae of specialist and generalist species. The former, H. erato , display a parsimonious ingestion rate of 0.74 ± 0.15 mg (fresh weight) min⁻¹ comprising 18% active feeding time, whereas S. frugiperda larvae show a more erratic and restrained feeding involving 4% of the time at 0.45 ± 0.14 mg min⁻¹. These S. frugiperda larvae ingest 124.4 ± 8.3 mg (fw) of the non-cyanogeneic Spinacia oleracea leaves in 24 h compared with only 74.7 ± 20.1 mg of P. capsularis in the same period. The total hydrogen cyanide released naturally from wild specimens of P. capsularis plants is in the range 326–3901 μg g⁻¹. Hydrogen cyanide evolution from macerated P. capsularis leaves takes place along a hyperbolic function with time and initial velocities of cyanide evolution are a linear function of total hydrogen cyanide. When feeding on P. capsularis leaves, H. erato releases only a minor fraction relative to total hydrogen cyanide (0.09%) and to the anticipated cyanide from the initial velocity (7%). By contrast, S. frugiperda evokes 5.8-fold more than the anticipated hydrogen cyanide release from the plant. The findings are interpreted as diverging strategies by generalist and specialist insects in the utilization of hydrogen cyanide in cyanogenic plants.