Replacement of benzidine by copper ethylacetoacetate and tetra base as spot-test reagent for hydrogen cyanide and cyanogen

Feigl, F.; Anger, V.

doi:10.1039/an9669100282

Cited by 186 publications

(103 citation statements)

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“…Plants were assayed for the presence/absence of cyanogenic constituents using a modified Feigl-Anger hydrogen cyanide (HCN) assay (Feigl and Anger 1966), as described by Olsen et al (2007). The presence/absence of functional alleles at Ac and Li can be inferred independently through the exogenous addition of either linamarase or linamarin, respectively, to an acyanogenic leaf sample (see Olsen et al 2007).…”

Section: Methodsmentioning

confidence: 99%

Evidence on the Molecular Basis of the Ac/ac Adaptive Cyanogenesis Polymorphism in White Clover (Trifolium repens L.)

2008

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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. C YANOGENESIS (cyanide release following tissue damage) occurs in .2650 plant species, including ferns, gymnosperms, monocots, and dicots (Seigler and Brinker 1993). In its most common form, cyanogenesis involves the interaction of two compounds, cyanogenic glycosides and their hydrolyzing enzymes; these are separated in intact tissue and brought into contact with tissue disruption. White clover (Trifolium repens L., Fabaceae) is polymorphic for cyanogenesis, with both cyanogenic and acyanogenic plants occurring in natural populations (Armstrong et al. 1913;Ware 1925). This polymorphism arises through the presence/absence of both of the underlying cyanogenic components. Inheritance of cyanogenesis in white clover follows a simple Mendelian two-locus, two-allele model (Coop 1940;Melville and Doak 1940;Corkill 1942): the gene Ac/ac controls the presence/absence of cyanogenic glucosides; and the independently segregating gene Li/li controls the presence/absence of linamarase, a cyanogenic b-glucosidase (reviewed by Hughes 1991). Plants that carry at least one dominant (functional) allele at both genes (Ac and Li) are cyanogenic, while the occurrence of two nonfunctional alleles at either gene confers the acyanogenic phenotype.White clover is a common legume of fields, lawns, and pastures in mesic temperate climates. It is a native of Eurasia but has been introduced worldwide as a forage crop and lawn plant. It is a perennial and an insectpollinated, obligate outcrosser; plants also spread vegetatively by stolons. Chromosome number and genetic map data indicate that white clover is an allotetraploid (e.g., Barrett et al. 200...

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Section: Methodsmentioning

confidence: 99%

Evidence on the Molecular Basis of the Ac/ac Adaptive Cyanogenesis Polymorphism in White Clover (Trifolium repens L.)

2008

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“…Cyanotypes were determined for sampled plants using a modified Feigl-Anger HCN assay as described elsewhere (Feigl and Anger, 1966;Olsen et al, 2007; see also Supplementary Methods). Linkage disequilibrium between Ac/ac and Li/li was calculated via Hill's maximum likelihood estimator (D; Hill, 1974) to test for evidence of epistatic selection within populations.…”

Section: Determination Of Cyanotypes and Inferred Cyanogenesis Allelementioning

confidence: 99%

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

Kooyers

Olsén

2013

Heredity

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Searching for the bull's eye: agents and targets of selection vary among geographically disparate cyanogenesis clines in white clover (Trifolium repens L.) NJ Kooyers 1 and KM OlsenThe 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 climateassociated 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.

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“…To enable processing of a large number of samples, we developed a microtiter plate-based screen for cyanide release using the cyanide-sensitive paper developed by Feigl and Anger (1966). Whatman 3MM paper is pretreated with a solution of tetra base (4,4'-methylenebis-(N,N-dimethylaniline)) and copper(II) ethylacetoacetate dissolved in chloroform.…”

Section: Isolation Of Cyanogenesis Deficient Mutants In L Japonicusmentioning

confidence: 99%

Genetic Screening Identifies Cyanogenesis-Deficient Mutants of Lotus japonicus and Reveals Enzymatic Specificity in Hydroxynitrile Glucoside Metabolism

et al. 2010

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Cyanogenesis, the release of hydrogen cyanide from damaged plant tissues, involves the enzymatic degradation of amino acid-derived cyanogenic glucosides (a-hydroxynitrile glucosides) by specific b-glucosidases. Release of cyanide functions as a defense mechanism against generalist herbivores. We developed a high-throughput screening method and used it to identify cyanogenesis deficient (cyd) mutants in the model legume Lotus japonicus. Mutants in both biosynthesis and catabolism of cyanogenic glucosides were isolated and classified following metabolic profiling of cyanogenic glucoside content. L. japonicus produces two cyanogenic glucosides: linamarin (derived from Val) and lotaustralin (derived from Ile). Their biosynthesis may involve the same set of enzymes for both amino acid precursors. However, in one class of mutants, accumulation of lotaustralin and linamarin was uncoupled. Catabolic mutants could be placed in two complementation groups, one of which, cyd2, encoded the b-glucosidase BGD2. Despite the identification of nine independent cyd2 alleles, no mutants involving the gene encoding a closely related b-glucosidase, BGD4, were identified. This indicated that BGD4 plays no role in cyanogenesis in L. japonicus in vivo. Biochemical analysis confirmed that BGD4 cannot hydrolyze linamarin or lotaustralin and in L. japonicus is specific for breakdown of related hydroxynitrile glucosides, such as rhodiocyanoside A. By contrast, BGD2 can hydrolyze both cyanogenic glucosides and rhodiocyanosides. Our genetic analysis demonstrated specificity in the catabolic pathways for hydroxynitrile glucosides and implied specificity in their biosynthetic pathways as well. In addition, it has provided important tools for elucidating and potentially modifying cyanogenesis pathways in plants.

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Replacement of benzidine by copper ethylacetoacetate and tetra base as spot-test reagent for hydrogen cyanide and cyanogen

Cited by 186 publications

References 0 publications

Evidence on the Molecular Basis of the Ac/ac Adaptive Cyanogenesis Polymorphism in White Clover (Trifolium repens L.)

Evidence on the Molecular Basis of the Ac/ac Adaptive Cyanogenesis Polymorphism in White Clover (Trifolium repens L.)

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

Genetic Screening Identifies Cyanogenesis-Deficient Mutants of Lotus japonicus and Reveals Enzymatic Specificity in Hydroxynitrile Glucoside Metabolism

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