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.     

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.     

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.     

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.     

Biochemical characterisation of the Li locus,which controls the activity of the cyanogenic β-glucosidase in Trifolium repens L.

The cyanogenic -glucosidase (linamarase) was purified from white clover leaf tissue. The enzyme is a homodimer with a molecular weight of 105 300–103 400 daltons estimated from molecular exclusion chromatography. The effect of buffer ions on the pH optimum and charge properties of the enzyme are presented. A combination of molecular exclusion chromatography and CM cellulose ion exchange chromatography purified linamarase 16 fold to a single 62 000 dalton polypeptide on SDS polyacrylamide gel electrophoresis. This polypeptide represented about 5% of the total soluble leaf protein and can be seen as a prominent band in SDS polyacrylamide gel electrophoresis of crude leaf extracts from Li Li plants. Screening backcross progeny showed that extracts from li li plants, which have no linamarase activity, lack this 62 000 dalton polypeptide. Linamarase is the major glycoprotein in white clover leaf extracts which binds to Concanavalin A-Sepharose.     

Detection of selection in populations of white clover (Trifolium repens L.)

Seed populations of white clover polymorphic for the presence/absence of both ovariogenic glucosides and the hydrolysing enzyme linamarase, were introduced into three natural populations. Over the first six months of life a significant increase in the frequency of linamarase containing individuals occurred. Estimated selection coefficients against plants lacking linamarase were in the region of 0.3. This result may have been due to selection at the enzyme locus alone, or to selection favouring cyanogenic individuals which possess both cyanogenic glucosides and enzyme.     

Restriction fragment length polymorphism segregation analysis of the Li locus in Trifolium repens L.

The Li locus in white clover controls the presence of cyanogenic -glucosidase (linamarase) activity in leaf tissue, such that plants homozygous for the null allele (li) have no linamarase activity in this tissue. The isolation of a cDNA clone from linamarase mRNA is described. The cDNA clone is used to further characterise alleles of the Li locus. Northern blot analysis shows that plants homozygous for the null allele (li li) produce very reduced levels of mRNA which hybridises to the cDNA. Heterozygous plants (Li li), which have intermediate levels of enzyme activity, produce intermediate levels of mRNA. Southern blot analysis of Hind III digested genomic DNA shows that the white clover genome contains three genes with homology to the linamarase cDNA and that at least two of these genes segregate independently. Analysis of the cosegregation of linamarase activity and the presence of genomic restriction fragments identifies the genomic sequence specifying linamarase structure and indicates either a structural or cis acting control function of the Li locus.     

Synthesis of the cyanogenic beta-glucosidase, linamarase, in white clover

The beta-glucosidase, linamarase, which specifically hydrolyzes cyanogenic substrates, linamarin and lotaustralin, in white clover, is synthesized in the early stages of leaf and seedling development in genetically competent plants. Plants, from natural populations, possessing at least one Li allele synthesize linamarase but plants with only li alleles do not, nor do they produce inactive but antigenically related linamarase. Linamarase is known to be a mannosyl glycoprotein, which in its active form is a dimer, with a subunit size of 62,000 Mr. We demonstrate that the antibiotic tunicamycin, which prevents N-acetyl-asparagine linked glycosylation, reduces in vivo synthesis of linarmarase. In vitro translation of mRNA from a Li Li plant yields a 59,000 Mr immunoprecipitated linamarase polypeptide which is modified to a 62,000 Mr product by the addition of dog pancreas microsomes. No anti-linamarase immunoprecipitable product is obtained from the in vitro translation products of mRNA from a li li plant.     

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.     

An analysis of the costs and benefits of the cyanogenic system in Trifolium repens L.

Summary The effect of the cyanogenic glucosides linamarin and lotaustralin and their hydrolyzing enzyme linamarase was studied in a B₂ generation segregating for the genes Ac and Li. Plants containing the glucosides are protected against grazing by snails both in the seedling stage and as adult plants. In seedlings, however, there is a direct effect on survival, whereas in adult plants the leaf area of plants containing linamarin/lotaustralin is less reduced under intense grazing. Linamarase has no effect on grazing by snails, possibly as a result of the presence of -glucosidase activity in the gut of these animals. The genes Ac and Li, or genes tightly linked to them, have other effects as well: plants possessing one dominant Ac allele produce fewer flowers than homozygous ac plants. I compared this difference in flower production to the metabolic cost of producing the cyanogenic glucosides. The energy content of the difference in flower head production far exceeded the metabolic cost of cyanoglucoside production in Acac plants. It is possible that the cost of maintaining a certain level of cyanoglucosides is much more important for the plant than the initial cost of biosynthesis. The importance of the effects of Ac and Li in the maintenance of cyanogenic polymorphism in white clover is discussed.     

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.     

In-situ localization of cyanogenic β-glucosidase (linamarase) gene expression in leaves of cassava (Manihot esculenta Cranz) using non-isotopic riboprobes

The release of hydrogen cyanide (cyanogenesis) from damaged plant tissue depends upon the sequential action of a β-glucosidase and an α-hydroxynitrilase on cyanoglucosides. The non-isotopic digoxigenin labelling system was used to visualize the presence of cyanogenic β-glucosidase (linamarase) mRNA in cells of young leaves of Manihot esculenta Cranz (cassava). Strong hybridization to antisense riboprobes produced from the cDNA clone pCAS5, indicates localization of linamarase gene expression in laticifers (latex vessels). This is supported by the demonstration of linamarase mRNA in exuded latex. In contrast, in-situ localization of the control gene pGLF4, showed expression in all leaf mesophyll cells. High levels of linamarase activity were demonstrated in the latex of leaf petioles and this activity was shown to be dependent on the presence of attached leaflets. Assays of α-hydroxynitrilase activity in exuded latex and whole leaves shows that, unlike linamarase, this enzyme is present at very low levels in latex and must be located elsewhere in the leaf.     

Developmental and Physiological Studies on the Cyanogenic Glucosides of White Clover, Trifolium repens L.

Cyanogenesis in Trifolium repens L. is under the control oftwo loci; Ac/ac and Li/Hi control cyanogenic glucoside and linamaraseproduction respectively. Results obtained show that neitherthe dominant allele (Ac) coding for cyanogenic glucoside productionnor the dominant allele (Li) coding for linamarase productionare expressed in roots, seeds or seedlings before shoot emergence.Both linamarase and cyanogenic glucoside are produced duringshoot growth and there is little turnover of cyanogenic glucosidein mature leaves. As the leaves senesce there is breakdown ofthe mechanism separating cyanogenic glucoside and linamarase,since cyanogenic glucoside is lost in plants of genotype AcAc Li Li but not in those of genotype Ac Ac Li Li. About 60%of the cyanogenic glucoside produced was lotaustralin, in shootsof plants which were fed with equal quantities of the precursoramino acids L-valine and L-isoleucine. In contrast, the proportionof cyanogenic glucoside as lotaustralin found in leaves of oneplant, was only 40%. Different plants were shown to producedifferent quantities of cyanogenic glucoside, and the amountproduced was dependent on temperature.     

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.     

Cyanotypic frequencies in adjacent and mixed populations of Trifolium occidentale Coombe and Trifolium repens L. are regulated by different mechanisms

The cyanogenic polymorphism in Trifolium repens is caused by the variation in two genes, the interaction of which produces four distinct cyanotypes. Along the Atlantic coasts of Bretagne, T. repens is sometimes found in populations mixed with the related species Trifolium occidentale, although the latter species usually occurs only in a narrow fringe along the coast, whereas T. repens is a more inland species. No plants of T. occidentale have ever been reported to have linamarase activity. Indeed, of 763 T. occidentale plants studied, none contained linamarase activity. However, the variation in the proportion of cyanotypes in T. repens was enormous, even between sites less than 2km apart. Our results confirm the presumption that T. repens and T. occidentale are indeed separate species. Both the fact that T. occidentale plants never contain linamarase activity, and the difference in proportion of plants with cyanoglucosides in mixed stands show that gene flow between the species must be rare. These dissimilar distributions strongly indicate that cyanotypic frequencies in adjacent and mixed populations of the very closely related species T. occidentale and T. repens are regulated by different mechanisms     

REVIEW ARTICLE: Cyanogenesis in cassava (Manihot esculenta Crantz)

Cassava is the most agronomically important of the cyanogeniccrops. Linamarin, the predominant cyanogenic glycoside in cassava,can accumulate to concentrations as high as 500 mg kg^–1fresh weight in roots and to higher levels in leaves. Recently,the pathway of linamarin synthesis and the cellular site oflinamarin storage have been determined. In addition, the cyanogenicenzymes, linamarase and hydroxynitrile lyase, have been characterizedand their genes cloned. These results, as well as studies onthe organ- and tissue-specific localization of linamarase andhydroxy-nitrile lyase, allow us to propose models for the regulationof cyanogenesis in cassava. There remain, however, many unansweredquestions regarding the tissue-specific synthesis, transport,and accumulation of cyanogenic glycosides. The resolution ofthe sequestions will facilitate the development of food processing,biochemical and transgenic plant approaches to reducing thecyanogen content of cassava foods. Key words: Cyanide, cyanogenic glycosides, linamarin, cyanogens     

Environmental factors affecting the cyanogenic potential of flax seedlings

The levels of cyanogenic glucosides (linamarin and lotaustralin) and the activity of linamarase were studied in 5-day old seedlings of oil flax (Linum usitatissimum L., cv. LCSD 200) under different environmental conditions. White light enhanced the cyanoglucosides content, and this effect depended on its intensity and the time of exposure. The level of cyanoglucosides rose with temperature, and it reached the highest level at the highest temperature (30 °C). Linamarase (EC. 3.2.1.21) activity was the highest at 20°C, especially in light-grown seedlings. Lower enzyme activity at the extreme temperature (15 and 30 °C) was observed. Water stress (low water potential, ω=−0.34 MPa) reduced by more than twice the cyanoglucoside level and linamarase activity. The possible protective, or/and regulatory roles of cyanogenic glucosides was discussed.     

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.     

Studies on Variability in White Clover: Growth Habits and Cyanogenic Glucosides

Growth habits and cyanogenesis were studied in a field experimentwith white clover (Trifolium repens L.) cv. Huia. Eighty controlplants propagated by seeds, and 80 clones of an in vivo selectedvariant were examined in mid-late August and late September.The temperature effect on cyanogenic glucoside levels was alsoexamined on in vitro grown plantlets of the variant in growthchambers. Results showed that the upright growth habit dominated in controlplants. The population of the variant plants was mainly composedof prostrate clones and a transition from prostrate to uprightgrowth habit occurred. Considerable variation was observed withregard to all measured morphological characters in both controland variant plants. Great variation was also noted in cyanogenicglucoside content in the leaf laminae of the control plants.Low cyanogenic plants, on the other hand, dominated in the variant.The number of low and high cyanogenic plants increased in thecontrol by the end of the growth season, after the first frosts.A one-way shift of cyanogenic (slight increase) was recordedin the variant. Similar levels of cyanogenic glucoside and linamarasewere determined in the in vitro and field-grown plants. Cyanogenicglucoside content slightly decreased with the age of the invitro plantlets, but variation in temperature did not causeany changes in their level. The response to the environmentalchanges, regarding cyanogenesis, appear to be genetically determined. Trifolium repens, in vitro, somaclonal variation, cyanogenic glucosides