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Cyanogenic glucosides are specialized metabolites produced by over 3000 species of higher plants from more than 130 families. The deployment of cyanogenic glucosides is influenced by biotic and abiotic factors in addition to being developmentally regulated, consistent with their roles in plant defense and stress mitigation. Despite their ubiquity, very little is known regarding the molecular mechanisms that regulate their biosynthesis. The biosynthetic pathway of dhurrin, the cyanogenic glucoside found in the important cereal crop sorghum (Sorghum bicolor (L.) Moench), was described over 20 years ago, and yet no direct regulator of the biosynthetic genes has been identified. To isolate regulatory proteins that bind to the promoter region of the key dhurrin biosynthetic gene of sorghum, SbCYP79A1, yeast one-hybrid screens were performed. A bait fragment containing 1204 base pairs of the SbCYP79A1 5′ regulatory region was cloned upstream of a reporter gene and introduced into Saccharomyces cerevisiae. Subsequently, the yeast was transformed with library cDNA representing RNA from two different sorghum developmental stages. From these screens, we identified SbGATA22, an LLM domain B-GATA transcription factor that binds to the putative GATA transcription factor binding motifs in the SbCYP79A1 promoter region. Transient assays in Nicotiana benthamiana show that SbGATA22 localizes to the nucleus. The expression of SbGATA22, in comparison with SbCYP79A1 expression and dhurrin concentration, was analyzed over 14 days of sorghum development and in response to nitrogen application, as these conditions are known to affect dhurrin levels. Collectively, these findings suggest that SbGATA22 may act as a negative regulator of SbCYP79A1 expression and provide a preliminary insight into the molecular regulation of dhurrin biosynthesis in sorghum. [ABSTRACT FROM AUTHOR]

Context: Wild sorghum taxa (Sorghum Moench) cover much of the northern Australian landscape and harbour potentially useful traits for crop improvement. Specialised metabolites such as phenolics, silicon and cyanogenic glucosides (which are toxic to grazing animals) are present in Sorghum and have been associated with drought tolerance. However, these taxa remain poorly studied, especially regarding intraspecific variation. Aims: To investigate the intraspecific diversity of wild sorghums growing along aridity clines, including morphology, biochemistry and genotype. We hypothesised that genotypic and phenotypic diversity would co-vary with niche diversity. Methods: Multiple accessions of Sorghum plumosum , S. stipoideum and S. timorense were grown under common conditions for 10 weeks. Concentrations of cyanogenic glucosides, phenolics and silicon were measured alongside five morphological traits. Accessions were genotyped using single-nucleotide polymorphisms. Key results: Several traits varied significantly within species, but no trait consistently co-varied with aridity across species. In S. timorense , there appeared to be genotypic differentiation among accessions, but this was not translated into phenotypic differences, which was likely to be due to similarities in climate. Conclusions: Our results challenge assumptions that phenotypic and genotypic diversity correlate with niche diversity and support the hypothesis that mature Australian wild sorghums store most of their cyanogenic glucosides in their roots. Implications: Wild sorghums harbour advantageous traits for crop improvement, such as niche diversity and low toxicity. Future wild sorghum research must acknowledge intraspecific diversity, and as many populations as possible should be conserved, because genetic diversity is not necessarily discernible from phenotype or provenance alone. Accessions of three wild sorghum species collected in Australia. Wild Sorghum species dominate parts of northern Australia and contain potentially useful traits for crop improvement, but the diversity within these species has not been sufficiently examined. We assessed phenotypic and genotypic diversity of three Sorghum species and found unexpected complexity in the relationships among the environment, genotype and phenotype. Our results challenge assumptions that diversity levels can be assumed from phenotype or environment alone, with implications for conservation and crop improvement. Photograph by Jack McCausland. [ABSTRACT FROM AUTHOR]

Cyanogenic glucosides are specialized metabolites produced by over 3000 species of higher plants from more than 130 families. The deployment of cyanogenic glucosides is influenced by biotic and abiotic factors in addition to being developmentally regulated, consistent with their roles in plant defense and stress mitigation. Despite their ubiquity, very little is known regarding the molecular mechanisms that regulate their biosynthesis. The biosynthetic pathway of dhurrin, the cyanogenic glucoside found in the important cereal crop sorghum (Sorghum bicolor (L.) Moench), was described over 20 years ago, and yet no direct regulator of the biosynthetic genes has been identified. To isolate regulatory proteins that bind to the promoter region of the key dhurrin biosynthetic gene of sorghum, SbCYP79A1, yeast one-hybrid screens were performed. A bait fragment containing 1204 base pairs of the SbCYP79A1 5′ regulatory region was cloned upstream of a reporter gene and introduced into Saccharomyces cerevisiae. Subsequently, the yeast was transformed with library cDNA representing RNA from two different sorghum developmental stages. From these screens, we identified SbGATA22, an LLM domain B-GATA transcription factor that binds to the putative GATA transcription factor binding motifs in the SbCYP79A1 promoter region. Transient assays in Nicotiana benthamiana show that SbGATA22 localizes to the nucleus. The expression of SbGATA22, in comparison with SbCYP79A1 expression and dhurrin concentration, was analyzed over 14 days of sorghum development and in response to nitrogen application, as these conditions are known to affect dhurrin levels. Collectively, these findings suggest that SbGATA22 may act as a negative regulator of SbCYP79A1 expression and provide a preliminary insight into the molecular regulation of dhurrin biosynthesis in sorghum.

Plants and phytophagous arthropods have coevolved in a long battle for survival. Plants respond to phytophagous feeders by producing a battery of antiherbivore chemical defences, while herbivores try to adapt to their hosts by attenuating the toxic effect of the defence compounds. Cyanogenic glucosides are a widespread group of defence chemicals that come from cyanogenic plants. Among the non-cyanogenic ones, the Brassicaceae family has evolved an alternative cyanogenic pathway to produce cyanohydrin as a way to expand defences. When a plant tissue is disrupted by an herbivore attack, cyanogenic substrates are brought into contact with degrading enzymes that cause the release of toxic hydrogen cyanide and derived carbonyl compounds. In this review, we focus our attention on the plant metabolic pathways linked to cyanogenesis to generate cyanide. It also highlights the role of cyanogenesis as a key defence mechanism of plants to fight against herbivore arthropods, and we discuss the potential of cyanogenesis-derived molecules as alternative strategies for pest control. [ABSTRACT FROM AUTHOR]

Seed germination is crucial for plant productivity, and the biochemical changes during germination affect seedling survival, plant health and yield. While the general metabolism of germination is extensively studied, the role of specialized metabolism is less investigated. We therefore analyzed the metabolism of the defense compound dhurrin during sorghum (Sorghum bicolor) grain germination and early seedling development. Dhurrin is a cyanogenic glucoside, which is catabolized into different bioactive compounds at other stages of plant development, but its fate and role during germination is unknown. We dissected sorghum grain into three different tissues and investigated dhurrin biosynthesis and catabolism at the transcriptomic, metabolomic and biochemical level. We further analyzed transcriptional signature differences of cyanogenic glucoside metabolism between sorghum and barley (Hordeum vulgare), which produces similar specialized metabolites. We found that dhurrin is de novo biosynthesized and catabolized in the growing embryonic axis as well as the scutellum and aleurone layer, two tissues otherwise mainly acknowledged for their involvement in release and transport of general metabolites from the endosperm to the embryonic axis. In contrast, genes encoding cyanogenic glucoside biosynthesis in barley are exclusively expressed in the embryonic axis. Glutathione transferase enzymes (GSTs) are involved in dhurrin catabolism and the tissue-resolved analysis of GST expression identified new pathway candidate genes and conserved GSTs as potentially important in cereal germination. Our study demonstrates a highly dynamic tissue- and species-specific specialized metabolism during cereal grain germination, highlighting the importance of tissue-resolved analyses and identification of specific roles of specialized metabolites in fundamental plant processes. • Dhurrin is de novo biosynthesized and catabolized in all sorghum grain sub-tissues. • Cyanogenic glucoside metabolism during grain germination is species-specific. • Monocot-specific glutathione transferases are highly expressed during germination. [ABSTRACT FROM AUTHOR]

This study aimed to determine the effect of fermentation using an isolated Levilactobacillus brevis strain on the antinutrients and the protein quality of cassava leaves. Cassava leaves from tajirika and kibandameno variety which are considered bitter and sweet varieties, respectively, were fermented using the probiotic isolate and spontaneous fermentation was used as a control. The antinutrients of interest were cyanide and phytic acid whereas the protein quality of the leaves was analyzed in terms of the total crude protein and in-vitro digestibility. These were analyzed using standard procedures namely; picrate paper method for cyanide, spectrophotometry for phytic acid, Kjedahl method for the crude protein, and trypsin and pepsin enzymes simulation for the actual human digestion process for the in vitro protein digestibility. The data obtained was analyzed using the SPSS software and means separated at p 37% with the L. brevis isolate for both cassava varieties which was higher than 20–33% for the spontaneous fermentation (control). The crude protein in both the varieties ranged between 28% and 32% and improved up to 43% on fermentation. Upon digestion of the cassava leaves, the in vitro protein digestibility improved from 63% up to 94%. The findings of this study therefore indicates that the Levilactobacillus brevis isolate can reduce cyanide and phytic acid as well as improve the protein quality by increasing the crude protein content and enhancing the digestibility of protein in cassava leaves of kibandameno and tajirika varieties, hence can be used to detoxify cassava leaves and for food formulation.

Domesticated sorghum (Sorghum bicolor [L.] Moench subsp. bicolor) diverts significant amounts of nitrogen away from primary metabolism to the synthesis of cyanogenic glucosides (CNglc) – specialized metabolites that release toxic hydrogen cyanide (HCN). Our aim was to identify the point in the genus Sorghum Moench at which plants gained the ability to maintain hazardous concentrations of cyanogenic glucosides in their leaves into maturity (HCN potential >0.4 mg g−1). This ability occurs in domesticated sorghum (in the subgenus Eusorghum), but not in wild taxa in other Sorghum subgenera.Eight accessions from the subgenus Eusorghum were grown in a common garden: an improved sorghum line, five sorghum landraces, the crop's wild progenitor (S. bicolor subsp. verticilliflorum [Steud.] de Wet ex Wiersema & J. Dahlb.) and wild Sorghum propinquum (Kunth) Hitchc. HCN potential was measured in plants (n = 80) at the three‐leaf stage and at 6 weeks old.All study accessions, including the wild taxa, had hazardous CNglc concentrations in the leaves at both the three‐leaf stage (mean HCN potentials > = 2.5 mg g−1) and at 6 weeks old (mean HCN potentials > = 0.68 mg g−1), greatly contrasting the much lower mature leaf HCN potentials previously found in wild Sorghum taxa outside subgenus Eusorghum (generally <= 0.01 mg g−1).Our results suggest that the ability to maintain hazardous leaf HCN potentials into maturity might have arisen during the divergence of Eusorghum from other Sorghum subgenera, rather than during the speciation or domestication of S. bicolor, and highlights the value of utilizing Sorghum taxa outside Eusorghum in efforts to improve the crop safety of sorghum. [ABSTRACT FROM AUTHOR]

The basic helix-loop-helix (bHLH) proteins are a large superfamily of transcription factors, and play a central role in a wide range of metabolic, physiological, and developmental processes in higher organisms. However, systematic investigation of bHLH gene family in cassava (Manihot esculenta Crantz) has not been reported. In the present study, we performed a genome-wide survey and identified 148 MebHLHs genes were unevenly harbored in 18 chromosomes. Through phylogenetic analyses along with Arabidopsis counterparts, these MebHLHs genes were divided into 19 groups, and each gene contains a similar structure and conserved motifs. Moreover, many cisacting regulatory elements related to various defense and stress responses showed in MebHLH genes. Interestingly, transcriptome data analyses unveiled 117 MebHLH genes during postharvest physiological deterioration (PPD) process of cassava tuberous roots, while 65 MebHLH genes showed significantly change. Meanwhile, the relative quantitative analysis of 15 MebHLH genes demonstrated that they were sensitive to PPD, suggesting they may involve in PPD process regulation. Cyanogenic glucosides (CGs) biosynthesis during PPD process was increased, silencing of MebHLH72 and MebHLH114 showed that linamarin content was significantly decreased in the leaves. To summarize, the genome-wide identification and expression profiling of MebHLH candidates pave a new avenue for uderstanding their function in PPD and CGs biosynthesis, which will accelerate the improvement of PPD tolerance and decrease CGs content in cassava tuberous roots. [ABSTRACT FROM AUTHOR]

Aposematic animals advertise their toxicity or unpalatability with bright warning coloration. However, acquiring and maintaining chemical defenses can be energetically costly, and consequent associations with other important traits could shape chemical defense evolution. Here, we have tested whether chemical defenses are involved in energetic trade‐offs with other traits, or whether the levels of chemical defenses are condition dependent, by studying associations between biosynthesized cyanogenic toxicity and a suite of key life‐history and fitness traits in a Heliconius butterfly under a controlled laboratory setting. Heliconius butterflies are well known for the diversity of their warning color patterns and widespread mimicry and can both sequester the cyanogenic glucosides of their Passiflora host plants and biosynthesize these toxins de novo. We find energetically costly life‐history traits to be either unassociated or to show a general positive association with biosynthesized cyanogenic toxicity. More toxic individuals developed faster and had higher mass as adults and a tendency for increased lifespan and fecundity. These results thus indicate that toxicity level of adult butterflies may be dependent on individual condition, influenced by genetic background or earlier conditions, with maternal effects as one strong candidate mechanism. Additionally, toxicity was higher in older individuals, consistent with previous studies indicating accumulation of toxins with age. As toxicity level at death was independent of lifespan, cyanogenic glucoside compounds may have been recycled to release resources relevant for longevity in these long‐living butterflies. Understanding the origins and maintenance of variation in defenses is necessary in building a more complete picture of factors shaping the evolution of aposematic and mimetic systems. [ABSTRACT FROM AUTHOR]

In 2009, Food Standards Australia New Zealand set a total cyanide content limit of 10 ppm for ready-to-eat cassava products to address food safety concerns about cyanogenic glucosides in cassava. This study surveys a range of cassava food products available in Melbourne, Australia, ten years after the implementation of these regulations. Of all the products tested, the mean cyanide content was greatest in ready-to-eat cassava chips (48.4 ppm), although imported ready-to-eat products had a higher mean cyanide content (95.9 ppm) than those manufactured in Australia (1.0 ppm). Cyanide was detected in frozen cassava products (grated mean = 12.9 ppm; whole root mean = 19.8 ppm), but was significantly reduced through processing according to packet instructions in both product types. Three methods were used to quantify total cyanide content: the evolved cyanide method, the picrate absorbance method and the picrate chart method, with satisfactory agreement between methods. The picrate absorbance and chart methods reported mean cyanide contents 13.7 ppm and 23.1 ppm higher, respectively, than the evolved cyanide method. Our results reaffirm the need for the ongoing testing of cassava food products, especially ready-to-eat products whose cyanide content will not be reduced before consumption. [ABSTRACT FROM AUTHOR]

Abstract Aposematic animals advertise their toxicity or unpalatability with bright warning coloration. However, acquiring and maintaining chemical defenses can be energetically costly, and consequent associations with other important traits could shape chemical defense evolution. Here, we have tested whether chemical defenses are involved in energetic trade‐offs with other traits, or whether the levels of chemical defenses are condition dependent, by studying associations between biosynthesized cyanogenic toxicity and a suite of key life‐history and fitness traits in a Heliconius butterfly under a controlled laboratory setting. Heliconius butterflies are well known for the diversity of their warning color patterns and widespread mimicry and can both sequester the cyanogenic glucosides of their Passiflora host plants and biosynthesize these toxins de novo. We find energetically costly life‐history traits to be either unassociated or to show a general positive association with biosynthesized cyanogenic toxicity. More toxic individuals developed faster and had higher mass as adults and a tendency for increased lifespan and fecundity. These results thus indicate that toxicity level of adult butterflies may be dependent on individual condition, influenced by genetic background or earlier conditions, with maternal effects as one strong candidate mechanism. Additionally, toxicity was higher in older individuals, consistent with previous studies indicating accumulation of toxins with age. As toxicity level at death was independent of lifespan, cyanogenic glucoside compounds may have been recycled to release resources relevant for longevity in these long‐living butterflies. Understanding the origins and maintenance of variation in defenses is necessary in building a more complete picture of factors shaping the evolution of aposematic and mimetic systems.

Plants and phytophagous arthropods have coevolved in a long battle for survival. Plants respond to phytophagous feeders by producing a battery of antiherbivore chemical defences, while herbivores try to adapt to their hosts by attenuating the toxic effect of the defence compounds. Cyanogenic glucosides are a widespread group of defence chemicals that come from cyanogenic plants. Among the non-cyanogenic ones, the Brassicaceae family has evolved an alternative cyanogenic pathway to produce cyanohydrin as a way to expand defences. When a plant tissue is disrupted by an herbivore attack, cyanogenic substrates are brought into contact with degrading enzymes that cause the release of toxic hydrogen cyanide and derived carbonyl compounds. In this review, we focus our attention on the plant metabolic pathways linked to cyanogenesis to generate cyanide. It also highlights the role of cyanogenesis as a key defence mechanism of plants to fight against herbivore arthropods, and we discuss the potential of cyanogenesis-derived molecules as alternative strategies for pest control.

Cyanogenic glucosides (CNglcs) play an important role in plant defense response; however, the mechanism of regulation of CNglc synthesis by the external environment and endogenous hormones is largely unclear. In this study, we found that jasmonates (JAs) promoted the synthesis of CNglcs by activating the expression of CNglc biosynthesis genes in Lotus japonicus. Several differentially expressed basic helix–loop–helix (bHLH) family genes related to the synthesis of CNglcs were identified by RNA-seq. LjbHLH7 can directly activate the expression of CYP79D3 gene, the first step of CNglc synthesis, by binding to the G-box sequence of its promoter. Transgenic plants overexpressing LjbHLH7 exhibited higher relative CNglc content and enhanced insect resistance compared with the wild type. Furthermore, the transcriptional activity of LjbHLH7 was suppressed by the interaction with the L. japonicus JASMONATE-ZIM DOMAIN protein LjJAZ4. Based on these results, we propose that LjbHLH7 acts as an activator and LjJAZ4 acts as a repressor of JA-induced regulation of CNglc biosynthesis in L. japonicus. [ABSTRACT FROM AUTHOR]

Main conclusion: Droughted sorghum had higher concentrations of ROS in both wildtype and dhurrin-lacking mutants. Dhurrin increased in wildtype genotypes with drought. Dhurrin does not appear to mitigate oxidative stress in sorghum. Sorghum bicolor is tolerant of high temperatures and prolonged droughts. During droughts, concentrations of dhurrin, a cyanogenic glucoside, increase posing a risk to livestock of hydrogen cyanide poisoning. Dhurrin can also be recycled without the release of hydrogen cyanide presenting the possibility that it may have functions other than defence. It has been hypothesised that dhurrin may be able to mitigate oxidative stress by scavenging reactive oxygen species (ROS) during biosynthesis and recycling. To test this, we compared the growth and chemical composition of S. bicolor in total cyanide deficient sorghum mutants (tcd1) with wild-type plants that were either well-watered or left unwatered for 2 weeks. Plants from the adult cyanide deficient class of mutant (acdc1) were also included. Foliar dhurrin increased in response to drought in all lines except tcd1 and acdc1, but not in the roots or leaf sheaths. Foliar ROS concentration increased in drought-stressed plants in all genotypes. Phenolic concentrations were also measured but no differences were detected. The total amounts of dhurrin, ROS and phenolics on a whole plant basis were lower in droughted plants due to their smaller biomass, but there were no significant genotypic differences. Up until treatments began at the 3-leaf stage, tcd1 mutants grew more slowly than the other genotypes but after that they had higher relative growth rates, even when droughted. The findings presented here do not support the hypothesis that the increase in dhurrin commonly seen in drought-stressed sorghum plays a role in reducing oxidative stress by scavenging ROS. [ABSTRACT FROM AUTHOR]

Bamboo shoot is highly nutritious and contains a plethora of health-promoting bioactive compounds. It is a valuable source of food for Asiatic countries but it contains some antinutrients such as cyanogenic glycosides, glucosinolates, tannins, oxalates and phytates which deter its consumption due to safety issues. The most predominant antinutrient in bamboo shoot is cyanogenic glycosides. It causes increase in blood glucose and lactic acid levels and a decrease in the ATP/ADP ratio indicating the shift from aerobic to anaerobic metabolism. The anti-nutrients such as phytate can cause vitamins and minerals deficiencies. Though anti-nutrients may have deleterious effect when present in high concentration, they may also exert beneficial health effects at low concentrations. In order to eliminate or reduce the level of anti-nutrients to barest minimum, appropriate processing techniques such as soaking, boiling, drying and fermentation can be used. The cyanogen content in bamboo shoots range from 36.32 to more than 1000 mg/kg. Impact of different processing techniques revealed that, fermentation is the best method for reducing the antinutrient content and improving the quality of bamboo shoots as well as increasing the shelf life of the shoots. [ABSTRACT FROM AUTHOR]

Main conclusion: Developmental and organ-specific expression of genes in dhurrin biosynthesis, bio-activation, and recycling offers dynamic metabolic responses optimizing growth and defence responses in Sorghum. Plant defence models evaluate the costs and benefits of resource investments at different stages in the life cycle. Poor understanding of the molecular regulation of defence deployment and remobilization hampers accuracy of the predictions. Cyanogenic glucosides, such as dhurrin are phytoanticipins that release hydrogen cyanide upon bio-activation. In this study, RNA-seq was used to investigate the expression of genes involved in the biosynthesis, bio-activation and recycling of dhurrin in Sorghum bicolor. Genes involved in dhurrin biosynthesis were highly expressed in all young developing vegetative tissues (leaves, leaf sheath, roots, stems), tiller buds and imbibing seeds and showed gene specific peaks of expression in leaves during diel cycles. Genes involved in dhurrin bio-activation were expressed early in organ development with organ-specific expression patterns. Genes involved in recycling were expressed at similar levels in the different organ during development, although post-floral initiation when nutrients are remobilized for grain filling, expression of GSTL1 decreased > tenfold in leaves and NITB2 increased > tenfold in stems. Results are consistent with the establishment of a pre-emptive defence in young tissues and regulated recycling related to organ senescence and increased demand for nitrogen during grain filling. This detailed characterization of the transcriptional regulation of dhurrin biosynthesis, bioactivation and remobilization genes during organ and plant development will aid elucidation of gene regulatory networks and signalling pathways that modulate gene expression and dhurrin levels. In-depth knowledge of dhurrin metabolism could improve the yield, nitrogen use efficiency and stress resilience of Sorghum. [ABSTRACT FROM AUTHOR]

Abstract Evolutionary convergence of color pattern in mimetic species is tightly linked with the evolution of chemical defenses. Yet, the evolutionary forces involved in natural variations of chemical defenses in aposematic species are still understudied. Herein, we focus on the evolution of chemical defenses in the butterfly tribe Heliconiini. These neotropical butterflies contain large concentrations of cyanogenic glucosides, cyanide‐releasing compounds acting as predator deterrent. These compounds are either de novo synthesized or sequestered from their Passiflora host plant, so that their concentrations may depend on host plant specialization and host plant availability. We sampled 375 wild Heliconiini butterflies across Central and South America, covering 43% species of this clade, and quantify individual variations in the different CGs using liquid chromatography coupled with tandem mass spectrometry. We detected new compounds and important variations in chemical defenses both within and among species. Based on the most recent and well‐studied phylogeny of Heliconiini, we show that ecological factors such as mimetic interactions and host plant specialization have a significant association with chemical profiles, but these effects are largely explained by phylogenetic relationships. Our results therefore suggest that shared ancestries largely contribute to chemical defense variation, pointing out at the interaction between historical and ecological factors in the evolution of Müllerian mimicry.

Chemical defences in animals are both incredibly widespread and highly diverse. Yet despite the important role they play in mediating interactions between predators and prey, extensive differences in the amounts and types of chemical compounds can exist between individuals, even within species and populations. Here we investigate the potential role of environment and development on the chemical defences of warningly coloured butterfly species from the tribe Heliconiini, which can both synthesize and sequester cyanogenic glycosides (CGs). We reared 5 Heliconiini species in captivity, each on a single species-specific host plant as larvae, and compared them to individuals collected in the wild to ascertain whether the variation in CG content observed in the field might be the result of differences in host plant availability. Three of these species were reared as larvae on the same host plant, Passiflora riparia, to further test how species, sex, and age affected the type and amount of different defensive CGs, and how they affected the ratio of synthesized to sequestered compounds. Then, focusing on the generalist species Heliconius numata, we specifically explored variation in chemical profiles as a result of the host plant consumed by caterpillars and their brood line, using rearing experiments carried out on two naturally co-occurring host plants with differing CG profiles. Our results show significant differences in both the amount of synthesized and sequestered compounds between butterflies reared in captivity and those collected in the field. We also found a significant effect of species and an effect of sex in some, but not all, species. We show that chemical defences in H. numata continue to increase throughout their life, likely because of continued biosynthesis, and we suggest that variation in the amount of synthesized CGs in this species does not appear to stem from larval host plants, although this warrants further study. Interestingly, we detected a significant effect of brood lines, consistent with heritability influencing CG concentrations in H. numata. Altogether, our results point to multiple factors resulting in chemical defence variation in Heliconiini butterflies and highlight the overlooked effect of synthesis capabilities, which may be genetically determined to some extent. [ABSTRACT FROM AUTHOR]

Chemical defences against predators underlie the evolution of aposematic coloration and mimicry, which are classic examples of adaptive evolution. Surprisingly little is known about the roles of ecological and evolutionary processes maintaining defence variation, and how they may feedback to shape the evolutionary dynamics of species. Cyanogenic Heliconius butterflies exhibit diverse warning color patterns and mimicry, thus providing a useful framework for investigating these questions. We studied intraspecific variation in de novo biosynthesized cyanogenic toxicity and its potential ecological and evolutionary sources in wild populations of Heliconius erato along environmental gradients, in common-garden broods and with feeding treatments. Our results demonstrate substantial intraspecific variation, including detectable variation among broods reared in a common garden. The latter estimate suggests considerable evolutionary potential in this trait, although predicting the response to selection is likely complicated due to the observed skewed distribution of toxicity values and the signatures of maternal contributions to the inheritance of toxicity. Larval diet contributed little to toxicity variation. Furthermore, toxicity profiles were similar along steep rainfall and altitudinal gradients, providing little evidence for these factors explaining variation in biosynthesized toxicity in natural populations. In contrast, there were striking differences in the chemical profiles of H. erato from geographically distant populations, implying potential local adaptation in the acquisition mechanisms and levels of defensive compounds. The results highlight the extensive variation and potential for adaptive evolution in defense traits for aposematic and mimetic species, which may contribute to the high diversity often found in these systems. [ABSTRACT FROM AUTHOR]

Aposematism is a phenomenon in which defended prey advertise their unprofitability to predators through conspicuous signals. Aposematic (warning) colouration is widespread across numerous insect orders but rarely occurs in insects that inhabit aquatic environments where conditions can impose constraints on visual signal transmission. In the semi‐aquatic insect order Plecoptera (stoneflies), only the terrestrial adult stage of Austroperla cyrene (Austroperlidae) from New Zealand has been suggested to possess aposematic colouration, associated with the presence of hydrogen cyanide and documented unpalatability to predators. Here, we review evidence for the occurrence of aposematic colouration in Australian and South American Austroperlidae based on a synthesis of taxonomic literature and studies examining feeding preferences of predators in aquatic environments. Contrasting black, white and yellow colouration occurs commonly in adult Austroperlidae, and nymphs of several surveyed species were found to be aversive to predators, together suggesting that aposematism may be widespread in this family. The wider prevalence of aposematism in Austroperlidae should be examined further by directly testing the palatability of various species to predators and by analysing the chemical compositions of their defensive secretions. Austroperlid stoneflies offer new potential for understanding the evolution of aposematism in ecological and phylogenetic contexts that have not yet been explored. [ABSTRACT FROM AUTHOR]

Fresh cassava is highly susceptible to rapid post-harvest physiological deterioration due to the high water activity. Therefore, foam mat drying was considered as an alternative drying technique for preservation. The drying kinetics of white-fleshed cassava foam (air volume fraction = 0.26, overrun = 36.0 %) and yellow-fleshed cassava foam (air volume fraction = 0.24, overrun = 32.2 %) were studied. The influence of temperature and foam thickness on moisture removal ratio (MR), drying rate, diffusivity and some important physicochemical properties of the cassava foams (CSF) during drying were researched and compared with those of non-foamed cassava pulp (NFP). The cassava foams were dried to foam powders at temperatures of 50 ' C, 65 ' C and 80 ' C and foam thicknesses of 6 mm, 8 mm and 10 mm. The CSF exhibited two falling rates. The CSF powders had significantly lower total cyanogenic glucosides content compared to the NFP powder, and a high retention of total carotenoids. The dry foam powders had higher lightness/luminosity (L *) values than dry pulp powders. Micrographs revealed interactions between starch granules and the hydrocolloids but microstructure was not affected by increasing temperatures. [ABSTRACT FROM AUTHOR]

[Display omitted] • Cyanogenic glucosides include amygdalin, linamarin, taxiphyllin, dhurrin, lotaustralin, etc. • Cyanogenic glucosides cause toxicity upon conversion to hydrogen cyanide (HCN) • Cyanogenic glucosides can be detected by NMR spectroscopy, HPLC, UHPLC, and GC techniques. Cyanogenic glucosides are bioactive compounds widely present in diverse plant-based foods, serving pivotal roles in storage and transport within plant systems. They possess inherent defensive properties against pathogens and herbivores, safeguarding plants from potential harm and decay. These compounds manifest in various forms, such as amygdalin, linamarin, taxiphyllin, dhurrin, lotaustralin, prunasin, linostatin, neolinostatin, triglochinin, and epilotaustralin, with variations in their distribution and concentration across plant species. However, converting cyanogenic glucosides into hydrogen cyanide (HCN) poses a dual concern. Firstly, it can lead to toxicity when ingested, and secondly, it interferes with the absorption of essential nutrients in the human body. This comprehensive review aims to elucidate the synthesis of cyanogenic glucosides in plant-based food products and plant systems. It also provides an in-depth analysis of various efficient detection methods. Furthermore, it explores a spectrum of strategies employed to mitigate the impact of cyanogenic glucosides in plant-based foods. These encompass soaking, crushing, grating, boiling, drying, autoclaving, ultrasound-assisted detoxification, and enzymatic-treated detoxification, with each method thoroughly examined for its effectiveness. This review aims to provide valuable insights into cyanogenic glucosides, from their origin and detection to the various detoxification approaches employed, ultimately promoting safer and informed dietary choices. [ABSTRACT FROM AUTHOR]

In 2009, Food Standards Australia New Zealand set a total cyanide content limit of 10 ppm for ready-to-eat cassava products to address food safety concerns about cyanogenic glucosides in cassava. This study surveys a range of cassava food products available in Melbourne, Australia, ten years after the implementation of these regulations. Of all the products tested, the mean cyanide content was greatest in ready-to-eat cassava chips (48.4 ppm), although imported ready-to-eat products had a higher mean cyanide content (95.9 ppm) than those manufactured in Australia (1.0 ppm). Cyanide was detected in frozen cassava products (grated mean = 12.9 ppm; whole root mean = 19.8 ppm), but was significantly reduced through processing according to packet instructions in both product types. Three methods were used to quantify total cyanide content: the evolved cyanide method, the picrate absorbance method and the picrate chart method, with satisfactory agreement between methods. The picrate absorbance and chart methods reported mean cyanide contents 13.7 ppm and 23.1 ppm higher, respectively, than the evolved cyanide method. Our results reaffirm the need for the ongoing testing of cassava food products, especially ready-to-eat products whose cyanide content will not be reduced before consumption.

Die Vielfalt an Verbindungen zeichnet die sekundären Pflanzeninhaltsstoffe aus. Cyanogene Glykoside entstehen aus Aminosäuren und stellen in Pflanzen eine Speicherform für Blausäure dar. Sie sind ungiftig, solange sie nicht mit den dementsprechenden spaltenden Enzymen in Kontakt kommen. Die Funktion der cyanogenen Glykoside wurde viel diskutiert, für mehrere Pflanzenarten wurde die Hypothese eines Abwehrmechanismus anerkannt. Sorghum wird aufgrund ihres Vertretungscharakters für Mais in Europa angebaut, besonders bei zunehmender Sommertrockenheit und zur Sicherung einer vielfältigen Fruchtfolge in einigen Gebieten. Weißklee (Trifolium repens) stellt trotz seiner sortenspezifisch schwankenden cyanogenen Glykosidgehalte eine bedeutende Futterpflanze dar. Aufgrund dessen wurde die Cyanogenese bei Weißklee intensiv untersucht, zwei Gene (Ac und Li) sind für die Cyanogenese verantwortlich. Die durch die Pflanzenzüchtung zur Verfügung stehenden Weißkleesorten mit niedrigen cyanogenen Glykosidgehalten werden in der Praxis bevorzugt genutzt.

Summary: Manihot esculenta (cassava) is a root crop originating from South America that is a major staple in the tropics, including in marginal environments. This study focused on South American and African germplasm and investigated the genetic architecture of hydrogen cyanide (HCN), a major component of root quality. HCN, representing total cyanogenic glucosides, is a plant defense component against herbivory but is also toxic for human consumption. We genotyped 3354 landraces and modern breeding lines originating from 26 Brazilian states and 1389 individuals were phenotypically characterized across multi‐year trials for HCN. All plant material was subjected to high‐density genotyping using genotyping by sequencing. We performed genome‐wide association mapping to characterize the genetic architecture and gene mapping of HCN. Field experiments revealed strong broad‐ and narrow‐sense trait heritability (0.82 and 0.41, respectively). Two major loci were identified, encoding for an ATPase and a MATE protein, and contributing up to 7 and 30% of the HCN concentration in roots, respectively. We developed diagnostic markers for breeding applications, validated trait architecture consistency in African germplasm and investigated further evidence for the domestication of sweet and bitter cassava. Fine genomic characterization revealed: (i) the major role played by vacuolar transporters in regulating HCN content; (ii) the co‐domestication of sweet and bitter cassava major alleles are dependent upon geographical zone; and (iii) the major loci allele for high HCN in M. esculenta Crantz seems to originate from its ancestor, M. esculenta subsp. flabellifolia. Taken together, these findings expand our insights into cyanogenic glucosides in cassava roots and its glycosylated derivatives in plants. [ABSTRACT FROM AUTHOR]

Humans hold a love–hate relationship with cyanogenic plants. Preferentially selected for domestication due to their heightened pest resistance, they can nevertheless threaten both human and livestock health if not consumed in moderation or adequately processed. Over 20% of crops are cyanogenic due to the stable accumulation of cyanogenic glucosides (CNglcs) and, while the biochemical pathways of these specialized metabolites are known, the same cannot be said regarding their underlying molecular pathways. In a comprehensive study, Chen et al. (2022) characterized a basic helix–loop–helix (bHLH) transcription factor that is responsive to methyl jasmonate and directly induces expression of the key CNglc biosynthetic gene in the model species Lotus japonicus. [ABSTRACT FROM AUTHOR]

Abstract Lima bean, Phaseolus lunatus, is a crop legume that produces the cyanogenic glucosides linamarin and lotaustralin. In the legumes Lotus japonicus and Trifolium repens, the biosynthesis of these two α‐hydroxynitrile glucosides involves cytochrome P450 enzymes of the CYP79 and CYP736 families and a UDP‐glucosyltransferase. Here, we identify CYP79D71 as the first enzyme of the pathway in P. lunatus, producing oximes from valine and isoleucine. A second CYP79 family member, CYP79D72, was shown to catalyze the formation of leucine‐derived oximes, which act as volatile defense compounds in Phaseolus spp. The organization of the biosynthetic genes for cyanogenic glucosides in a gene cluster aided their identification in L. japonicus. In the available genome sequence of P. vulgaris, the gene orthologous to CYP79D71 is adjacent to a member of the CYP83 family. Although P. vulgaris is not cyanogenic, it does produce oximes as volatile defense compounds. We cloned the genes encoding two CYP83s (CYP83E46 and CYP83E47) and a UDP‐glucosyltransferase (UGT85K31) from P. lunatus, and these genes combined form a complete biosynthetic pathway for linamarin and lotaustralin in Lima bean. Within the genus Phaseolus, the occurrence of linamarin and lotaustralin as functional chemical defense compounds appears restricted to species belonging to the closely related Polystachios and Lunatus groups. A preexisting ability to produce volatile oximes and nitriles likely facilitated evolution of cyanogenesis within the Phaseolus genus.

Sorghum bicolor (L.) Moench produces the nitrogen-containing natural product dhurrin that provides chemical defense against herbivores and pathogens via the release of toxic hydrogen cyanide gas. Drought can increase dhurrin in shoot tissues to concentrations toxic to livestock. As dhurrin is also a remobilizable store of reduced nitrogen and plays a role in stress mitigation, reductions in dhurrin may come at a cost to plant growth and stress tolerance. Here, we investigated the response to an extended period of water limitation in a unique EMS-mutant adult cyanide deficient class 1 (acdc1) that has a low dhurrin content in the leaves of mature plants. A mutant sibling line was included to assess the impact of unknown background mutations. Plants were grown under three watering regimes using a gravimetric platform, with growth parameters and dhurrin and nitrate concentrations assessed over four successive harvests. Tissue type was an important determinant of dhurrin and nitrate concentrations, with the response to water limitation differing between above and below ground tissues. Water limitation increased dhurrin concentration in the acdc1 shoots to the same extent as in wild-type plants and no growth advantage or disadvantage between the lines was observed. Lower dhurrin concentrations in the acdc1 leaf tissue when fully watered correlated with an increase in nitrate content in the shoot and roots of the mutant. In targeted breeding efforts to down-regulate dhurrin concentration, parallel effects on the level of stored nitrates should be considered in all vegetative tissues of this important forage crop to avoid potential toxic effects.

Objective: To make a tentative assessment of the consumption of cassava in three countries in South-east Asia and the cyanogenic potential (CNp) of the crop as a possible food safety issue.Design: We used data from the Ministry of Health in Vietnam and Statistics Authorities in Indonesia and Philippines (mean household consumption per province) to assess cassava consumption. Conversions of units were needed to facilitate the comparison of cassava consumption between countries. The most up-to-date data available regarding both cassava consumption and the CNp of cassava grown in the respective countries were assessed.Settings: Vietnam, Indonesia and Philippines.Participants: Respondents from provinces in Vietnam (nineteen), Indonesia (thirty-three) and Philippines (eighty-one) were asked to complete a recall questionnaire detailing either the previous 24-h' or the 7-d' cassava consumption.Results: Among the three countries, available data indicated that the highest median cassava-consumption figures percapita were from Indonesia and the Philippines (9·01 and 7·28 g/capita per d, respectively), with Vietnam having the least (1·14 g/capita per d). Published information regarding the CNp of cassava in the three countries was limited.Conclusions: While the findings of the present study are somewhat limited by a lack of available information regarding both the extent of cassava consumption and the CNp of cassava consumed in the three countries, it appears likely that cyanogen intake arising from cassava consumption among the three countries exceeds the FAO/WHO Provisional Maximum Tolerable Daily Intake, although any risk to public health appears limited to a minority of provinces in each country. [ABSTRACT FROM AUTHOR]

Lima bean, Phaseolus lunatus, is a crop legume that produces the cyanogenic glucosides linamarin and lotaustralin. In the legumes Lotus japonicus and Trifolium repens, the biosynthesis of these two α‐hydroxynitrile glucosides involves cytochrome P450 enzymes of the CYP79 and CYP736 families and a UDP‐glucosyltransferase. Here, we identify CYP79D71 as the first enzyme of the pathway in P. lunatus, producing oximes from valine and isoleucine. A second CYP79 family member, CYP79D72, was shown to catalyze the formation of leucine‐derived oximes, which act as volatile defense compounds in Phaseolus spp. The organization of the biosynthetic genes for cyanogenic glucosides in a gene cluster aided their identification in L. japonicus. In the available genome sequence of P. vulgaris, the gene orthologous to CYP79D71 is adjacent to a member of the CYP83 family. Although P. vulgaris is not cyanogenic, it does produce oximes as volatile defense compounds. We cloned the genes encoding two CYP83s (CYP83E46 and CYP83E47) and a UDP‐glucosyltransferase (UGT85K31) from P. lunatus, and these genes combined form a complete biosynthetic pathway for linamarin and lotaustralin in Lima bean. Within the genus Phaseolus, the occurrence of linamarin and lotaustralin as functional chemical defense compounds appears restricted to species belonging to the closely related Polystachios and Lunatus groups. A preexisting ability to produce volatile oximes and nitriles likely facilitated evolution of cyanogenesis within the Phaseolus genus. [ABSTRACT FROM AUTHOR]

The potential of cassava (Manihot esculenta Crantz.) for simultaneous Hg and Au phytoextraction was explored by investigating Hg and Au localization in cassava roots through Micro-Proton Induced X-Ray Emission, High-Resolution Transmission Electron Microscopy (HR-TEM) and X-Ray Diffractometry (XRD). The effect of Hg and Au in the cyanogenic glucoside linamarin distribution was also investigated using Matrix Assisted Laser Desorption Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (MALDI-FT-ICR-MS) imaging. Hg was located mainly in the root vascular bundle of plants grown in 50 or 100 μmol L−1 Hg solutions. Au was localized in the epidermis and cortex or in the epidermis and endodermis for 50 and 100 μmol L−1 Au solutions, respectively. For 50 μmol L−1 solutions of both Hg and Au, the two metals were co-localized in the epidermis. When the Hg concentrations were increased to 100 μmol L−1, Au was still localized to a considerable extent in the epidermis while Hg was located in all root parts. HR-TEM and XRD revealed that Au nanoparticles were formed in cassava roots. MALDI-FT-ICR-MS imaging showed linamarin distribution in the roots of control and plants and metal-exposed plants thus suggesting that linamarin might be involved in Hg and Au uptake and distribution. [ABSTRACT FROM AUTHOR]

Ａｍｉｎｏ ａｃｉｄｓ-ｄｅｒｉｖｅｄ ｃｙａｎｏｇｅｎｉｃ ｇｌｕｃｏｓｉｄｅｓ ｃａｔａｌｙｚｉｎｇ ｂｙ ｐｌａｎｔ ｃｙｔｏｃｈｒｏｍｅ Ｐ４５０ ｅｎｚｙｍｅｓ ａｒｅ ｐｌａｎｔ ｓｅｃｏｎｄａｒｙ ｍｅｔａｂｏｌｉｓｍꎬ ｗｈｉｃｈ ｉｓ ｒｅｌａｔｅｄ ｔｏ ｐｌａｎｔ ｄｅｆｅｎｓｅ ａｎｄ ａｎｔｉ-ｓｔｒｅｓｓ. Ａ ｇｅｎｅ ｏｆ ｃｙｔｏｃｈｒｏｍｅ Ｐ４５０(ｎａｍｅｌｙ ＭｏＣＹＰ７１ꎬ ＧｅｎＢａｎｋ ＩＤ ｉｓ ＭＫ１７２８５８) ｆｒｏｍ Ｍａｌａｎｉａ ｏｌｅｉｆｅｒａ ｗａｓ ｉｓｏｌａｔｅｄ ａｎｄ ｃｌｏｎｅｄ ｔｈｒｏｕｇｈ ａｎａｌｙｚｉｎｇ ｔｈｅ ｔｒａｎｓｃｒｉｐｔｏｍｅ ｏｆ Ｍ. ｏｌｅｉｆｅｒａꎬ ｔｈｅｎ ｉｔｓ ｆｕｎｃｔｉｏｎ ｗａｓ ａｎａｌｙｚｅｄ ｂｙ ｂｉｏｉｎｆｏｒｍａｔｉｃｓ ａｎａｌｙｓｉｓꎬ ａｎｄ ｔｈｅ ｅｘｐｒｅｓｓｉｏｎ ｏｆ ＭｏＣＹＰ７１ ｉｎ ｄｉｆｆｅｒｅｎｔ ｄｅｖｅｌｏｐｍｅｎｔａｌ ｐｅｒｉｏｄｓ ｏｆ ｆｒｕｉｔ ｗａｓ ｄｅｔｅｃｔｅｄ. Ｔｈｅ ｒｅｓｕｌｔｓ ｗｅｒｅ ａｓ ｆｏｌｌｏｗｓ: ＭｏＣＹＰ７１ ｇｅｎｅ ｈａｓ １ ５７２ ｂｐꎬ ｅｎｃｏｄｅｓ ５２３ ａｍｉｄ ａｃｉｄｓꎬ ａｎｄ ｉｔｓ ｓｅ- ｑｕｅｎｃｅ ｏｆ ｃＤＮＡ ｈａｓ ８８％ ｉｄｅｎｔｉｔｙ ｗｉｔｈ ｔｈｅ ＣＹＰ７１ ｃＤＮＡ ｏｆ Ｃｏｆｆｅａ ｅｕｇｅｎｉｏｉｄｅｓ(ＸＭ_０２７３１９２８２) ａｎｄ Ｃｏｆｆｅａ ａｒａｂｉｃａ (ＸＭ_０２７２１３４５６)ꎻ Ｔｈｅ ｒｅｌａｔｉｖｅ ｍｏｌｅｃｕｌａｒ ｗｅｉｇｈｔ ｏｆ ＭｏＣＹＰ７１ ｐｒｏｔｅｉｎ ｗａｓ ５８ ９７６.５４ꎬ ｔｈｅ ｔｈｅｏｒｅｔｉｃａｌ ｐＩ ｗａｓ ８.１０ꎬ ｔｈｅ ｍｏｌｅｃｕｌａｒ ｆｏｒｍｕｌａ ｗａｓ Ｃ２６７５ Ｈ４１８４ Ｎ７０４ Ｏ７４４ Ｓ２７ꎬ ｔｈｅ ｉｎｓｔａｂｉｌｉｔｙ ｉｎｄｅｘ(Ⅱ) ｗａｓ ４０.８４ꎬ ａｎｄ ｔｈｅ ｐｒｏｔｅｉｎ ｂｅｌｏｎｇｅｄ ｔｏ ａｎ ｉｎｓｔａｂｌｅ ｐｒｏｔｅｉｎꎻ Ｔｈｅｒｅ ｈａｄ ｎｏ ｓｉｇｉｎａｌ ｐｅｐｔｉｄｅ ｉｎ ｔｈｉｓ ｐｒｏｔｅｉｎ ｓｅｑｕｅｎｃｅꎬ ａｎｄ ｔｈｅ ｐｒｏｔｅｉｎ ｅｘｉｓｔｓ ｉｎ ｔｈｅ ｓｅｃｒｅｔｉｏｎ ｐａｔｈｗａｙ ａｎｄ ｃｏｎ- ｔａｉｎｅｄ ｔｗｏ ｔｒａｎｓｍｅｍｂｒａｎｅ ｓｔｒｕｃｔｕｒｅꎬ ｌｏｃａｔｅｄ ｉｎ ｔｈｅ ｐｒｏｔｅｉｎ ｓｅｑｕｅｎｃｅｓ ｏｆ ２０－３７ ａｎｄ ３１１－３３３ꎬ ａｎｄ ｔｈｅ ｔｒａｎｓｍｅｍｂｒａｎｅ ｓｉｔｅｓ ａｎｃｈｏｒｅｄ ｉｎ ｔｈｅ ｏｒｇａｎｅｌｌｅ ｍｅｍｂｒａｎｅꎻ ＭｏＣＹＰ７１ ｈａｄ ａｌｌ ｔｈｅ ｃｏｎｓｅｒｖｅｄ ｄｏｍａｉｎ ｏｆ ＣＹＰ ｆａｍｉｌｙꎬ ｃｏｎｔａｉｎｉｎｇ ｐｒｏｌｉｎｅ- ｒｉｃｈ ｒｅｇｉｏｎ (ＰＰＳＰＰＲＬＰ)ꎬ Ｋ ｈｅｌｉｘ ｒｅｇｉｏｎ(ＫＥＴＦＲ)ꎬ Ｉ ｈｅｌｉｘ ｒｅｇｉｏｎ(ＧＧＩＤＴＳ)ꎬ ＰＥＲＦ ｄｏｍａｉｎ(ＰＥＲＦ) ａｎｄ ｔｈｅ ｍａｉｎ ｉｄｅｎｔｉｆｉｅｄ ｆｅａｔｕｒｅ ｈｅｍｅ-ｂｉｎｄｉｎｇ ｒｅｇｉｏｎ (ＦＧＡＧＲＲＩＣＰＧ)ꎬ ａｎｄ ｔｈｅ ｐｒｏｔｅｉｎ ＭｏＣＹＰ７１ ａｎｄ ｔｈｅ ＣＹＰ７１Ｅ ｐｒｏｔｅｉｎ ｉｎ Ｔｈｅｏ- ｂｒｏｍａ ｃａｃａｏꎬ Ｄｕｒｉｏ ｚｉｂｅｔｈｉｎｕｓꎬ Ｓｏｒｇｈｕｍ ｂｉｃｏｌｏｒ(ＧｅｎＢａｎｋ ＩＤ ｉｓ ＥＯＸ９２９０８.１ꎬ ＸＰ_０２２７７３８７５.１ａｎｄ ＡＡＣ３９３１８.１ꎬ ｒｅ- ｓｐｅｃｔｉｖｅｌｙ) ｃｌｕｓｔｅｒｅｄ ｉｎｔｏ ｏｎｅ ｃｌａｎꎻ Ｔｈｅ ｅｘｐｒｅｓｓｉｏｎ ｌｅｖｅｌ ｏｆ ＭｏＣＹＰ７１ ｇｅｎｅ ｒｅｄｕｃｅｄ ｇｒａｄｕａｌｌｙ ｉｎ ｔｈｅ ｆｉｒｓｔ ｔｈｒｅｅ ｍｏｎｔｈｓ ａｆｔｅｒ ｂｌｏｓｓｏｍ ｆａｄｉｎｇꎬ ｐａｒｔｉｃｕｌａｒｌｙꎬ １ｓｔ ｍｏｎｔｈ ｔｈｅ ｆｒｕｉｔ ａｆｔｅｒ ｂｌｏｓｓｏｍ ｆａｄｉｎｇ> ２ｎｄ ｍｏｎｔｈ> ３ｒｄ ｍｏｎｔｈꎬ ｈｏｗｅｖｅｒꎬ ｉｔｓ ｅｘ- ｐｒｅｓｓｉｏｎ ｌｅｖｅｌ ｉｎｃｒｅａｓｅｄ ｓｈａｒｐｌｙ ｉｎ ４ｔｈ ｍｏｎｔｈ. Ｔｈｅ ｐｒｅｓｅｎｔ ｓｔｕｄｙ ｈａｄ ａ ｉｍｐｏｒｔａｎｔ ｓｉｇｎｉｆｉｃａｎｃｅ ｔｏ ｄｅｆｅｎｓｅ ｔｏ ｉｎｓｅｃｔ ｐｅｓｔｓꎬ ｔｉｓｓｕｅ ｒｉｐｅｎｉｎｇ ａｎｄ ｓｅｃｏｎｄａｒｙ ｍｅｔａｂｏｌｉｓｍ ｄｉｇｇｉｎｇ ｉｎ Ｍａｌａｎｉａ ｏｌｅｉｆｅｒａ. [ABSTRACT FROM AUTHOR]

Evolutionary convergence of color pattern in mimetic species is tightly linked with the evolution of chemical defenses. Yet, the evolutionary forces involved in natural variations of chemical defenses in aposematic species are still understudied. Herein, we focus on the evolution of chemical defenses in the butterfly tribe Heliconiini. These neotropical butterflies contain large concentrations of cyanogenic glucosides, cyanide‐releasing compounds acting as predator deterrent. These compounds are either de novo synthesized or sequestered from their Passiflora host plant, so that their concentrations may depend on host plant specialization and host plant availability. We sampled 375 wild Heliconiini butterflies across Central and South America, covering 43% species of this clade, and quantify individual variations in the different CGs using liquid chromatography coupled with tandem mass spectrometry. We detected new compounds and important variations in chemical defenses both within and among species. Based on the most recent and well‐studied phylogeny of Heliconiini, we show that ecological factors such as mimetic interactions and host plant specialization have a significant association with chemical profiles, but these effects are largely explained by phylogenetic relationships. Our results therefore suggest that shared ancestries largely contribute to chemical defense variation, pointing out at the interaction between historical and ecological factors in the evolution of Müllerian mimicry. [ABSTRACT FROM AUTHOR]

Sorghum bicolor (L.) Moench produces the nitrogen-containing natural product dhurrin that provides chemical defense against herbivores and pathogens via the release of toxic hydrogen cyanide gas. Drought can increase dhurrin in shoot tissues to concentrations toxic to livestock. As dhurrin is also a remobilizable store of reduced nitrogen and plays a role in stress mitigation, reductions in dhurrin may come at a cost to plant growth and stress tolerance. Here, we investigated the response to an extended period of water limitation in a unique EMS-mutant adult cyanide deficient class 1 (acdc1) that has a low dhurrin content in the leaves of mature plants. A mutant sibling line was included to assess the impact of unknown background mutations. Plants were grown under three watering regimes using a gravimetric platform, with growth parameters and dhurrin and nitrate concentrations assessed over four successive harvests. Tissue type was an important determinant of dhurrin and nitrate concentrations, with the response to water limitation differing between above and below ground tissues. Water limitation increased dhurrin concentration in the acdc1 shoots to the same extent as in wild-type plants and no growth advantage or disadvantage between the lines was observed. Lower dhurrin concentrations in the acdc1 leaf tissue when fully watered correlated with an increase in nitrate content in the shoot and roots of the mutant. In targeted breeding efforts to down-regulate dhurrin concentration, parallel effects on the level of stored nitrates should be considered in all vegetative tissues of this important forage crop to avoid potential toxic effects. [ABSTRACT FROM AUTHOR]

Many defended species use conspicuous visual warning signals to deter potential predators from attacking. Traditional theory holds that these signals should converge on similar forms, yet variation in visual traits and the levels of defensive chemicals is common, both within and between species. It is currently unclear how the strength of signals and potency of defences might be related: conflicting theories suggest that aposematic signals should be quantitatively honest, or, in contrast, that investment in one component should be prioritized over the other, while empirical tests have yielded contrasting results. Here, we advance this debate by examining the relationship between defensive chemicals and signal properties in a family of aposematic Lepidoptera, accounting for phylogenetic relationships and quantifying coloration from the perspective of relevant predators. We test for correlations between toxin levels and measures of wing colour across 14 species of day‐flying burnet and forester moths (Lepidoptera: Zygaenidae), protected by highly aversive cyanogenic glucosides, and find no clear evidence of quantitative signal honesty. Significant relationships between toxin levels and coloration vary between sexes and sampling years, and several trends run contrary to expectations for signal honesty. Although toxin concentration is positively correlated with increasing luminance contrast in forewing pattern in 1 year, higher toxin levels are also associated with paler and less chromatically salient markings, at least in females, in another year. Our study also serves to highlight important factors, including sex‐specific trends and seasonal variation, that should be accounted for in future work on signal honesty in aposematic species. [ABSTRACT FROM AUTHOR]

Cyanogenic glucosides (CG), the monoglycosides linamarin and lotaustralin, as well as the diglucosides linustatin and neolinustatin, have been identified in flax. The roles of CG and hydrogen cyanide (HCN), specifically the product of their breakdown, differ and are understood only to a certain extent. HCN is toxic to aerobic organisms as a respiratory inhibitor and to enzymes containing heavy metals. On the other hand, CG and HCN are important factors in the plant defense system against herbivores, insects and pathogens. In this study, fluctuations in CG levels during flax growth and development (using UPLC) and the expression of genes encoding key enzymes for their metabolism (valine N-monooxygenase, linamarase, cyanoalanine nitrilase and cyanoalanine synthase) using RT-PCR were analyzed. Linola cultivar and transgenic plants characterized by increased levels of sulfur amino acids were analyzed. This enabled the demonstration of a significant relationship between the cyanide detoxification process and general metabolism. Cyanogenic glucosides are used as nitrogen-containing precursors for the synthesis of amino acids, proteins and amines. Therefore, they not only perform protective functions against herbivores but are general plant growth regulators, especially since changes in their level have been shown to be strongly correlated with significant stages of plant development.

• There is growing evidence for the convergent evolution of physically linked gene clusters encoding chemical defense pathways. Metabolic clusters are proposed to evolve because they ensure co-inheritance of all required genes where the defense is favored, and prevent inheritance of toxic partial pathways where it is not. This hypothesis rests on the assumption that clusters evolve in species where selection favors intraspecific polymorphism for the defense; however, they have not been examined in polymorphic species. • We examined metabolic cluster evolution in relation to an adaptive polymorphism for cyanogenic glucoside (CNglc) production in clover. Using 163 accessions, we performed CNglc assays, BAC sequencing, Southern hybridizations and molecular evolutionary analyses. • We find that the CNglc pathway forms a 138-kb cluster in white clover, and that the adaptive polymorphism occurs through presence/absence of the complete cluster. Component genes are orthologous to those in the distantly related legume Lotus japonicus. • These findings provide empirical support for the co-inheritance hypothesis, and they indicate that adaptive CNglc variation in white clover evolves through recurrent deletions of the entire pathway. They further indicate that the shared ancestor of many important legume crops was likely cyanogenic and that this defense was lost repeatedly over the last 50 Myr. [ABSTRACT FROM AUTHOR]

Abstract: The distinctive black and red wing pattern of six‐spot burnet moths (Zygaena filipendulae, L.) is a classic example of aposematism, advertising their potent cyanide‐based defences. While such warning signals provide a qualitatively honest signal of unprofitability, the evidence for quantitative honesty, whereby variation in visual traits could provide accurate estimates of individual toxicity, is more equivocal. Combining measures of cyanogenic glucoside content and wing color from the perspective of avian predators, we investigate the relationship between coloration and defences in Z. filipendulae, to test signal honesty both within and across populations. There were no significant relationships between mean cyanogenic glucoside concentration and metrics of wing coloration across populations in males, yet in females higher cyanogenic glucoside levels were associated with smaller and lighter red forewing markings. Trends within populations were similarly inconsistent with quantitative honesty, and persistent differences between the sexes were apparent: larger females, carrying a greater total cyanogenic glucoside load, displayed larger but less conspicuous markings than smaller males, according to several color metrics. The overall high aversiveness of cyanogenic glucosides and fluctuations in color and toxin levels during an individual's lifetime may contribute to these results, highlighting generally important reasons why signal honesty should not always be expected in aposematic species. [ABSTRACT FROM AUTHOR]

Premise of the Study: The maintenance of adaptive polymorphisms within species requires fitness trade‐offs reflecting selection for each morph. Cyanogenesis, the ability to produce hydrogen cyanide (HCN) after tissue damage, occurs in >3000 plant species and exists as a discrete polymorphism in white clover. This polymorphism is spatially distributed in recurrent clines, with higher frequencies of cyanogenic plants in warmer climates. The HCN autotoxicity hypothesis proposes that cyanogenic plants are selected against where frosts are common, as freezing liberates HCN and could impair cellular respiration. Methods: We tested the HCN autotoxicity hypothesis using a freezing chamber to examine survival, tissue damage, and physiological recovery as assessed via chlorophyll fluorescence following mild and severe freezing treatments. We utilized 65 genotypes from a single polymorphic population to eliminate effects of population structure. Key Results: Cyanogenic plants did not differ from acyanogenic plants in survival, tissue damage, or recovery following freezing. However, plants producing either of the two required cyanogenic precursors had lower survival and tissue damage after freezing than plants lacking both precursors. Conclusions: These results suggest that freezing‐induced HCN toxicity is unlikely to be responsible for the maintenance of the cyanogenesis polymorphism in white clover. However, energetic trade‐offs associated with costs of producing the cyanogenic precursors may confer a fitness benefit to acyanogenic plants under stressful climatic conditions. The lack of evidence for HCN toxicity suggests that cyanogenic clover uses physiological mechanisms mediated by β‐cyanoalanine synthase and alternative oxidase to maintain cellular function in the presence of HCN. [ABSTRACT FROM AUTHOR]

Summary: Cyanogenic glucosides are nitrogen‐containing specialized metabolites that provide chemical defense against herbivores and pathogens via the release of toxic hydrogen cyanide. It has been suggested that cyanogenic glucosides are also a store of nitrogen that can be remobilized for general metabolism via a previously unknown pathway. Here we reveal a recycling pathway for the cyanogenic glucoside dhurrin in sorghum (Sorghum bicolor) that avoids hydrogen cyanide formation. As demonstrated in vitro, the pathway proceeds via spontaneous formation of a dhurrin‐derived glutathione conjugate, which undergoes reductive cleavage by glutathione transferases of the plant‐specific lambda class (GSTLs) to produce p‐hydroxyphenyl acetonitrile. This is further metabolized to p‐hydroxyphenylacetic acid and free ammonia by nitrilases, and then glucosylated to form p‐glucosyloxyphenylacetic acid. Two of the four GSTLs in sorghum exhibited high stereospecific catalytic activity towards the glutathione conjugate, and form a subclade in a phylogenetic tree of GSTLs in higher plants. The expression of the corresponding two GSTLs co‐localized with expression of the genes encoding the p‐hydroxyphenyl acetonitrile‐metabolizing nitrilases at the cellular level. The elucidation of this pathway places GSTs as key players in a remarkable scheme for metabolic plasticity allowing plants to reverse the resource flow between general and specialized metabolism in actively growing tissue. [ABSTRACT FROM AUTHOR]

Almond bitterness is the most important trait for breeding programs since bitter-kernelled seedlings are usually discarded. Amygdalin and its precursor prunasin are hydrolyzed by specific enzymes called β-glucosidases. In order to better understand the genetic control of almond bitterness, some studies have shown differences in the location of prunasin hydrolases (PH, the β-glucosidase that degrades prunasin) in sweet and bitter genotypes. The aim of this work was to isolate and characterize different PHs in sweet- and bitter-kernelled almonds to determine whether differences in their genomic or protein sequences are responsible for the sweet or bitter taste of their seeds. RNA was extracted from the tegument, nucellus and cotyledon of one sweet (Lauranne) and two bitter (D05–187 and S3067) almond genotypes throughout fruit ripening. Sequences of nine positive Phs were then obtained from all of the genotypes by RT-PCR and cloning. These clones, from mid ripening stage, were expressed in a heterologous system in tobacco plants by agroinfiltration. The PH activity was detected using the Feigl-Anger method and quantifying the hydrogen cyanide released with prunasin as substrate. Furthermore, β-glucosidase activity was detected by Fast Blue BB salt and Umbelliferyl method. Differences at the sequence level (SNPs) and in the activity assays were detected, although no correlation with bitterness was found. [ABSTRACT FROM AUTHOR]

Oximes (R 1 R 2 C=NOH) are nitrogen-containing chemical constituents that are formed in species representing all kingdoms of life. In plants, oximes are positioned at important metabolic bifurcation points between general and specialized metabolism. The majority of plant oximes are amino acid-derived metabolites formed by the action of a cytochrome P450 from the CYP79 family. Auxin, cyanogenic glucosides, glucosinolates, and a number of other bioactive specialized metabolites including volatiles are produced from oximes. Oximes with the E configuration have high biological activity compared with Z-oximes. Oximes or their derivatives have been demonstrated or proposed to play roles in growth regulation, plant defense, pollinator attraction, and plant communication with the surrounding environment. In addition, oxime-derived products may serve as quenchers of reactive oxygen species and storage compounds for reduced nitrogen that may be released on demand by the activation of endogenous turnover pathways. As highly bioactive molecules, chemically synthesized oximes have found versatile uses in many sectors of society, especially in the agro- and medical sectors. This review provides an update on the structural diversity, occurrence, and biosynthesis of oximes in plants and discusses their role as key players in plant general and specialized metabolism. [ABSTRACT FROM AUTHOR]

The natural products cyanogenic glycosides (CNglcs) are present in various forage plant species including Sorghum spp., Trifolium spp., and Lotus spp. The release of toxic hydrogen cyanide (HCN) from endogenous CNglcs, which is known as cyanogenesis, leads to a serious problem for animal consumption while as defensive secondary metabolites, CNglcs play multiple roles in plant development and responses to adverse environment. Therefore, it is highly important to fully uncover the molecular mechanisms of CNglc biosynthesis and regulation to manipulate the contents of CNglcs in forage plants for fine-tuning the balance between defensive responses and food safety. This review summarizes recent studies on the production, function, polymorphism, and regulation of CNglcs in forage plants, aiming to provide updated knowledge on the ways to manipulate CNglcs for further beneficial economic effects. [ABSTRACT FROM AUTHOR]