Cyanogenic Glucoside Dhurrin Research Articles

A Convenient Route to Large-Scale Chemical Synthesis of p-Hydroxyphenylacetaldehyde Oxime and Its p-β-d-Glucopyranoside: Key Intermediates and Products in Plant Specialized Metabolism.

Oximes are unrecognized chameleons in general and specialized plant metabolism. E- and Z-p-hydroxyphenylacetaldehyde oxime are key intermediates in the biosynthesis of the cyanogenic glucoside dhurrin produced in sorghum. Nevertheless, none of the geometrical oxime isomers accumulate in the plant. Herein, we report a convenient route to the chemical synthesis of E- and Z-p-hydroxyphenylacetaldehyde oxime and its biologically produced p-β-d-glucopyranoside using p-hydroxyphenylacetic acid as a starting material. This starting material is also available in radiolabeled forms. All reaction steps proceeded with excellent yield under mild conditions, operational facility, and scalability.

Structure-guided engineering of key amino acids in UGT85B1 controlling substrate and stereo-specificity in aromatic cyanogenic glucoside biosynthesis.

SUMMARYCyanogenic glucosides are important defense molecules in plants with useful biological activities in animals. Their last biosynthetic step consists of a glycosylation reaction that confers stability and increases structural diversity and is catalyzed by the UDP‐dependent glycosyltransferases (UGTs) of glycosyltransferase family 1. These versatile enzymes have large and varied substrate scopes, and the structure–function relationships controlling scope and specificity remain poorly understood. Here, we report substrate‐bound crystal structures and rational engineering of substrate and stereo‐specificities of UGT85B1 from Sorghum bicolor involved in biosynthesis of the cyanogenic glucoside dhurrin. Substrate specificity was shifted from the natural substrate (S)‐p‐hydroxymandelonitrile to (S)‐mandelonitrile by combining a mutation to abolish hydrogen bonding to the p‐hydroxyl group with a mutation to provide steric hindrance at the p‐hydroxyl group binding site (V132A/Q225W). Further, stereo‐specificity was shifted from (S) to (R) by substituting four rationally chosen residues within 6 Å of the nitrile group (M312T/A313T/H408F/G409A). These activities were compared to two other UGTs involved in the biosynthesis of aromatic cyanogenic glucosides in Prunus dulcis (almond) and Eucalyptus cladocalyx. Together, these studies enabled us to pinpoint factors that drive substrate and stereo‐specificities in the cyanogenic glucoside biosynthetic UGTs. The structure‐guided engineering of the functional properties of UGT85B1 enhances our understanding of the evolution of UGTs involved in the biosynthesis of cyanogenic glucosides and will enable future engineering efforts towards new biotechnological applications.

Transcript profiles of wild and domesticated sorghum under water-stressed conditions and the differential impact on dhurrin metabolism

Main conclusionAustralian native species of sorghum contain negligible amounts of dhurrin in their leaves and the cyanogenesis process is regulated differently under water-stress in comparison to domesticated sorghum species.Cyanogenesis in forage sorghum is a major concern in agriculture as the leaves of domesticated sorghum are potentially toxic to livestock, especially at times of drought which induces increased production of the cyanogenic glucoside dhurrin. The wild sorghum species endemic to Australia have a negligible content of dhurrin in the above ground tissues and thus represent a potential resource for key agricultural traits like low toxicity. In this study we investigated the differential expression of cyanogenesis related genes in the leaf tissue of the domesticated species Sorghum bicolor and the Australian native wild species Sorghum macrospermum grown in glasshouse-controlled water-stress conditions using RNA-Seq analysis to analyse gene expression. The study identified genes, including those in the cyanogenesis pathway, that were differentially regulated in response to water-stress in domesticated and wild sorghum. In the domesticated sorghum, dhurrin content was significantly higher compared to that in the wild sorghum and increased with stress and decreased with age whereas in wild sorghum the dhurrin content remained negligible. The key genes in dhurrin biosynthesis, CYP79A1, CYP71E1 and UGT85B1, were shown to be highly expressed in S. bicolor. DHR and HNL encoding the dhurrinase and α-hydroxynitrilase catalysing bio-activation of dhurrin were also highly expressed in S. bicolor. Analysis of the differences in expression of cyanogenesis related genes between domesticated and wild sorghum species may allow the use of these genetic resources to produce more acyanogenic varieties in the future.

Cyanogenesis in the Sorghum Genus: From Genotype to Phenotype.

Domestication has resulted in a loss of genetic diversity in our major food crops, leading to susceptibility to biotic and abiotic stresses linked with climate change. Crop wild relatives (CWR) may provide a source of novel genes potentially important for re-gaining climate resilience. Sorghum bicolor is an important cereal crop with wild relatives that are endemic to Australia. Sorghum bicolor is cyanogenic, but the cyanogenic status of wild Sorghum species is not well known. In this study, leaves of wild species endemic in Australia are screened for the presence of the cyanogenic glucoside dhurrin. The direct measurement of dhurrin content and the potential for dhurrin-derived HCN release (HCNp) showed that all the tested Australian wild species were essentially phenotypically acyanogenic. The unexpected low dhurrin content may reflect the variable and generally nutrient-poor environments in which they are growing in nature. Genome sequencing of six CWR and PCR amplification of the CYP79A1 gene from additional species showed that a high conservation of key amino acids is required for correct protein function and dhurrin synthesis, pointing to the transcriptional regulation of the cyanogenic phenotype in wild sorghum as previously shown in elite sorghum.

Variation in production of cyanogenic glucosides during early plant development: A comparison of wild and domesticated sorghum

Domestication has narrowed the genetic diversity found in crop wild relatives, potentially reducing plasticity to cope with a changing climate. The tissues of domesticated sorghum (Sorghum bicolor), especially in younger plants, are cyanogenic and potentially toxic. Species of wild sorghum produce lower levels of the cyanogenic glucoside (CNglc) dhurrin than S. bicolor at maturity, but it is not known if this is also the case during germination and early growth. CNglcs play multiple roles in primary and specialised metabolism in domesticated sorghum and other crop plants. In this study, the temporal and spatial distribution of dhurrin in wild and domesticated sorghum at different growth stages was monitored in leaf, sheath and root tissues up to 35 days post germination using S. bicolor and the wild species S. brachypodum and S. macrospermum as the experimental systems. Growth parameters were also measured and allocation of plant total nitrogen (N%) to both dhurrin and nitrate (NO3−) was calculated. Negligible amounts of dhurrin were produced in the leaves of the two wild species compared to S. bicolor. The morphology of the two wild sorghums also differed from S. bicolor, with the greatest differences observed for the more distantly related S. brachypodum. S. bicolor had the highest leaf N% whilst the wild species had significantly higher root N%. Allocation of nitrogen to dhurrin in aboveground tissue was significantly higher in S. bicolor compared to the wild species but did not differ in the roots across the three species. The differences in plant morphology, dhurrin content and re-mobilisation, and nitrate/nitrogen allocation suggest that domestication has affected the functional roles of dhurrin in sorghum.

Production of the cyanogenic glycoside dhurrin in yeast

Cyanogenic glycosides are defense compounds found in a wide range of plant species, including many crops. We demonstrate that the cyanogenic glucoside dhurrin, naturally found in sorghum, can be produced at high titers in Saccharomyces cerevisiae, constituting the first report of cyanogenic glycoside production in a microbe. Genetic modifications to increase the supply of the dhurrin precursor tyrosine enabled dhurrin production in excess of 80 mg/L. The dhurrin-producing yeast strain was used as a chassis to investigate previously uncharacterized enzymes identified close to the biosynthetic gene cluster containing the dhurrin pathway enzymes. This work shows the potential of heterologous expression in yeast to facilitate investigations of plant cyanogenic glycoside pathways.

Investigation into the role of DNA methylation in cyanogenesis in sorghum (Sorghum bicolor L. Moench)

Sorghum bicolor produces the cyanogenic glucoside dhurrin, a secondary metabolite integral to plant defence and stress responses. Dhurrin production is both developmentally and environmentally regulated in S. bicolor, with high levels of variation within and between lines. Such phenotypic variation may result from polymorphic differences or epigenetic modifications in genes associated with cyanogenesis. In this study the chemical 5-Azacytidine was used to assess S. bicolor’s response to genome-wide demethylation, which had not previously been investigated in the context of cyanogenic glucoside regulation. Morphological changes, the expression levels of key genes involved in dhurrin synthesis and turnover, and the cyanogenic potential (HCNp) of leaf tissues were analysed. Treatment resulted in alterations in dhurrin synthesis, gene expression, and dhurrin levels, suggesting that DNA methylation is involved in the regulation of HCNp in the initial stages of S. bicolor development. Previously identified EMS mutants from the adult cyanide deficient class (acdc) have been found to exhibit altered dhurrin concentrations during development. This study shows that acdc mutants possess a CΔT change in the promoter of CYP79A1, a key gene in dhurrin synthesis, and that this mutation is stably inherited and associated with the acdc phenotype. To further investigate the role of epigenesis in dhurrin production, we determine the methylation status of the 250 bp region surrounding the CΔT mutation site in wild-type and mutant plants over two stages of development.

Glutathione transferases catalyze recycling of auto-toxic cyanogenic glucosides in sorghum.

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 invitro, 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.

Label-free Raman hyperspectral imaging analysis localizes the cyanogenic glucoside dhurrin to the cytoplasm in sorghum cells

Localisation of metabolites in sorghum coleoptiles using Raman hyperspectral imaging analysis was compared in wild type plants and mutants that lack cyanogenic glucosides. This novel method allows high spatial resolution in situ localization by detecting functional groups associated with cyanogenic glucosides using vibrational spectroscopy. Raman hyperspectral imaging revealed that dhurrin was found mainly surrounding epidermal, cortical and vascular tissue, with the greatest amount in cortical tissue. Numerous “hotspots” demonstrated dhurrin to be located within both cell walls and cytoplasm adpressed towards the plasmamembrane and not in the vacuole as previously reported. The high concentration of dhurrin in the outer cortical and epidermal cell layers is consistent with its role in defence against herbivory. This demonstrates the ability of Raman hyperspectral imaging to locate cyanogenic glucosides in intact tissues, avoiding possible perturbations and imprecision that may accompany methods that rely on bulk tissue extraction methods, such as protoplast isolation.

Counting the costs: nitrogen partitioning in Sorghum mutants.

Long-standing growth/defence theories state that the production of defence compounds come at a direct cost to primary metabolism when resources are limited. However, such trade-offs are inherently difficult to quantify. We compared the growth and nitrogen partitioning in wild type Sorghum bicolor (L.) Moench, which contains the cyanogenic glucoside dhurrin, with unique mutants that vary in dhurrin production. The totally cyanide deficient 1 (tcd1) mutants do not synthesise dhurrin at all whereas mutants from the adult cyanide deficient class 1 (acdc1) have decreasing concentrations as plants age. Sorghum lines were grown at three different concentrations of nitrogen. Growth, chemical analysis, physiological measurements and expression of key genes in biosynthesis and turnover were determined for leaves, stems and roots at four developmental stages. Nitrogen supply, ontogeny, tissue type and genotype were all important determinants of tissue nitrate and dhurrin concentration and turnover. The higher growth of acdc1 plants strongly supports a growth/defence trade-off. By contrast, tcd1 plants had slower growth early in development, suggesting that dhurrin synthesis and turnover may be beneficial for early seedling growth rather than being a cost. The relatively small trade-off between nitrate and dhurrin suggests these may be independently regulated.

Responses of Synechocystis sp. PCC 6803 to heterologous biosynthetic pathways

BackgroundThere are an increasing number of studies regarding genetic manipulation of cyanobacteria to produce commercially interesting compounds. The majority of these works study the expression and optimization of a selected heterologous pathway, largely ignoring the wholeness and complexity of cellular metabolism. Regulation and response mechanisms are largely unknown, and even the metabolic pathways themselves are not fully elucidated. This poses a clear limitation in exploiting the rich biosynthetic potential of cyanobacteria.ResultsIn this work, we focused on the production of two different compounds, the cyanogenic glucoside dhurrin and the diterpenoid 13R-manoyl oxide in Synechocystis PCC 6803. We used genome-scale metabolic modelling to study fluxes in individual reactions and pathways, and we determined the concentrations of key metabolites, such as amino acids, carotenoids, and chlorophylls. This allowed us to identify metabolic crosstalk between the native and the introduced metabolic pathways. Most results and simulations highlight the metabolic robustness of cyanobacteria, suggesting that the host organism tends to keep metabolic fluxes and metabolite concentrations steady, counteracting the effects of the heterologous pathway. However, the amino acid concentrations of the dhurrin-producing strain show an unexpected profile, where the perturbation levels were high in seemingly unrelated metabolites.ConclusionsThere is a wealth of information that can be derived by combining targeted metabolite identification and computer modelling as a frame of understanding. Here we present an example of how strain engineering approaches can be coupled to ‘traditional’ metabolic engineering with systems biology, resulting in novel and more efficient manipulation strategies.

The CYP79A1 catalyzed conversion of tyrosine to (E)-p-hydroxyphenylacetaldoxime unravelled using an improved method for homology modeling

The vast diversity and membrane-bound nature of plant P450s makes it challenging to study the structural characteristics of this class of enzymes especially with respect to accurate intermolecular enzyme-substrate interactions. To address this problem we here apply a modified hybrid structure strategy for homology modeling of plant P450s. This allows for structural elucidation based on conserved motifs in the protein sequence and secondary structure predictions. We modeled the well-studied Sorghum bicolor cytochrome P450 CYP79A1 catalyzing the first step in the biosynthesis of the cyanogenic glucoside dhurrin. Docking experiments identified key regions of the active site involved in binding of the substrate and facilitating catalysis. Arginine 152 and threonine 534 were identified as key residues interacting with the substrate. The model was validated experimentally using site-directed mutagenesis. The new CYP79A1 model provides detailed insights into the mechanism of the initial steps in cyanogenic glycoside biosynthesis. The approach could guide functional characterization of other membrane-bound P450s and provide structural guidelines for elucidation of key structure-function relationships of other plant P450s.

The biosynthetic gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor contains its co-expressed vacuolar MATE transporter.

Genomic gene clusters for the biosynthesis of chemical defence compounds are increasingly identified in plant genomes. We previously reported the independent evolution of biosynthetic gene clusters for cyanogenic glucoside biosynthesis in three plant lineages. Here we report that the gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor additionally contains a gene, SbMATE2, encoding a transporter of the multidrug and toxic compound extrusion (MATE) family, which is co-expressed with the biosynthetic genes. The predicted localisation of SbMATE2 to the vacuolar membrane was demonstrated experimentally by transient expression of a SbMATE2-YFP fusion protein and confocal microscopy. Transport studies in Xenopus laevis oocytes demonstrate that SbMATE2 is able to transport dhurrin. In addition, SbMATE2 was able to transport non-endogenous cyanogenic glucosides, but not the anthocyanin cyanidin 3-O-glucoside or the glucosinolate indol-3-yl-methyl glucosinolate. The genomic co-localisation of a transporter gene with the biosynthetic genes producing the transported compound is discussed in relation to the role self-toxicity of chemical defence compounds may play in the formation of gene clusters.

Fusion of Ferredoxin and Cytochrome P450 Enables Direct Light-Driven Biosynthesis.

Cytochrome P450s (P450s) are key enzymes in the synthesis of bioactive natural products in plants. Efforts to harness these enzymes for in vitro and whole-cell production of natural products have been hampered by difficulties in expressing them heterologously in their active form, and their requirement for NADPH as a source of reducing power. We recently demonstrated targeting and insertion of plant P450s into the photosynthetic membrane and photosynthesis-driven, NADPH-independent P450 catalytic activity mediated by the electron carrier protein ferredoxin. Here, we report the fusion of ferredoxin with P450 CYP79A1 from the model plant Sorghum bicolor, which catalyzes the initial step in the pathway leading to biosynthesis of the cyanogenic glucoside dhurrin. Fusion with ferredoxin allows CYP79A1 to obtain electrons for catalysis by interacting directly with photosystem I. Furthermore, electrons captured by the fused ferredoxin moiety are directed more effectively toward P450 catalytic activity, making the fusion better able to compete with endogenous electron sinks coupled to metabolic pathways. The P450-ferredoxin fusion enzyme obtains reducing power solely from its fused ferredoxin and outperforms unfused CYP79A1 in vivo. This demonstrates greatly enhanced electron transfer from photosystem I to CYP79A1 as a consequence of the fusion. The fusion strategy reported here therefore forms the basis for enhanced partitioning of photosynthetic reducing power toward P450-dependent biosynthesis of important natural products.

Drought-induced changes in nitrogen partitioning between cyanide and nitrate in leaves and stems of sorghum grown at elevated CO2 are age dependent

Sorghum [Sorghum bicolor (L.) Moench] is the world’s fifth most important crop, grown for forage, grain, and as a biofuel. Fast growing and drought tolerant, it is increasingly being planted as a climate change-ready alternative to maize. All parts of the sorghum plant except the grain contain the cyanogenic glucoside dhurrin, which breaks down to release hydrogen cyanide (prussic acid) when plant tissue is disrupted. Fresh forage, hay and silage may be toxic to stock when derived from plants that are young, droughted or heavily fertilized. Sorghum also stores nitrate, which can cause nitrite toxicity. The impact of elevated CO2 on dhurrin and nitrate concentration is unknown. It is important to understand how global environmental change will affect composition in order to be able to predict the safety of the crop in coming decades. Sorghum was grown experimentally at elevated CO2 in two free-air CO2 enrichment (FACE) experiments at ambient and elevated CO2 (ca. 550ppm) and either irrigated regularly or only once after sowing in consecutive years and sampled at different stages of development. Since FACE-grown sorghum has been shown to have improved water status we hypothesized that they would contain less dhurrin. We found the most important factors governing cyanide concentration were (in decreasing order): plant age, irrigation treatment and tissue type. For nitrate, tissue type was by far the most important factor, followed by plant age, and then irrigation treatment. The concentration of CO2 in the atmosphere had no significant effect on the total nitrogen concentration, or the concentrations of cyanide and nitrate. As sorghum is becomes more widely used for forage, it will be important to have simple methods to assess the cyanide levels in the field or to develop new, low cyanogenic varieties to ensure that it is safe for grazing.

The bifurcation of the cyanogenic glucoside and glucosinolate biosynthetic pathways.

The biosynthetic pathway for the cyanogenic glucoside dhurrin in sorghum has previously been shown to involve the sequential production of (E)- and (Z)-p-hydroxyphenylacetaldoxime. In this study we used microsomes prepared from wild-type and mutant sorghum or transiently transformed Nicotianabenthamiana to demonstrate that CYP79A1 catalyzes conversion of tyrosine to (E)-p-hydroxyphenylacetaldoxime whereas CYP71E1 catalyzes conversion of (E)-p-hydroxyphenylacetaldoxime into the corresponding geometrical Z-isomer as required for its dehydration into a nitrile, the next intermediate in cyanogenic glucoside synthesis. Glucosinolate biosynthesis is also initiated by the action of a CYP79 family enzyme, but the next enzyme involved belongs to the CYP83 family. We demonstrate that CYP83B1 from Arabidopsisthaliana cannot convert the (E)-p-hydroxyphenylacetaldoxime to the (Z)-isomer, which blocks the route towards cyanogenic glucoside synthesis. Instead CYP83B1 catalyzes the conversion of the (E)-p-hydroxyphenylacetaldoxime into an S-alkyl-thiohydroximate with retention of the configuration of the E-oxime intermediate in the final glucosinolate core structure. Numerous microbial plant pathogens are able to detoxify Z-oximes but not E-oximes. The CYP79-derived E-oximes may play an important role in plant defense.

Metabolic consequences of knocking outUGT85B1, the gene encoding the glucosyltransferase required for synthesis of dhurrin inSorghum bicolor(L. Moench)

Many important food crops produce cyanogenic glucosides as natural defense compounds to protect against herbivory or pathogen attack. It has also been suggested that these nitrogen-based secondary metabolites act as storage reserves of nitrogen. In sorghum, three key genes, CYP79A1, CYP71E1 and UGT85B1, encode two Cytochrome P450s and a glycosyltransferase, respectively, the enzymes essential for synthesis of the cyanogenic glucoside dhurrin. Here, we report the use of targeted induced local lesions in genomes (TILLING) to identify a line with a mutation resulting in a premature stop codon in the N-terminal region of UGT85B1. Plants homozygous for this mutation do not produce dhurrin and are designated tcd2 (totally cyanide deficient 2) mutants. They have reduced vigor, being dwarfed, with poor root development and low fertility. Analysis using liquid chromatography-mass spectrometry (LC-MS) shows that tcd2 mutants accumulate numerous dhurrin pathway-derived metabolites, some of which are similar to those observed in transgenic Arabidopsis expressing the CYP79A1 and CYP71E1 genes. Our results demonstrate that UGT85B1 is essential for formation of dhurrin in sorghum with no co-expressed endogenous UDP-glucosyltransferases able to replace it. The tcd2 mutant suffers from self-intoxication because sorghum does not have a feedback mechanism to inhibit the initial steps of dhurrin biosynthesis when the glucosyltransferase activity required to complete the synthesis of dhurrin is lacking. The LC-MS analyses also revealed the presence of metabolites in the tcd2 mutant which have been suggested to be derived from dhurrin via endogenous pathways for nitrogen recovery, thus indicating which enzymes may be involved in such pathways.

The necrotrophic effector protein SnTox3 re-programs metabolism and elicits a strong defence response in susceptible wheat leaves.

BackgroundThe fungus Stagonospora nodorum is a necrotrophic pathogen of wheat. It causes disease by secreting proteinaceous effectors which interact with proteins encoded by dominant susceptibility genes in the host. The outcome of these interactions results in necrosis, allowing the fungus to thrive on dead plant material. The mechanisms of these effectors though are poorly understood. In this study, we undertake a comprehensive transcriptomics, proteomic and metabolomic approach to understand how a susceptible wheat cultivar responds to exposure to the Stagonospora nodorum effector protein SnTox3.ResultsMicroarray and proteomic studies revealed that SnTox3 strongly induced responses consistent with those previously associated with classical host defence pathways including the expression of pathogenicity-related proteins and the induction of cell death. Collapse of the photosynthetic machinery was also apparent at the transcriptional and translational level. SnTox3-infiltrated wheat leaves also showed a strong induction of enzymes involved in primary metabolism consistent with increases in hexoses, amino acids and organic acids as determined by primary metabolite profiling. Methionine and homocysteine metabolism was strongly induced upon exposure to SnTox3. Pathogenicity in the presence of homocysteine was inhibited confirming that the compound has a role in plant defence. Consistent with the strong defence responses observed, secondary metabolite profiling revealed the induction of several compounds associated with plant defence, including the phenylpropanoids chlorogenic acid and feruloylquinic acid, and the cyanogenic glucoside dhurrin. Serotonin did not accumulate subsequent to SnTox3 infiltration.ConclusionsThese data support the theory that the SnTox3 effector protein elicits a host cell death response to facilitate the pathogen’s necrotrophic infection cycle. Our data also demonstrate that the mechanism of SnTox3 appears distinct from the previously characterised Stagonospora nodorum effector SnToxA. Collectively, this comprehensive analysis has advanced our understanding of necrotrophic effector biology and highlighted the complexity of effector-triggered susceptibility.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0215-5) contains supplementary material, which is available to authorized users.

Effects of PEG-induced osmotic stress on growth and dhurrin levels of forage sorghum

Sorghum (Sorghum bicolor L. Moench) is a valuable forage crop in regions with low soil moisture. Sorghum may accumulate high concentrations of the cyanogenic glucoside dhurrin when drought stressed resulting in possible cyanide (HCN) intoxication of grazing animals. In addition, high concentrations of nitrate, also potentially toxic to ruminants, may accumulate during or shortly after periods of drought. Little is known about the degree and duration of drought-stress required to induce dhurrin accumulation, or how changes in dhurrin concentration are influenced by plant size or nitrate metabolism. Given that finely regulating soil moisture under controlled conditions is notoriously difficult, we exposed sorghum plants to varying degrees of osmotic stress by growing them for different lengths of time in hydroponic solutions containing polyethylene glycol (PEG). Plants grown in medium containing 20% PEG (−0.5 MPa) for an extended period had significantly higher concentrations of dhurrin in their shoots but lower dhurrin concentrations in their roots. The total amount of dhurrin in the shoots of plants from the various treatments was not significantly different on a per mass basis, although a greater proportion of shoot N was allocated to dhurrin. Following transfer from medium containing 20% PEG to medium lacking PEG, shoot dhurrin concentrations decreased but nitrate concentrations increased to levels potentially toxic to grazing ruminants. This response is likely due to the resumption of plant growth and root activity, increasing the rate of nitrate uptake. Data presented in this article support a role for cyanogenic glucosides in mitigating oxidative stress.

Evaluation of the activity of dhurrin and sorghum towards Meloidogyne incognita

The epidermal cells of leaf and roots of sorghum (Sorghum bicolor), sudangrass (S. sudanensis) and sorghum-sudangrass hybrids (S. bicolor × S. sudanensis) contain the cyanogenic glucoside dhurrin, which can degrade into hydrogen cyanide (HCN), well known for its toxicity to many organisms, including nematodes. In vitro bioassays were carried out with the aim of evaluating the nematicidal and the nematistatic effects of dhurrin concentrations on second-stage juveniles (J2) of Meloidogyne incognita, and the effectiveness of dhurrin in decreasing or preventing hatching of M. incognita. Furthermore, Sorghum hybrid cv. Super Dolce 10, selected for its dhurrin content, and Eruca sativa sel. Nemat were used in two glasshouse experiments to study the host-nematode relationship and the development of M. incognita life cycle in the roots. In the in vitro bioassays, Sorghum hybrid cv. Super Dolce 10 showed an inhibitory effect on hatching, a slow nematicidal effect on J2 and no nematistatic activity. In the glasshouse experiments, both Sorghum hybrid cv. Super Dolce 10 and E. sativa sel. Nemat proved to be a poor to non-host of M. incognita, with reproduction factor (R) always <1, compared with Solanum lycopersicum cv. UC82, an excellent host (R = 43). In both bioactive accessions two generations were completed in 15 weeks (four cycles in S. lycopersicum), but with the production of very few egg masses, while root infestation was also always very low, with gall index (G.I.) < 1, compared with S. lycopersicum (G.I. = 4). In this study, the effectiveness of E. sativa sel. Nemat in controlling M. incognita confirmed data from previous work.