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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. Abstract 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.

Significance Statement The high cyanogenic glucoside content in some cassava varieties prevents herbivory but can be toxic for human consumption. The identification of an intracellular transporter gene and its allelic variation allow us to identify cultivars with up to 30% reduced cyanogenic glucoside content in cassava root., 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.

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.

Chemical defences are key components in insect–plant interactions, as insects continuously learn to overcome plant defence systems by, e.g., detoxification, excretion or sequestration. Cyanogenic glucosides are natural products widespread in the plant kingdom, and also known to be present in arthropods. They are stabilised by a glucoside linkage, which is hydrolysed by the action of β-glucosidase enzymes, resulting in the release of toxic hydrogen cyanide and deterrent aldehydes or ketones. Such a binary system of components that are chemically inert when spatially separated provides an immediate defence against predators that cause tissue damage. Further roles in nitrogen metabolism and inter- and intraspecific communication has also been suggested for cyanogenic glucosides. In arthropods, cyanogenic glucosides are found in millipedes, centipedes, mites, beetles and bugs, and particularly within butterflies and moths. Cyanogenic glucosides may be even more widespread since many arthropod taxa have not yet been analysed for the presence of this class of natural products. In many instances, arthropods sequester cyanogenic glucosides or their precursors from food plants, thereby avoiding the demand for de novo biosynthesis and minimising the energy spent for defence. Nevertheless, several species of butterflies, moths and millipedes have been shown to biosynthesise cyanogenic glucosides de novo, and even more species have been hypothesised to do so. As for higher plant species, the specific steps in the pathway is catalysed by three enzymes, two cytochromes P450, a glycosyl transferase, and a general P450 oxidoreductase providing electrons to the P450s. The pathway for biosynthesis of cyanogenic glucosides in arthropods has most likely been assembled by recruitment of enzymes, which could most easily be adapted to acquire the required catalytic properties for manufacturing these compounds. The scattered phylogenetic distribution of cyanogenic glucosides in arthropods indicates that the ability to biosynthesise this class of natural products has evolved independently several times. This is corroborated by the characterised enzymes from the pathway in moths and millipedes. Since the biosynthetic pathway is hypothesised to have evolved convergently in plants as well, this would suggest that there is only one universal series of unique intermediates by which amino acids are efficiently converted into CNglcs in different Kingdoms of Life. For arthropods to handle ingestion of cyanogenic glucosides, an effective detoxification system is required. In butterflies and moths, hydrogen cyanide released from hydrolysis of cyanogenic glucosides is mainly detoxified by β-cyanoalanine synthase, while other arthropods use the enzyme rhodanese. The storage of cyanogenic glucosides and spatially separated hydrolytic enzymes (β-glucosidases and α-hydroxynitrile lyases) are important for an effective hydrogen cyanide release for defensive purposes. Accordingly, such hydrolytic enzymes are also present in many cyanogenic arthropods, and spatial separation has been shown in a few species. Although much knowledge regarding presence, biosynthesis, hydrolysis and detoxification of cyanogenic glucosides in arthropods has emerged in recent years, many exciting unanswered questions remain regarding the distribution, roles apart from defence, and convergent evolution of the metabolic pathways involved.

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.

Release of bud dormancy in perennial woody plants is a temperature-dependent process and thus flowering in these species is heavily affected by climate change. The lack of cold winters in temperate growing regions often results in reduced flowering and low fruit yields. This is likely to decrease the availability of fruits and nuts of the Prunus spp. in the near future. In order to maintain high yields, it is crucial to gain detailed knowledge on the molecular mechanisms controlling the release of bud dormancy. Here, we studied these mechanisms using sweet cherry (Prunus avium L.), a crop where the agrochemical hydrogen cyanamide (HC) is routinely used to compensate for the lack of cold winter temperatures and to induce flower opening. In this work, dormant flower buds were sprayed with hydrogen cyanamide followed by deep RNA sequencing, identifying three main expression patterns in response to HC. These transcript level results were validated by quantitative real time polymerase chain reaction and supported further by phytohormone profiling (ABA, SA, IAA, CK, ethylene, JA). Using these approaches, we identified the most up-regulated pathways: the cytokinin pathway, as well as the jasmonate and the hydrogen cyanide pathway. Our results strongly suggest an inductive effect of these metabolites in bud dormancy release and provide a stepping stone for the characterization of key genes in bud dormancy release.

Cyanogenic glucosides are among the most widespread defense chemicals of plants. Upon plant tissue disruption, these glucosides are hydrolyzed to a reactive hydroxynitrile that releases toxic hydrogen cyanide (HCN). Yet many mite and lepidopteran species can thrive on plants defended by cyanogenic glucosides. The nature of the enzyme known to detoxify HCN to β-cyanoalanine in arthropods has remained enigmatic. Here we identify this enzyme by transcriptome analysis and functional expression. Phylogenetic analysis showed that the gene is a member of the cysteine synthase family horizontally transferred from bacteria to phytophagous mites and Lepidoptera. The recombinant mite enzyme had both β-cyanoalanine synthase and cysteine synthase activity but enzyme kinetics showed that cyanide detoxification activity was strongly favored. Our results therefore suggest that an ancient horizontal transfer of a gene originally involved in sulfur amino acid biosynthesis in bacteria was co-opted by herbivorous arthropods to detoxify plant produced cyanide. DOI: http://dx.doi.org/10.7554/eLife.02365.001, eLife digest Hydrogen cyanide is a poison that is deadly for most forms of life. Also known as prussic acid, it has killed countless humans throughout history in accidents and during the Holocaust. Hydrogen cyanide is also used by plants to defend themselves against insects and other herbivorous animals. Many plants produce chemicals called cyanogenic glycosides that can be converted into hydrogen cyanide when the plant is eaten. This is an ancient and efficient defense against all sorts of herbivores, including humans. For instance, cassava is a key source of food in sub-Saharan Africa and South America, but it contains cyanogenic glucosides and is highly toxic if eaten in unprocessed form. However, some insects and mites can thrive on cyanogenic plants, often to the extent of becoming pests on these plants. Certain moths, such as burnet moths, have gone further and now depend on cyanogenic glucosides for their own defenses against predators such as birds. How these mites and insects are capable of fending off cyanide toxicity has long remained a mystery. Now Wybouw et al. have identified a mite enzyme that detoxifies hydrogen cyanide to produce a compound called beta-cyanoalanine. Remarkably, the DNA that encodes this enzyme did not evolve in animals but originally belonged to a bacterium. Wybouw et al. show that the gene was transferred to the genome of the spider mite Tetranychus urticae perhaps a few hundred million years ago. An equivalent gene was also found in moths and butterflies, which explains why these insects can thrive on plants that produce hydrogen cyanide. This lateral gene transfer from bacteria to animals is a remarkable coalition of two kingdoms against another, and illustrates a new aspect of the chemical warfare between plants and animals. This study also increases our awareness of the importance of laterally transferred genes in the genomes of higher organisms. DOI: http://dx.doi.org/10.7554/eLife.02365.002

A spectrophotometric method for determining cyanogenic glucosides (CGs) was validated in accordance with IUPAC and ICH requirements for new methods. The method demonstrates good linearity in the range of 0.1-0.88 μgg-1 hydrocyanic acid (HCN). The accuracy of the method ranges between 95.2% and 96.4%. The precision of the method, reflected as the relative standard deviation of the replicates, is lower than 12%. The method was compared with the most used method to detect CGs in cassava roots, the one established by Essers et al. Both methods were applied to quantify CGs in commercial cassava products. The new method is a useful and reliable tool for monitoring levels of CGs in cassava commercial products. En este trabajo se valida un método espectrofotométrico para la determinación de glucósidos cianogénicos (CGs) de acuerdo a los requerimientos de IUPAC y ICH para nuevos métodos. El método demuestra una buena linealidad en el rango de 0,1-0,88 μgg-1 de ácido cianhídrico (HCN). La exactitud del método está entre 95,2 y 96,4%. La precisión del método reflejada como la desviación estándar relativa de los replicados es menor al 12%. El método se compara con el método más usado para la detección de CGs en raíces de yuca, el método de Essers et al. Ambos métodos se usan en la cuantificación de CGs en productos comerciales de yuca. El nuevo método es una herramienta útil y confiable para monitorear los niveles de CGs en productos comerciales de yuca.