ABSTRACT Dhurrin, a cyanogenic glucoside, plays an important role in Sorghum bicolor physiology and defense. The concentration of dhurrin in sorghum is influenced by both nitrogen status and stage of plant organ development. While nitrogen resupply activates the expression of genes for dhurrin biosynthesis, the molecular mechanisms underlying this regulation remain unclear. In this study, we investigated the transcriptional response of sorghum to nitrogen resupply following growth under nitrogen‐limiting conditions. Using a time‐course design, we measured hydrogen cyanide potential (HCNp), growth, and nitrate content at 0‐, 2‐, 6‐, 12‐, 24‐, 36‐, 48‐, and 60‐h after resupply and collected tissue for RNAseq analysis in parallel for analysis of gene expression and construction of gene regulatory networks (GRNs). HCNp (mg g −1 DW) increased significantly in leaf and stem tissues following nitrogen resupply, with increases in the leaf partially driven by continued declines in controls under ongoing nitrogen stress. Expression of the dhurrin pathway genes was upregulated in leaves from 24 h after nitrogen resupply, with diel expression patterns observable over the remaining time points. No upregulation was observed in roots or stems, suggesting that developmental context overrides environmental cues. GRN analysis identified candidate transcription factors regulating dhurrin biosynthesis genes, including members of the MYB, bZIP, and GARP‐type transcription factor families. Some of these candidate transcription factors may be involved in relieving senescence‐associated suppression of dhurrin biosynthesis and link nitrogen signaling to pathway activation. These findings provide new insight into the nitrogen‐responsive regulation of dhurrin in sorghum, highlighting candidate regulators for future functional characterization.

In Australia, Sorghum bicolor is frequently produced under dryland cropping systems, making these crops particularly susceptible to stress. Sorghum also contains the cyanogenic glucoside dhurrin, a toxic metabolite which is a major concern where sorghum is grown as livestock forage. The wild Australian relatives of S. bicolor, including species within the Chaetosorghum and Stiposorghum subgenera, contain minimal concentrations of dhurrin compared to domesticated sorghum. As the core biosynthetic enzymes are conserved, they provide a valuable comparative system to explore differences in the regulation of dhurrin biosynthesis. This study examined the transcriptional and metabolic response of Sorghum macrospermum to nitrogen resupply following nitrogen-limiting growth. Over a 48-hour time-course, changes in nitrate and dhurrin accumulation, and expression of key genes involved in nitrogen metabolism, dhurrin biosynthesis, and candidate transcriptional regulators were measured. In S. macrospermum, dhurrin accumulation and upregulation of the biosynthetic gene SmCYP79A1 occurred in root tissues from 12 to 48 hours post-resupply. Very low dhurrin levels and negligible SmCYP79A1 expression were observed in leaves and stems. Notably, nitrate levels remained stable over time, suggesting distinct nitrogen management strategies. Candidate regulators previously implicated in dhurrin biosynthesis in S. bicolor were not detected in the roots of S. macrospermum, indicating that different regulatory mechanisms drive the root-specific response. These findings highlight the evolutionary divergence in nitrogen and dhurrin regulation between domesticated and wild sorghum species. They further highlight S. macrospermum as a potential genetic resource for developing low-cyanide and nitrogen-use efficient sorghum with improved resilience to suit dryland cropping systems.

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.

The cyanogenic glucoside, dhurrin, present in Sorghum bicolor is thought to have multiple functions, including in defence against herbivory. The hormone methyl jasmonate (MeJA) is also induced by herbivory and is key to instigating defence processes in plants. To investigate whether dhurrin is induced in response to herbivore attack and also to the associated presence of MeJA, sorghum plants were either wounded or exogenous MeJA was applied. We show that specific wounding (pin board and perforation) and the application of MeJA increases dhurrin concentration in leaves and sheath tissue 12 h after treatment. Quantitative PCR shows that the expression of two genes, SbCYP79A1 and SbUGT85B1, involved in the synthesis of dhurrin are significantly induced by exogenous MeJA and by wounding. Analysis of 2 kb of sequence upstream of the start codon of SbCYP79A1 identifies several cis-acting elements that have been linked to MeJA induction. A promoter deletion series, coupled to GFP, and transiently expressed in Nicotiana benthamiana suggests that there are potentially three sequence motifs (~-925 to -976) involved in the binding of transcription factors that result in increased expression of SbCYP79A1 and the synthesis of dhurrin in response to MeJA.

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.

Australian 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.

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.

Sorghum [Sorghum bicolor (L.) Moench] is an important food crop with a diverse gene pool residing in its wild relatives. A total of 15 sorghum accessions from the unexploited wild gene pool of the Sorghum genus, representing the five subgenera, were sequenced, and the complete chloroplast genomes and 99 common single‐copy concatenated nuclear genes were assembled. Annotation of the chloroplast genomes identified a total of 81 protein‐coding genes, 38 tRNA, and four rRNA genes. The gene content and gene order among the species was identical. A total of 153 nonsynonymous amino acid changes in 40 genes were identified across the species. Phylogenetic analysis of both the whole chloroplast genome and nuclear genes revealed a similar topology with two distinct clades within the genus. The species within the subgenera Eusorghum, Chaetosorghum, and Heterosorghum clustered in one clade, whereas the species within the subgenera Parasorghum and Stiposorghum clustered in a second clade. However, the subgenera Parasorghum and Stiposorghum were not monophyletic, suggesting the need for further research to resolve the relationships within this group. The close relationship between the two monotypic subgenera Chaetosorghum and Heterosorghum suggests that species within these subgenera could be considered as one group. This analysis provides an improved understanding of the genetic relationships within the Sorghum genus and defines diversity in wild sorghum species that may be useful for crop improvement.

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.

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.

In plants, the production of secondary metabolites is considered to be at the expense of primary growth. Sorghum produces a cyanogenic glycoside (dhurrin) that is believed to act as its chemical defence. Studies have shown that acyanogenic plants are smaller in size compared to the wildtype. This study aimed to investigate whether the small plant size is due to delayed germination or due to the lack of dhurrin derived nitrogen. A novel plant system consisting of totally cyanide deficient class 1 (tcd1) and adult cyanide deficient 1 (acdc1) mutant lines was employed. The data for germination, plant height and developmental stage during seedling development and final plant reproductive fitness was recorded. The possible role of phytohormones in recovering the wildtype phenotype, especially in developmentally acyanogenic acdc1 line, was also investigated. The data on plant growth have shown that the lack of dhurrin is disadvantageous to growth, but only at the early developmental stage. The tcd1 plants also took longer to mature probably due to delayed flowering. None of the tested hormones were able to recover the wildtype phenotype. We conclude that the generation of dhurrin is advantageous for plant growth, especially at critical growth stages like germinating seed by providing a ready source of reduced nitrogen.

Using a Sorghum bicolor cultivar and seven wild Sorghum species endemic to Australia as our experimental system, we monitored their different responses to drought by assessing growth and morphological, physiological and biochemical parameters. Drought stress significantly decreased height, biomass, the maximum potential quantum efficiency of photosystem II, photosynthetic rate and relative water content in S. bicolor, while several of the wild species were much more tolerant. Drought significantly increased dhurrin concentration in aboveground tissue in S. bicolor but not in the wild species. Root dhurrin content was unaffected by drought in S. bicolor, in contrast to the varied responses observed in the wild species. Sorghum macrospermum and S. brachypodum maintained relatively high growth and photosynthetic performance under drought, with negligible aboveground dhurrin content. These wild species are promising candidates for sorghum crop improvement.

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.

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.

The majority of flowering-plant species can survive complete air-dryness in their seed and/or pollen. Relatively few species (‘resurrection plants’) express this desiccation tolerance in their foliage. Knowledge of the regulation of desiccation tolerance in resurrection plant foliage is reviewed. Elucidation of the regulatory mechanism in resurrection grasses may lead to identification of genes that can improve stress tolerance and yield of major crop species. Well-hydrated leaves of resurrection plants are desiccation-sensitive and the leaves become desiccation tolerant as they are drying. Such drought-induction of desiccation tolerance involves changes in gene-expression causing extensive changes in the complement of proteins and the transition to a highly-stable quiescent state lasting months to years. These changes in gene-expression are regulated by several interacting phytohormones, of which drought-induced abscisic acid (ABA) is particularly important in some species. Treatment with only ABA induces desiccation tolerance in vegetative tissue of Borya constricta Churchill. and Craterostigma plantagineum Hochstetter. but not in the resurrection grass Sporobolus stapfianus Gandoger. Suppression of drought-induced senescence is also important for survival of drying. Further research is needed on the triggering of the induction of desiccation tolerance, on the transition between phases of protein synthesis and on the role of the phytohormone, strigolactone and other potential xylem-messengers during drying and rehydration.

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.

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.

Rising sea levels are threatening agricultural production in coastal regions due to inundation and contamination of groundwater. The development of more salt-tolerant crops is essential. Cassava is an important staple, particularly among poor subsistence farmers. Its tolerance to drought and elevated temperatures make it highly suitable for meeting global food demands in the face of climate change, but its ability to tolerate salt is unknown. Cassava stores nitrogen in the form of cyanogenic glucosides and can cause cyanide poisoning unless correctly processed. Previous research demonstrated that cyanide levels are higher in droughted plants, possibly as a mechanism for increasing resilience to oxidative stress. We determined the tolerance of cassava to salt at two different stages of development, and tested the hypothesis that cyanide toxicity would be higher in salt-stressed plants. Cassava was grown at a range of concentrations of sodium chloride (NaCl) at two growth stages: tuber initiation and tuber expansion. Established plants were able to tolerate 100mM NaCl but in younger plants 40mM was sufficient to retard plant growth severely. Nutrient analysis showed that plants were only able to exclude sodium at low concentrations. The foliar cyanogenic glucoside concentration in young plants increased under moderate salinity stress but was lower in plants grown at high salt. Importantly, there was no significant change in the cyanogenic glucoside concentration in the tubers. We propose that the mechanisms for salinity tolerance are age dependent, and that this can be traced to the relative cost of leaves in young and old plants.

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.