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.

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.