Divergent nitrogen regulation of dhurrin biosynthesis in <i>Sorghum macrospermum</i> , a crop wild relative of Sorghum

Abstract 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.