078 | Stress-driven Dhurrin accumulation in sorghum revealed by physiological and biochemical markers

Alessio Giannilivigni¹, Manuela Giraldo-Acosta², Flavia Girolami³, Stefano Giantin⁴, Luca Carisio⁴, Carlo Nebbia³, Marino Bañón Arnao², Alberico Franzin⁴, Giuseppe Mannino¹. | ¹Department of Life Sciences and Systems Biology, Plant Physiology Unit, University of Turin, Italy; ²Department of Plant Biology Plant Physiology, University of Murcia, Spain; ³Department of Veterinary Sciences, University of Turin, Grugliasco [TO], Italy; ⁴Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta IZSPLV, Turin, Italy; ⁵Green Has Italia Spa, Plant Physiology Unit, Canale [CN], Italy.

Abstract

Dhurrin is a cyanogenic glycoside naturally occurring in Sorghum bicolor, representing a major safety concern for animal feeding, as its hydrolysis into toxic hydrogen cyanide can lead to the release of hydrogen cyanide and causing poisoning in livestock. Dhurrin accumulation is highly variable and strongly influenced by environmental conditions, particularly abiotic stresses. In this work, we investigated how drought, salinity, heat, and their combinations modulate physiological, biochemical, and metabolic pathways associated with stress resilience and cyanogenic glycoside biosynthesis. Plants were grown under controlled conditions (FC100), subjected to individual stress (drought, FC40; salt, NaCl; or heat stress, 40C) or a combination of them (FC40+40C; FC40+NaCl; 40C+NaCl). An integrated set of biochemical, transcriptomic and metabolic indicators was selected to comprehensively assess the impact of the applied stresses, including photosynthetic pigments, osmo-protectants, oxidative markers, phytohormones, fatty acids and secondary metabolites, which were quantitatively analyzed using UV/Vis assays, qRT-PCR analysis, and HPLC–MS/MS profiling. Our results demonstrate that drought and salinity markedly enhanced proline and abscisic acid levels, accompanied by shifts in chlorophyll composition and increased antioxidant capacity, whereas heat stress induced the strongest oxidative imbalance, reflected in elevated H₂O₂. Dhurrin accumulation increased under salinity and reached its maximum under combined stresses, particularly heat plus drought or heat plus salinity, revealing a synergistic effect on cyanogenic potential. Flavonoid profiling further highlighted distinct stress-specific metabolic signatures, confirmed by multivariate clustering that separated individual stresses from their combinations. Overall, these findings identify reliable physiological and metabolic markers associated with stress-induced dhurrin biosynthesis and provide mechanistic insights relevant for cyanogenic risk assessment, agronomic management, and the development of sorghum genotypes with improved resilience and reduced toxicity.