A factorial multi-layer analysis reveals non-additive sulfur-iron interactions shaping metabolic and transcriptional responses in durum wheat.

Reciprocal iron-sulfur regulation in durum wheat converges on a few metabolic hubs that integrate nutrient status with plant growth and performance. Iron and sulfur availabilities were factorially modulated in hydroponically grown wheat to establish how each nutrient and their interaction shape growth, ionome, amino acid and organic acid metabolism, and the expression of key S- and Fe-homeostasis genes. Sulfur availability strongly shaped plant responses to iron availability, determining biomass allocation, ionomic patterns, and the expression of sulfate transporters, sulfur-assimilatory enzymes, and iron homeostasis genes. Metabolomics highlighted citrate, O-acetylserine, and methionine as root metabolites most consistently associated with combined Fe × S treatments. Correlation analysis linked these variables with markers of sulfate transport and assimilation, defining a reproducible interaction signature across biological layers. Sulfur deficiency, especially when combined with low iron, also increased free asparagine accumulation in vegetative tissues, indicating a strong reallocation in nitrogen metabolism under dual stress. Sulfur availability is a major modulator of iron-responsive phenotypic, transcriptional, and metabolic traits in durum wheat and provides a systems-level framework for future functional studies of Fe/S crosstalk.