Phosphorus counters arsenic phytotoxicity in wheat by activating riboflavin-mediated defense response rather than reducing arsenic accumulation

Soil arsenic (As) contamination represents a global concern, as its accumulation leads to crop yield reduction and human health risks through food chain. Although the protective role of phosphorus (P) against As phytotoxicity has been widely documented, underlying mechanisms remained debated. This study systematically investigated the function of P in alleviating As toxicity in wheat Triticum aestivum L. by integrating individual uptake and growth parameters, physiological indices, visualized analysis, and metabolomic response. It was found that P supplementation (80 μm) significantly ameliorated As-induced (2.5 μm) root growth inhibition, oxidative stress, and membrane damage, with relative root elongation increased from 61.4% to 99.3% and MDA content reduced from 6.63 to 3.79 nmol/mg. Notably, under As (2.5 μm) treatment, As accumulation (0.156 to 0.149 mg/g) did not differ significantly with elevated P uptake ranging from 5.32 to 7.46 mg/g in wheat roots, suggesting toxic mitigation effect of P is not by suppressing As uptake. Non-targeted metabolomics revealed As exposure primarily disrupted biological pathways related to amino acids synthesis and activation, accompanied by dysregulation of the involved metabolites, such as arginine, alanine, tryptophan, threonine, and histidine, indicating that nitrogen metabolism disturbance at molecular level shaped the adverse effects at organismal level. Remarkably, these disturbances were eliminated at the highest P level, further supporting the ameliorative effect of P on As toxicity. Concurrently, P upregulated the defense-related riboflavin metabolism pathway, which are essential for multiple fundamental processes of plant. These findings demonstrated that the presence of P helped in counteracting As-induced harm not by limiting uptake but by enhancing stress defense response, thereby advancing our understanding of how P modifies As toxicity.