Fe₃O₄ nanoparticles alleviate florfenicol-induced phytotoxicity in soybean seedling through multi-level regulation of photosynthesis, carbon metabolism, and nutrient homeostasis.

The threat posed by antibiotic contamination to agricultural ecosystems requires effective alleviation strategies. This study characterized the multi-level regulatory network by which Fe₃O₄ nanoparticles (NPs) restore soybean carbon metabolism under florfenicol stress. In a pot experiment across three growth stages (VE, V1, V2), florfenicol suppressed photosynthetic systems, reducing chlorophyll content by 19-65%, Fv/Fm by 30-56%, and Rubisco activity by 31-36%. Fe₃O₄-NPs treatment (40-80 mg/kg) reversed these effects, increasing chlorophyll content (up to 88%), enhancing chlorophyll fluorescence parameters (Fv/Fm, Y(II), ETR), and reducing non-photochemical energy dissipation (Y(NO)). Mechanistically, Fe₃O₄-NPs up-regulated key genes involved in chlorophyll synthesis, photosystem II, Rubisco subunits, sucrose synthesis, and sugar transport; and restored Rubisco, sucrose phosphate synthase, sucrose synthase, and amylase activities. Metabolomic and KEGG analyses revealed enrichment of metabolites in pathways related to core carbon metabolism and defense-related signaling, with potential involvement of hormone signaling pathways in leaves. Fe₃O₄-NPs restored the accumulation of key nutrients-which serve as cofactors for Rubisco (Mg), glycolytic enzymes (K), electron transport (Cu, Fe), and antioxidant defense (Cu, Zn)-linking nutrient homeostasis to photosynthetic carbon assimilation and energy metabolism. These findings establish a multi-level regulatory framework for Fe₃O₄-NP-mediated alleviation of antibiotic stress, offering a potential strategy for safeguarding crop productivity in antibiotic-contaminated soils.