Mitigating cadmium toxicity in rice through tandem application of zinc oxide nanoparticles and Serendipita indica as revealed by multi-omics and NMT-based ion flux analysis

Cadmium (Cd) contamination in agricultural soils poses a significant risk to crop growth and food safety. Zinc oxide nanoparticles (ZnO-NPs) and the endophytic fungus Serendipita indica (S. indica) both have demonstrated their potential in ameliorating Cd stress in plants, but their combined effects and underlying mechanisms are still underexplored. This study investigates how the combined use of S. indica and ZnO-NPs can counteract Cd toxicity in two rice genotypes with different Cd accumulation tendencies. Two genotypes, Heizhan-43 (Hz43) and Yinni-801 (Yi801), were treated with Cd, ZnO-NPs, and S. indica, both individually and in combination. Non-invasive Micro-test technology (NMT) was employed to measure real-time ion fluxes in rice roots. Integrated analyses using MetaboAnalyst's Joint-Pathway Analysis module were conducted to assess metabolic and transcriptomic responses. The combined treatments significantly reduced Cd accumulation in both shoots and roots, with Hz43 showing a greater reduction in root Cd content. Nutrients like zinc and phosphorus were increased in Yi801 shoots, and the stress-responsive metabolites, such as proline and glycine betaine, were induced more in shoots than roots, particularly in Hz43. As revealed by NMT, Yi801 showed higher Cd^{2 +} and Ca²⁺ influxes under stress conditions, indicating that it is more responsive to the S. indica and ZnO-NPs treatments compared to Hz43, which exhibits a more moderate influx response. Transcriptomic and metabolomic analyses revealed enhanced peroxidase-related gene expression, reduced oxidative burst, and modulation of key metabolic pathways, including carbon metabolism in Hz43 and unsaturated fatty acid biosynthesis in Yi801. Additionally, the phenylpropanoid pathway was significantly upregulated, contributing to increased flavonoid and lignin production, hence enhancing plant defense mechanisms. The combined application of S. indica and ZnO-NPs effectively mitigates Cd toxicity, enhances nutrient uptake and promotes plant growth, offering a sustainable strategy for Cd-contaminated fields to ensure food safety and environmental health in the long run.