Zinc oxide nanoparticles and 24-epibrassinolide mitigate coexisting lead and cadmium toxicity in rice under elevated CO2: roles of metal transporters, antioxidant defense, and nutrient homeostasis

Abstract

Elevated CO₂ and heavy metal toxicity threaten global rice production and grain quality. Zinc oxide nanoparticles (ZnO-NPs) and 24-epibrassinolide (EBR) have been shown to individually mitigate heavy metal uptake in crops; their synergistic potential effect in the context of coexisting heavy metals, particularly under elevated CO₂ conditions, remains unexplored. This study investigated the efficiency of foliar ZnO-NPs (50 mg L⁻¹) and EBR (10⁻⁸ M), both individually and in combination, in reducing cadmium (Cd) and lead (Pb) accumulation and maintaining nutritional homeostasis in rice (Oryza sativa L.) cultivated in Cd–Pb co-contaminated soil under elevated CO₂ (600 μmol mol⁻¹). Results showed that elevated CO₂ exacerbated grain Cd accumulation (11.21%) while reducing Pb (19.09%) and significantly reduced nutrient content despite a non-significant increase in rice biomass as compared to the untreated control treatment under ambient CO₂. The combined application of ZnO-NPs and EBR was most effective, significantly reducing Cd (69.60%) and Pb (87.71%) in rice grain by enhancing photosynthesis, antioxidant enzyme activity (SOD, POD, CAT, APX), and lowering oxidative stress (MDA, H₂O₂, EL). At the molecular level, the combined treatment substantially downregulated the expression levels of metal transporter genes (OsHMA2, OsHMA6, OsNRAMP5) and upregulated the expression of Fe transporter gene OsIRT1, thereby improving Fe and Zn homeostasis. Additionally, nutrient analysis further revealed that ZnO-NPs and EBR co-application reversed heavy metal and elevated CO₂ induced nutrient deficits, significantly increasing grain nutrient content: Zn (163.71%), Fe (257.08%), Mn (213.37%), Mg (189.80%), Ca (313.75%), K (304.94%), and Cu (204.25%). Overall, these findings provide novel mechanistic insights into the combined application of ZnO-NPs and EBR for mitigating Cd–Pb toxicity under elevated CO₂, offering a climate-resilient strategy for safe and high-quality rice production.