Calcium oxide nanoparticles ameliorate cadmium toxicity in alfalfa seedlings by depriving its bioaccumulation, enhancing photosystem II functionality and antioxidant gene expression

Cadmium (Cd) is a highly toxic and carcinogenic pollutant that poses significant risks to living organisms and the environment, as it is absorbed by the plant roots and accumulates in different parts of crop during its production. A promising sustainable strategy to counteract these threats to use calcium oxide nanoparticles (CaO-NPs) as soil supplements in fodder crops. This approach has shown notable morpho-physiological and biochemical improvements under metal toxicity conditions. However, the specific mechanisms driving Cd tolerance, particularly at physio-biochemical level and antioxidant related genes expression in fodder crops including alfalfa remain unexplored. CaO-NPs supplementation can trigger various signaling pathways that lead to enhance the photosynthetic pigments formation, stomatal conductance, CO₂ assimilation rate and quantum yield of photosystem II. In this study, we evaluated various doses of CaO-NPs (0, 25, 50, and 100 mg kg⁻¹) for their efficacy in reducing Cd bioavailability and toxicity in alfalfa plants. Our results demonstrated that Ca²⁺ and Cd²⁺, which share the same ionic radius, compete for ion transport through channels. The small size and high availability of CaO-NPs facilitate their rapid translocation within plant tissues, reducing metal uptake by 61 % in shoots and 30 % in roots. Notably, application of CaO-NPs at 100 mg kg⁻¹ significantly increased shoot length (44 %) and root length (35 %) as compared to Cd-treated control plants. The highest dose of CaO-NPs also improved photosynthetic efficiency and gas exchange attributes including gs, Tr, Pn and Ci by 66 %, 27 %, 33 % and Ci 21 %, respectively, compared with the Cd treated control. Moreover, CaO-NPs (at 100 mg kg⁻¹) alleviated metal-induced oxidative stress by boosting antioxidant enzyme activities like superoxide dismutase (25 %) peroxidase (42 %), catalase (72 %) and ascorbate peroxidase (87 %) and diminishing reactive oxygen species (ROS) production when compared with sole Cd treatment. Scanning and transmission electron microscopy revealed that CaO-NPs positively impacted stomatal conductance and mitigated Cd toxicity in leaf ultrastructure. Additionally, the highest dose of CaO-NPs markedly upregulated the expression of antioxidant-related genes, MsCu/Zn SOD, MtPOD, MtCAT, and MtAPX in roots and shoots by 0.67 and 1.03 fold-change (FC), 0.61 and 0.53 FC, 0.54 and 0.88 FC, and 0.46 and 0.66 FC, respectively. In conclusion, CaO-NPs demonstrate significant potential for environmentally friendly mitigation of Cd stress in alfalfa by reducing its uptake, thereby supporting sustainable agriculture.