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.

Comparative role of calcium oxide nanoparticles and calcium bulk fertilizer to alleviate cadmium toxicity by modulating oxidative stress, photosynthetic performance and antioxidant-defense genes expression in alfalfa.

Cadmium (Cd) toxicity poses a significant threat to soil health and sustainable food production. Its bioaccumulation in plant tissues induces phytotoxicity by affecting physiological and biochemical attributes, leading to a reduction in plant biomass and production. Recently, nanotechnology has emerged as a promising approach for addressing heavy metal toxicity in an eco-friendly manner to enhance crop production. However, the comparative role of foliar applied calcium oxide nanoparticles (CaO-NPs) and bulk calcium fertilizer under Cd stress in alfalfa remains unexplored. Herein, we studied the ameliorative role of CaO-NPs and bulk calcium (50 and 100 mg L-1) to alleviate Cd stress (30 mg kg-1) in alfalfa seedlings. Plants exposed to Cd exhibited significant decreases in morpho-physiological traits, gas exchange attributes, and pigment contents as well as increase in Cd bioaccumulation in plant tissues. Notably, exogenous application of CaO-NPs ameliorates the toxic impact of Cd by enhancing plant biomass (45%), fluorescence efficiency and gaseous exchange attributes. The maximum dose of CaO-NPs induced Cd-tolerance response accompanied by a significant increase in antioxidative enzyme activities, such as superoxide dismutase (SOD; 29%), peroxidase (POD; 41%), catalase (CAT; 36%) and ascorbate peroxidase (APX; 49%), which play positive roles in ROS scavenging. TEM examination further revealed the protective role of these NPs in averting Cd-induced damage to leaf ultrastructure and mesophyll cells. Furthermore, CaO-NPs had a substantial influence on both Cd and Ca2+ accumulation in plant tissues, while qRT‒PCR analysis demonstrated higher expression of antioxidant defense genes viz. Cu/ZnSOD (0.38 fold change (FC)), MtPOD (0.51 FC), MtCAT (0.61 FC) and MtAPX (0.79 FC) under CaO-NPs application, over Cd control. Overall, our findings suggested that exogenous CaO-NPs could be effective in alleviating the adverse effects of Cd on alfalfa seedlings to ensure food safety and support sustainable agriculture.