Interactive effect of biochar and nano-calcium oxide on alfalfa cadmium detoxification: Boosting antioxidant defense, regulating gene expression, and remediating soil

Cadmium (Cd) contamination in soil severely impacts crop productivity, poses significant environmental and health risks, and necessitates effective remediation techniques. The combined application of wheat straw biochar (BC) and calcium oxide nanoparticles (nCaO) presents a promising approach to reducing Cd bioavailability in soil and mitigating its toxicity in alfalfa seedlings - an aspect not previously reported. The integration of calcium oxide nanoparticles-biochar (nCaO-BC) into soil effectively immobilizes Cd via adsorption and increasing OH − content in the soil solution, which promotes Cd(OH) 2 precipitation. The nCaO-BC raised soil pH (0.62 units) and reduced bioavailable Cd (77 %) compared to the Cd-contaminated control (Ck-Cd). Consequently, Cd concentration in alfalfa roots and shoots decreased by 54 % and 83 %, respectively. Furthermore, nCaO-BC amendment markedly enhanced plant biomass, stimulated antioxidant activity, and upregulated key antioxidant and calcium transporter genes including [ MsAPX (3.36-fold), MtCAT (1.48-fold), MtPOD (1.68-fold), MsCu/Zn SOD (2.20-fold), MtGSH (1.83-fold), and MtMDHAR (2.29-fold)], and MtCDPK1 (2.91-fold). Conversely, it downregulated Cd transporter genes MtHMA3 (2.01-fold) and MtNRAMP5 (1.75-fold) over the Ck-Cd treatment. Confocal microscopy further confirmed that nCaO-BC effectively preserved cellular integrity by mitigating oxidative stress. Moreover, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) investigation demonstrated reduced Cd toxicity to stomata, guard cells, and mesophyll cell ultrastructure following nCaO-BC treatment. These findings provide novel insights into sustainable agricultural practices, demonstrating the potential of nCaO-BC to reduce Cd mobility within the soil-plant system, thus offering a viable solution to the persistent challenge of heavy metal contamination in agricultural soils. • Cadmium (Cd) pollution in agricultural soils threatens crop yield and quality. • nCaO and BC amendments improved morphophysiological traits and reduced Cd translocation under Cd stress. • nCaO-BC activated the antioxidative enzyme system, protecting alfalfa from oxidative damage. • nCaO-BC enhanced gaseous exchange, improved stomatal performance and upregulated defense-related gene expression. • SEM, TEM, and confocal microscopy revealed the protective role of nCaO-BC against Cd stress.