Hydrogen Peroxide Is Involved in Methane-Alleviated Cadmium Toxicity in Alfalfa (Medicago sativa L.) Seedlings by Enhancing Cadmium Chelation onto Root Cell Walls

Although previous studies have demonstrated that methane (CH₄) can mitigate the toxicity of cadmium (Cd) in alfalfa seedlings, the CH₄-rich water used in these studies may create hypoxic conditions, potentially influencing the experimental outcomes. Therefore, this study aimed to investigate whether CH₄ can reduce Cd toxicity in alfalfa seedlings without the interference of hypoxia and to analyze its underlying mechanisms. Here, it was observed that supplementing oxygen with saturated CH₄-rich water can significantly alleviate the inhibition of 75 μM CdCl₂ on the growth of alfalfa (<i>Medicago sativa</i> L.) seedlings. Less Cd accumulation was also observed in both root and shoot parts, which could be explained by the CH₄-altered cell wall components in alfalfa seedling roots, including covalent and ionic soluble pectin, and the degree of demethylation in pectin, thus enabling a higher proportion of Cd binding to the cell walls and reducing the entry of Cd into the cells. The above actions of CH₄ were accompanied by an increase in hydrogen peroxide (H₂O₂) content and NADPH oxidase activity, which could be blocked by the addition of the NADPH oxidase inhibitor diphenylene iodonium (DPI). Taken together, these results implied that exogenously applied CH₄ could alleviate Cd toxicity in alfalfa seedlings by enhancing Cd chelation onto the root cell walls, which might be closely associated with NADPH oxidase-dependent H₂O₂ signals. These findings could provide insight into the mechanism through which CH₄ alleviates Cd toxicity in alfalfa plants.