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

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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 (Medicago sativa 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.