Methane alleviates alfalfa cadmium toxicity via decreasing cadmium accumulation and reestablishing glutathione homeostasis.

Although methane (CH4) generation triggered by some environmental stimuli, displays the protective response against oxidative stress in plants, whether and how CH4 regulates plant tolerance against cadmium stress is largely unknown. Here, we discovered that cadmium (Cd) stimulated the production of CH4 in alfalfa root tissues. The pretreatment with exogenous CH4 could alleviate seedling growth inhibition. Less amounts of Cd accumulation was also observed. Consistently, in comparison with Cd stress alone, miR159 transcript was down-regulated by CH4, and expression levels of its target gene ABC transporter was increased. By contrast, miR167 transcript was up-regulated, showing a relatively negative correlation with its target gene Nramp6. Meanwhile, Cd-triggered redox imbalance was improved by CH4, evidenced by the reduced lipid peroxidation and hydrogen peroxide accumulation, as well as the induction of representative antioxidant genes. Further results showed that Cd-triggered decrease of the ratio of reduced/oxidized (homo)glutathione was rescued by CH4. Additionally, CH4-triggered alleviation of seedling growth was sensitive to a selective inhibitor of glutathione biosynthesis. Overall, above results revealed that CH4-alleviated Cd accumulation at least partially, required the modulation of heavy metal transporters via miR159 and tniR167. Finally, the role of glutathione homeostasis elicited by CH4 was preliminarily suggested.