Methane alleviates copper-induced seed germination inhibition and oxidative stress in Medicago sativa

Recent results discovered the protective roles of methane (CH₄) against oxidative stress in animals. However, the possible physiological roles of CH₄ in plants are still unknown. By using physiological, histochemical and molecular approaches, the beneficial role of CH₄ in germinating alfalfa seeds upon copper (Cu) stress was evaluated. Endogenous production of CH₄ was significantly increased in Cu-stressed alfalfa seeds, which was mimicked by 0.39 mM CH₄. The pretreatment with CH₄ significantly alleviated the inhibition of seed germination and seedling growth induced by Cu stress. Cu accumulation was obviously blocked as well. Meanwhile, α/β amylase activities and sugar contents were increased, all of which were consistent with the alleviation of seed germination inhibition triggered by CH₄. The Cu-triggered oxidative stress was also mitigated, which was confirmed by the decrease of lipid peroxidation and reduction of Cu-induced loss of plasma membrane integrity in CH₄-pretreated alfalfa seedlings. The results of antioxidant enzymes, including ascorbate peroxidase (APX), superoxide dismutase (SOD), catalase (CAT), and guaiacol peroxidase (POD) total or isozymatic activities, and corresponding transcripts (APX1/2, Cu/Zn SOD and Mn-SOD), indicated that CH₄ reestablished cellular redox homeostasis. Further, Cu-induced proline accumulation was partly impaired by CH₄, which was supported by the alternation of proline metabolism. Together, these results indicated that CH₄ performs an advantageous effect on the alleviation of seed germination inhibition caused by Cu stress, and reestablishment of redox homeostasis mainly via increasing antioxidant defence.