Methane-rich water alleviates NaCl toxicity during alfalfa seed germination

• MRW alleviates NaCl-induced alfalfa seed germination inhibition.
• NaCl-induced oxidative stress is alleviated by MRW.
• A hypothesis is that heme oxygenase-1 is partially involved in MRW responses.

In this report, we investigated the beneficial role of methane-rich water (MRW) in the alleviation of salt toxicity in germinating alfalfa seeds. Upon NaCl stress, a progressive production of intracellular CH4 during seed germination was observed, which was mimicked by the pretreatment with 30% MRW. Both MRW and an inducer of heme oxygenase-1 (HO-1; a ubiquitous enzyme catalyzing degradation of heme to produce carbon monoxide) could up-regulate HO-1 gene expression, followed by the improvement of the inhibition of seed germination and seedling growth triggered by NaCl. Meanwhile, NaCl-induced lipid peroxidation and reactive oxygen species overaccumulation were reduced by MRW. This result was supported by the increases of total and isozymatic activities of representative antioxidant enzymes, and the up-regulation of corresponding transcripts. Above MRW cytoprotective responses were HO-1-dependent, since corresponding MRW-induced changes were sensitive to the potent inhibitor of HO-1, but reversed by the cotreatment with one of its catalytic by-products, carbon monoxide (CO) aqueous solution. Additionally, ion homeostasis was reestablished by MRW. Together, our evidence reveals that MRW alleviates NaCl-induced inhibition of seed germination and oxidative stress, partially by the up-regulation of HO-1.