Methane control of cadmium tolerance in alfalfa roots requires hydrogen sulfide

Hydrogen sulfide (H₂S) is well known as a gaseous signal in response to heavy metal stress, while methane (CH₄), the most prevalent greenhouse gas, confers cadmium (Cd) tolerance. In this report, the causal link between CH₄ and H₂S controlling Cd tolerance in alfalfa (Medicago sativa) plants was assessed. Our results observed that the administration of CH₄ not only intensifies H₂S metabolism, but also attenuates Cd-triggered growth inhibition in alfalfa seedlings, which were parallel to the alleviated roles in the redox imbalance and cell death in root tissues. Above results were not observed in roots after the removal of endogenous H₂S, either in the presence of either hypotaurine (HT; a H₂S scavenger) or _DL-propargylglycine (PAG; a H₂S biosynthesis inhibitor). Using in situ noninvasive microtest technology (NMT) and inductively coupled plasma mass spectroscopy (ICP-MS), subsequent results confirmed the participation of H₂S in CH₄-inhibited Cd influx and accumulation in roots, which could be explained by reestablishing glutathione (GSH) pool (reduced/oxidized GSH and homoglutathione) homeostasis and promoting antioxidant defence. Overall, our results clearly revealed that H₂S operates downstream of CH₄ enhancing tolerance against Cd stress, which are significant for both fundamental and applied plant biology.