The Mechanism Of Melatonin And Methane-alleviated Cadmium Toxicity Via Reestablishment Of Glutathione Homeostasis In Plants

The heavy metal cadmium?Cd?is one of the widespread severe environmental pollutants with detrimental effects on plants and human health.Excessive cadmium uptake and accumulation by plants may cause retarded growth,inhibit root elongation,and induce oxidative stress.To cope with Cd toxicity,plants have developed complicated and efficient mechanisms including restraint of Cd influx and promote of Cd efflux,Cd chelation,antioxidant system consisting of metabolites like glutathione and antioxidant enzymes.Recently melatonin and methane,two endogenous metabolites in both animals and plants,have been found to exhibit protective roles against unfavorable environmental conditions.However,the molecular mechanisms of melatonin and in cadmium tolerance are still largely unexplored,and the signaling role of methane as well as.Thses make us to explore the possible beneficial roles and underlying molecular mechanisms of melatonin and methane in plants confronting Cd tolerance.Firstly,we evaluated the effects of melatonin on alfalfa?Medicago sativa?seedlings under Cd stress.The content of melatonin increased significantly in alfalfa roots with CdSO4 treatment in a dose and time-dependent ways.Furthermore,pretreatment with melatonin alleviated Cd-induced inhibition of root growth and reduced Cd accumulation in roots.Melatonin also reestablished of redox homeostasis in alfalfa seedling roots exposed to Cd by up regulating Mn-SOD,POD,APX1 transcripts and corresponding enzyme activities.Glutathione?GSH?is a key antioxidant metabolite under Cd stress.Further analysis showed melatonin could increase?h?GSH content by up-regulating GSH biosynthesis related genes but lower the?h?GSSG?h?content,thus maintaining a relative higher?h?GSH/?h?GSSG?h?ratio in alfalfa roots.However,the protective effects of melatonin were blocked by BSO?a selective inhibitor of GSH biosynthesis?,suggesting that GSH was involved in melatonin-mediated Cadmium tolerance.In order to confirm the role of GSH in Melatonin-mediated Cd tolerance,we identified Arabidopsis T-DNA insertional snat mutant?SALK₀33944,which displayed impaired Melatonin accumulation?and obtained transgenic SNAT over-expression Arabidopsis lines?pCAMBIA1302,35S:SNAT?.The snat mutant was more sensitive to Cd stress than WT.In contrast,overexpression of SNAT conferred Cd tolerance in Arabidopsis seedlings.The Arabidopsis cad2 deficient mutant?in a key GSH biosynthetic enzyme GSH1 deficient?showed more Cd accumulation than WT plants.Melatonin partially alleviated the grow inhibition,Cd accumulation,oxidative damage and GSH depletion by Cd stress in both WT and snat plants.However,melatonin dose not worked in cad2 mutants,indicating that GSH biosynthesis play a crucial role in melatonin-induced Cd tolerance in Arabidopsis.Methane is a ubiquitous small gaseous molecule in various organisms.In animals,it has been found that methane exhibited anti-inflammatory and antioxidative actions.Therefore,we explored the roles of methane in alfalfa seedlings under Cd stress,and the possible biochemical and molecular mechanisms.In this study,we observed that methane pretreated alfalfa seedlings exhibited Cd tolerance by increased seedling growth,lower Cd content in roots.Less accumulation of Cd was correlated with a substantial increase and decrease in ABC transporter and Nramp6 mRNAs.Opposite responses were observed in the changes of miR159 and miR167 transcripts.Furthermore,exogenous methane treatment further increased Cd-induced methane production in alfalfa roots.Methane treatment protected alfalfa seedlings from Cd caused oxidative stress by regulating antioxidative enzymes such as SOD,POD and APX both in transcriptional and enzymatic activity levels.Crucially,methane treatment increased GSH content and reestablished intracellular redox homeostasis.However,the selective inhibitor of GSH biosynthesis BSO reversed methane alleviated growth inhibition and cell death triggered by Cd.Taken together,our results indicate that melatonin and methane could alleviate plant Cd toxicity by reinforcing antioxidant system especially GSH homeostasis.These findings will help to invent an effective way to enhance plant Cd tolerance in agriculture.