The Mechanisms Of Nitric Oxide Involved In Methane-mitigated Cadmium Toxicity In Plants

Methane(CH4)and nitric oxide(NO)are vital gas signal molecules or bioregulators,not only regulating the physiological and pathological processes in animals,but also important for various physiological functions in plants,including the induction of adventitious roots,enhancement tolerance against abiotic stress.However,the corresponding causal connection in cadmium(Cd)-toxicity response is still elusive.This work investigated the effects CH4 and NO pretreatment on alfalfa(Medicago sativa L.)physiological and biochemical charcteristics,as well as the interation between CH4 and NO exposed to Cd stress.Meanwhile,genetic evidences supported acquired results.The main results were as follows:1.Cd treatment successively stimulated the production of CH4 and NO in alfala seeding roots.Pretreatment with different concentrations CH4 differentially mitigated Cd toxicity,with a maximal response at 1.30 mM.Exogenous CH4 and sodium nitroprusside(SNP)not only strengthened NO production in Cd-stressed plants,but also alleviated the growth inhibition of seedings and reduced Cd accumulation in root tissues.At the same time,the addition of CH4 and NO stimulated antioxidant defence in stressed plants,confirmed by the histochemical staining for reactive oxygen species(ROS)content and lipid peroxidation,related antioxidant enzyme activities and corresponding transcripts.Thus,redox balance was reestablished.2.Consistently,reduced glutathione(GSH)content was increased by modulating the expression of GSH-related genes,but lower level of oxidized glutathione(GSSG),keeping a higher ratio of GSH/GSSG,indicating that glutathione homeostasis was reestablished.All of the above changes were dramatically abolished by applying NO scavenger.Fluorescence staining and spectrophotometric analysis showed that CH4 pretreatment could significantly promote endogenous NO production in roots when applied Cd or not,which was obviously blocked by the removel of NO.The decrease of NO content could aggravate Cd toxicity to alfalfa seedings.3.Genetic evidence showed that,unlike the response in wild-type(WT)Arabidopsis,CH4 supplementation could not rescued the redox and glutathione imbalance by Cd stress in NO defective mutants(noa1,nia1,and nia1/2).After adding SNP,the antioxidant ability and glutathione homeostasis were greatly restored.4.The effect of tungstate(nitrate reductase(NR)inhibitor)on seedlings growth was assessed.Compared to the Cd alone,fresh weight inhibition was significantly aggravated by tungstate,which markedly impaired CH4-attenuated Cd stress.Interestingly,changes in NR activities showed the similar tendencies to NO levels.Above results suggested that NR was an important pathway for NO production.Meanwhile,the increase of NR activity was closely related to rising NO levels in response to CH4 and Cd stress.In summary,the study indicated that NR-dependent NO was a downstream signal,involved in CH4-mitigated cadmium toxicity with reestablishing redox and glutathione homeostasis.