| Three identified gasous signaling molecules,i.e.nitric oxide,carbon monoxide and hydrogen sulfide,are found to play an important role in animals and plants' growth and developmental processes,as well as in the defences against abiotic stresses.The gasous signaling molecule induced endogenously could regulate abundant gene expression,or amplify the signaling cascade,which further actives the intracellular antioxidative and detoxification systems.Our results showed that treatment with HO-1 inducer or hydrogen sulfide could regulate glutathione homeostasis and antioxidative system under cadmium stress.Furthermore,we also examined the mechanism of their induction under cadmium exposure,as well as their role in cadmium tolerance.The main results were summarized as follows:1.Different concentration of Cd(0-500 ?mol/L)treatment decreased reduced glutathione concentration and increased the HO-1 gene expression and protein level in dose-dependent manner.Induction of HO-1 gene expression and its protein level by Cd was mimicked by a GSH depletor diethylmaleate(DEM)and enhanced by the inhibitor of GSH biosynthesis L-buthionine-sulfoximine(BSO),which confirmed the HO-1 induction is associated with GSH depletion.At the same time,HO-2 gene expression was not significant changed.Additionally,the induction of HO-1 transcript was inhibited by the transcriptional inhibitor actinomycin D or protein synthesis inhibitor cycloheximide(especially).Fourthermore,HO-1 up-regulation could be reversed by exogenous supplement of GSH or ascorbic acid(AsA).2.SA induced HO-1 gene expression and HO activity(especially)in alfalfa seedling roots could be inhibited by HO-1 inhibitor zinc protoporphyrin(ZnPPIX).ZnPPIX blocked the above actions,and the aggravated effects triggered by SA plus ZnPPIX was reversed when carbon monoxide,the catalytic by-products of HO-1,was added.Experiment by using HPLC with fluorescence detection showed that HO-1 induction increased the content of low-molecular-weight thiols in alfalfa seedling roots.Furthermore,HO-1 induction also increased the antioxidant enzyme activity such as SOD,POD and APX.Real-time RT-PCR analysis showed that the expression of APX1/2 and genes in volved in glutathione metabolism was increased by HO-1 induction.Significant enhancement of the ratios of reduced/oxidized homoglutathione(hGSH),ascorbic acid(ASA)/dehydroascorbate(DHA),and NAD(P)H/NAD(P)+ were observed,consistent with a decreased reactive oxygen species(ROS)distribution in the root tips.Additionally,HO-1 induction also decreased the Cd concentration in alfalfa seedling roots under Cd exposure.3.Analyses of hydrogen sulfide(H2S)content and associated enzyme activity have demonstrated that cadmium exposure significantly increased the metabolism and production of H2S.Cadmium induced the depletion of reduced(homo)glutathione((h)GSH)and the decrease of(h)GSH/(h)GSSG(h)ratio,thus causing the induction of H2S production.These results were confirmed by the fact that the addition of N-acetyl-L-cysteine,a precursor of GSH,reestablished the redox balance,which was accompanied by the decrease of H2S production.4.Application of H2S donor(NaHS),H2S synthetic associated inhibitor(PAG)and inhibitor of GSH biosynthesis have detect that,NaHS increased the(h)GSH content and decreased the(h)GSH/(h)GSSG(h)ratio in the roots of Medicago sativa.NaHS treatment increased the expression of genes in(h)GSH metabolism glutamylcysteine synthetase(ECS),glutathione synthetase(GS),homoglutathione synthetase(hGS),and glutathione reductase(GR),as well as genes in ROS metabolism,SOD,APX and glutathione peroxidase(GPX).Thus decreased cadmium concentration and keeping ROS under control which results to the cadmium tolerance in roots of Medicago sativa.Experiments with donors and inhibitors showed the possible cross talk between H2S and HO/CO pathway.Application of H2S donor increased the HO-1 gene expression which demonstrated that HO/CO is involved in H2S signaling pathway. |
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