The Roles Of Hydrogen Gas In Regulating Cadmium Tolerance In Chinese Cabbage

It has been recently reported that H2 could ameliorate the detrimental effects of various abiotic stresses in plants,such as salinity,drought,heavy mental,irradiation,etc.,while,the underlying mechanism and signal transduction pathway involved in this process are still unknown.In the present study,we used Chinese cabbage seedlings as experimental material and explored the role of H2 on cadmium stress and the related mechanisms.The main results are shown below.1.Our results showed that the addition of 50%saturation HRW(hydrogen-rich water)significantly alleviated the Cd toxic symptoms,including the improvement of both root elongation and seedling growth inhibition.These responses were consistent with a significant decrease of Cd accumulation in roots and shoots,which was further confirmed by the histochemical staining.Molecular evidence illustrated that Cd-induced up-regulations of BcIRT1 and Nrampl genes,responsible for Cd absorption,were blocked by HRW.By contrast,Cd-induced up-regulation of BcHMA3 gene which regulates Cd sequestration into the root vacuoles was substantially strengthened by HRW.Furthermore,compared with those in Cd-stress alone,the expressions of BcHMA2 and BcHMA4 which have function in the transportation of Cd to xylem were repressed by co-treatment with HRW.Meanwhile,HRW could enhance the activities of antioxidant enzymes,including superoxide dismutase,guaiacol peroxidase,catalase and ascorbate peroxidase.These results were further confirmed by the alleviation of oxidative damage,as indicated by the decrease of thiobarbituric acid reactive substances(TBARS)and reactive oxygen species(ROS)production.Together,this study suggested that the improvement of Cd tolerance by HRW was associated with reduced Cd uptake and increased antioxidant defense capacities.Therefore,the application of HRW may be a promising strategy to improve Cd tolerance of Chinese cabbage.2.We demonstrated that both 50 ?M CdCl2 and 50%-saturated HRW induced an increase of endogenous H2 in Chinese cabbage seedlings,and HRW alleviated Cd toxicity related to growth inhibition and oxidative damage.Seedlings supplied with HRW exhibited increased root length and reduced lipid peroxidation,similar to plants receiving GSH post-treatment.Additionally,seedlings post-treated with HRW accumulated higher levels of reduced glutathione(GSH)and ascorbic acid(AsA)and showed increased GST and GPX activities in roots.Molecular evidence illustrated that the expression of genes such as BcGS,BcGRl and BcGR2,which were down-regulated following the addition of Cd,GSH or BSO,could be reversed to varying degrees by the addition of HRW.Based on these results,we propose that H2 might be an important regulator for enhancing the tolerance of Chinese cabbage seedlings against Cd,mainly by governing reduced glutathione homeostasis.3.In this part,the effects of H2 and NO on alleviating the cadmium(Cd)stress and their relationship during this process were investigated.Seedlings treated with 100 ?M SNP(a donor of NO)or pretreated with 50%HRW(hydrogen-rich water)showed a significant increase in root length and fresh weight under Cd stress.Cd treatment induced much higher endogenous levels of NO and H2,36%and 66%higher,respectively.When treated with NO scavenger(PTIO)or NO biosynthesis enzyme inhibitors(L-NAME and Gln),the accumulation of NO was substantially inhibited.Besides,HRW-induced alleviation under Cd stress was disappeared.In addition,under Cd stress,HRW pretreatment significantly enhanced the NO accumulation,and together up-regulated the activity of NR and expression of BcNR.Proteomic analysis identified 29 proteins responded H2 and NO-induced amelioration of Cd stress,and nearly half of them were involved in oxidation-reduction process(23%)and antioxidant enzymes(19%).In addition,four of six proteins belonging to antioxidant enzymes were clustered together,and compared with Cd treatment,application of SNP or HRW simultaneously up-regulated the expression levels of all these 6 proteins which were inhibited again when PTIO were applied under Cd condition.Consistent with this,HRW and SNP treatment both reduced the concentration of reactive oxygen species(ROS),but increased the AsA content and activity of POD and SOD in seedling roots.While HRW-induced these changes were inhibited by PTIO,L-NAME and Gln.These results imply that under Cd toxicity,pretreatment of HRW induces the H2 accumulation,biosynthesized or permeated,that further increases the endogenous content of NO in seedlings through NR pathway.Finally,H2 and NO together improve the antioxidant system of seedlings against Cd toxicity.4.The aim of this study is to investigate the effects of HRW treatment on the response of Chinese cabbage seedlings to ROS,ion disturbance induced by Cd and Cd uptake.The results showed that after the H2 treatment,the number of death cell caused by Cd,H2O2 and Cu/A was decreased,and the K+loss induced by H2O2 and Cu/A was reduced significantly.Cd treatment resulted in severe loss of K+,H+,Ca2+ from the cabbage seedling roots,and with the increase of Cd,the loss was enhanced gradually.Compared with control,changes of ion fluxes induced by 50 and 100 pM Cd addition were much less in HRW pretreatment roots.In detail,the ion loss from HRW pretreatment roots induced by 50 ?M Cd was similar as that in control roots treated with 20?M Cd.The absorption kinetics revealed that with the extension of time,the uptake rate of Cd in the roots was decreased gradually.At 12 h Cd treatment,the uptake rate of Cd was nearly 0,and after that the roots began to exclude Cd2+.HRW treatment decreased the uptake rate of Cd by roots,which also could be resulted in by H2O2 with low concentration,while when DPI was added,the reduced Cd uptake rate induced by HRW was disappeared.The additional experiments with Arabidopsis mutant showed that the roots of RbohD mutant had a much higher Cd uptake rate than wild type,and HRW treatment could not reduce this uptake rate as in wild type.All these results suggested that H2 could decrease the K+loss under ROS treatment,protect plant cells against ROS damage and at the same time lessen the Cd-induced ion disturbance.In addition,H2O2 plays an important role in Cd uptake and is involved in the regulation of Cd absorption by H2.