The Mechanism Of Hydrogen Sulfide Regulating Chilling Tolerance And Its Signal Transduction In Cucumber

Cucumber(Cucumis sativus L.)is a kind of cold-sensitive and light-loving plant.In north China,it is mainly cultivated through the winter in solar-greenhouses,where low temperature is frequent,which leads to serious effect on growth,yield and quality of cucumber.Hydrogen sulfide(H2S)is a kind of endogenous gas signal molecule.It plays an important role in regulating plant growth and development,as well as in response to abiotic stress.However,the signal transduction way,downstream cascade reaction of H2 S and the interaction of H2 S with other signal molecules under adversity are not clear.In this experiment,the cucumber seeds were soaked with sodium hydrosulfide(NaHS,H2 S donor),sodium nitroprusside(SNP,NO donor),calcium chloride(CaCl2,Ca2+ donor),hydrogen peroxide(H2O2,ROS donor)and hypotaurine(HT,H2 S scavenger),hemoglobin(Hb,NO scavenger),ethylenebis(oxyethylenenitrilo)tetraacetic acid(EGTA,Ca2+ chelating agent),chlorpromazine(CPZ,CaM antagonist),1,1-Dimethyl-thiourea(DMTU,H2O2 scavenger),and the deionized water treatment was used as the control.We studied the response of H2 S,NO,Ca2+ and ROS to chilling stress.The interaction of H2 S and NO,Ca2+ or ROS signals,and the regulation mechanism of H2 S signal on photosynthetic carbon assimilation in improving chilling tolerance of cucumber were also investigated.The main results are as follows:1.The proper concentration of NaHS,SNP,CaCl2 and H2O2 could promote growth of cucumber seedlings.Seedlings treated with NaHS+Hb,NaHS+EGTA,NaHS+CPZ and NaHS+DMTU all showed obviously less amount of growth in stems,leaves and roots,compared with the NaHS treatment.CaCl2+HT treated seedlings also revealed less amount of growth than the CaCl2 treatment,but no remarkable difference was observed between H2O2+HT and H2O2 treatments.This indicates that there were interaction between H2 S and NO or H2 S and Ca2+ in promoting the growth in cucumber seedlings.H2O2 participated in the regulation of H2 S in the growth of cucumber seedlings.2.The endogenous H2 S,NO,Ca2+ and H2O2 in cucumber seedlings were induced by low temperature,and decreased after 6 h ~12 h.SNP and CaCl2 could promoted H2 S production,enhanced the activity and mRNA abundances of L/D-cysteine desulfhydrase(CDes)in cucumber seedlings under chilling stress.The inductive effect of low temperature on H2 S,CDes activity and mRNA abundances was weakened when NO or Ca2+ was eliminated.Furthermore,the mRNA abundances of calmodulin(CaM)and CBL-interacting protein kinase(CIPK5)were declined significantly when H2 S was scavenged.H2O2 and DMTU which was the synthesis scavenger of H2O2 did little effect on H2 S content and CDes activity and mRNA abundances,but H2O2 content and NADPH oxidase(Nox)expression were decreased when H2 S was eliminated.These data suggested that H2 S might be participate the signal transduction induce by low temperature in the upstream of H2O2,while there are “crosstalk” mechanisms between H2 S and NO or Ca2+.3.NaHS,SNP,CaCl2 and H2O2 promoted the generation of cucumber seeds under suboptimal temperature.The NaHS,SNP,CaCl2 and H2O2 significantly alleviated the injury symptoms caused by chilling stress,and also led to a significantly decrease in the H2O2 accumulation,superoxide anion(O2·-)production rate and electrolyte leakage(EL)compared to the water-pretreated seedlings.The function of H2 S to alleviate chilling stress was weakened when NO,Ca2+ and H2O2 were scavenged.The effect of Ca2+ on chilling tolerance was also weakened when H2 S was eliminated,but that of H2O2 had not been influenced.These results indicated that H2 S had an interaction with NO and Ca2+ in the inducing process of chilling tolerance in cucumber seedlings,while H2O2 was a participant in this process.4.NaHS,and SNP,CaCl2,H2O2 increased photosynthetic rate(Pn),stomatal conductance(Gs),transpiration rate(Tr),maximum photochemical efficiency of PSII in darkness(φPSII),maximum photochemical efficiency of PSII in darkness(Fv/Fm),but decreased intercellular CO2 concentrantion(Ci)under chilling stress.Compared with the water treated seedlings,the NaHS,and SNP,CaCl2,H2O2 treated seedlings showed an increase in the activities and mRNA abundances of ribulose bisphosphate carboxylic enzyme(RuBPCase),sedoheptulose-1,7-bisphosphatase(SBPase),fructose-1,6-bisphosphate aldolase(FBA),transketolase(TK).Scavenged the NO,Ca2+ or H2O2,the positive effect of H2 S on the photosynthesis could be weakened.Meanwhile,the function of Ca2+ and H2O2 was also weakened when H2 S was eliminated.These results demonstrated that H2 S could alleviate the negative effect of chilling stress on photosynthetic carbon assimilation,and consequently increase Pn.NO,Ca2+ and H2O2 involved in the regulation of H2 S on photosynthesis in cucumber seedlings under chilling stress.5.NaHS,SNP,CaCl2 and H2O2 increased the activities and mRNA abundances of superoxide dismutase(SOD),peroxidase(POD),catalase(CAT),ascorbate peroxidase(APX),glutathione reductase(GR)in cucumber seedlings during chilling stress.Seedlings of NaHS,SNP,CaCl2 and H2O2 treatments also showed an increase in the contents of ascorbic acid(AsA)and reduced glutathione(GSH),as well as the ratio of AsA/DHA and GSH/GSSG,whereas revealed a decrease in the content of malondiadehyde(MDA),compared with the water treatment.The positive effect of H2 S on the activities of antioxidant enzymes was weakened by eliminating NO,Ca2+,H2O2.Similarly,the function of Ca2+ was weakened by scavenging the H2 S,but that of H2O2 had not been markedly influenced.These data suggested that H2 S might be as an upstream signal of H2O2 and interact with NO and Ca2+ in regulating antioxidant system of cucumber seedlings under chilling stress.