Redox Signals Involved In The Regulation Of Hydrogen Sulfide On Photosynthesis In Cucumber Under Chilling Stress

Hydrogen sulfide(H₂S)and hydrogen peroxide(H₂O₂)are two crucial signaling molecules that participate in various physiological processes,and can alleviate the damage caused by abiotic stresses.However,how the synergy of H₂S and H₂O₂ regulates photosynthesis have rarely been studied,and the role of redox signaling in H2S-induced up regulation of CO2assimilation is unclear.This study aimed to reveal the roles of H₂O₂ and redox signal in H₂S-induced photosynthetic carbon assimilation and chilling tolerance in cucumber(Cucumis sativus L).‘Jinyou 35'seedlings were used as the material,sodium hydrosulfide hydrate(Na HS,an H₂S donor),H₂O₂,reduced glutathione(GSH),reduced ascorbic acid(As A)and their scavengers or inhibitors were applied as foliar sprayed at the two-leaf seedling stage.Deionized water(H₂O)treatment was used as the control.The main results are as follows:1.Both proper concentration of Na HS and H₂O₂ increased the net photosynthetic rate(P_n),light-saturated photosynthetic rate(A_sat),rubisco maximum carboxylation efficiency(V_c,max)and Ru BP maximum regeneration rate(J_max),as well as the activities and gene expressions of Calvin-cycle key enzymes under chilling stress.They also improved the actual photochemical efficiency of PS?(?_PS?),maximum photochemical efficiency of PS?(F_v'/F_m'),photosynthetic index(PIABS),the excitation efficiency of electron transport beyond QA(?0)and PS?reaction center activity(?I/I0)under chilling stress.In addition,Na HS and H₂O₂ enhanced the activities of sucrose phosphate synthase(SPS),invertase(AI)and sucrose synthase(SS),and increased the accumulations of sucrose,glucose,fructose,starch and total sugar.H₂S-and H₂O₂-treated seedlings showed a significant increase in relative m RNA expression of C-repeat binding factor(CBF1)and inducer of CBF expression(ICE).Na HS significantly enhanced the relative gene expression of respiratory burst oxidase homolog(RBOH),which in turn elevated endogenous H₂O₂ accumulation in cucumber seedlings.However,H₂O₂had little effect on gene expression of L-/D-cysteine desulfhydrase(L-/D-CDes)and endogenous H₂S level.These data suggested that Na HS alleviated the negative effects of chilling stress on photosynthesis by improving photosynthetic carbon assimilation,carbon metabolism,and photoprotection for both PS?and PS?in cucumber seedlings.The H₂S-induced adaptive response of photosynthesis to chilling stress was suppressed by N,N-dimethylthiourea(DMTU,a H₂O₂ scavenger)or diphenyleneiodonium chloride(DPI,a H₂O₂ generation inhibitor).H₂O₂may act as a downstream signal in H₂S-induced protection of the photosynthetic apparatus in cucumber seedlings under chilling stress.2.Transcription profiling analysis revealed that Na HS significantly up-regulated 397 genes under chilling stress compared with the control,and 13.9%of differentially expressed genes(DEGs)were associated with glutathione pathways.Moreover,transcription profiling analysis revealed that the glutathione-associated genes glutathione S-transferase(GST),maleylacetoacetate isomerase(MAAI),ascorbate peroxidase(APX),glutathione reductase(GR),glutamine synthetase(GS)and monodehydroascorbate ascorbate reductase(MDHAR)were significantly up-regulated in Na HS-treated cucumber seedlings,compared to the H₂O-treated seedlings.We then found that Na HS treatment showed obviously higher GSH/GSSG compared with the H₂O treatment during chilling stress condition,but the increase in GSH/GSSG was blocked by buthionine sulfoximine(BSO,an inhibitor of glutathione)or 6-aminonicotinamide(6-AN,a specific pentose inhibitor and thus inhibits the NADPH production).Na HS increased the P_n,A_sat,activities and m RNA expressions of Calvin-cycle key enzymes,as well as the photosystem activities compared with the control seedlings.GSH also up-regulated the CBF1 and ICE m RNA abundances.The Na HS-induced adaptive response of photosynthesis to chilling stress were suppressed by application of BSO and 6-AN.Thus,novel results highlighted that glutathione might be involved in H₂S-induced photosynthetic carbon assimilation and chilling tolerance in cucumber seedlings through the H₂O₂ signaling pathway.This result indicated that glutathione plays an important role in H₂S-induced chilling tolerance in cucumber seedlings.3.Na HS caused a significant increase in reduced ascorbic acid(As A)content and As A/DHA,as well as the activities and m RNA expressions of APX,GR,dehydroascorbate reductase(DHAR)and MDHAR in cucumber seedlings.The seedlings treated with As A showed a significantly higher P_n and A_sat,activities and gene expressions of the key enzymes in Calvin-cycle,as well as the photosystem activities compared with the control seedlings.As A also up-regulated the CBF1 and ICE m RNA abundances,and therefore improved the photosynthetic function and chilling tolerance in cucumber seedlings.However,the positive effects of Na HS on the photosynthetic function and cold tolerance gene were reduced or reversed by the inhibitor of ascorbic acid lycorine,and suggesting that ascorbic acid were also involved in the regulation of H₂S in photosynthetic carbon assimilation and chilling tolerance.4.Compared with the H₂O-treated seedlings,Na HS-treated seedlings showed increases in the As A and GSH contents,GSH/GSSG and of As A/DHA ratios,as well as the activities of APX,GR,DHAR and MDHAR,glutamate cysteine synthase(GCS)and GST in chloroplast of cucumber seedlings under chilling stress.This data further confirmed that H₂S could strengthen the photosynthetic carbon assimilation and chilling stress by regulation the redox state of glutathione and ascorbic acid.