Effects Of H₂S On D1 Protein Turnover In Wheat Under Drought Stress And Cloning Of Associated Genes

Drought is one of the major abiotic stress that affects crop yield.In recent years,several researches showed that hydrogen sulfide?H₂S?can improve drought tolerance of plants.In order to explore the function and response mechanism of H₂S under drought stress,this paper investigated the effects of exogenous H₂S on PSII photochemical activity and D1 protein turnover in two wheat varieties?Luohan No.6?LH-6,drought-tolerance varieties?and Chinese spring?droughtsensitive varieties??,TaLCD and TaDCD?responsible for H₂S produce?and the D1 protein turnoverrelated genes(TaDeg1,TaDeg2,TaDeg5,TaDeg7,TaDeg8,TaFts H1/5,TaFts H₂/8 and TaPBCP were cloned.In addition,TaLCD and TaDCD genes were transformated into Arabidopsis thaliana through Agrobacterium-mediated method,and obtained several transgenic plantlets.The main results are as follows:1.Hydrogen sulfide alleviated the damage in wheat seedlings under drought stress.Under drought stress,compared to water sprayed group,Na HS?an H₂S donor?treatment-wheat seeding grew better,leaf relative water content?RWC?significantly increased,thiobarbituric acid-reactive substance?TBARS?and hydrogen peroxide?H₂O2?content obviously decreased,activities of antioxidant enzymes such as superoxide dismutase?SOD?and catalase?CAT?also notably improved.These results indicated that H₂S enhanced the antioxidant and promoted Reactive oxygen species?ROS?scavenging in wheat,thus alleviating the oxidative stress induced by drought in wheat.2.Hydrogen sulfide relieved the inhibition of photosynthesis in the wheat leaf under drought stress.After the applying of Na HS,the maximum photochemical efficiency?Fv/Fm?,the actual photochemical efficiency?? PSII?,electron transport rate?ETR?and light chemical quenching coefficient?q P?in wheat seedlings were significantly increased and the coefficient of photochemical quenching?q N?obviously decreased compared and with that without Na HS treatment.All the chlorophyll fluorescence parameters were restored to a level close to the control group,which suggested that exogenous H₂S can relieve drought induced PSII damage and maintain the photochemical efficiency of PSII in plants,therefore ensuring the progress of photosynthesis.3.H₂S promoted the D1 protein turnover and protected the PSII repair cycle.D1 protein is the most vulnerable protein in PSII reaction center.Under drought stress,when treated with Na HS,the contents of D1 protein and phosphorylated D1 protein in wheat leaves were both significantly lower than that group without Na HS treatment,while the transcription level of Psb A?D1 protein synthesis gene?was higher.It may be related to the higher expression of STN8?D1 protein phosphorylation gene?and D1 protein degradation-related genes?Deg1,Deg5,Deg8,Fts H₂/8 and Fts H1/5?.These results indicated that H₂S reduced the damage of PSII under drought stress owing to accelerating the turnover of D1 protein,rather than raising the content of the D1 protein.4.This paper cloned the genes related to H₂S synthesis and D1 protein turnover which provided the basis to studying the molecular mechanism of H₂S action.The full or part sequences of TaLCD,TaDCD and D1 protein degradation genes TaDeg1,TaDeg2,TaDeg5,TaDeg7,TaDeg8,TaFts H1/5,TaFts H₂/8 and TaPBCP were obtained in silico cloning.Then designed the homologous primers according to Arabidopsis thaliana and Brochypodium distachyon,wheat?Chinese spring?c DNA was used as the template were for PCR,eventually CDS sequences of TaLCD?1398 bp?,TaDCD?1263bp?,TaDeg1?1284 bp?,TaDeg2?1803 bp?,TaDeg5?936 bp?,TaDeg7?3276 bp?,TaDeg8?1338 bp?,TaFts H1/5?2063 bp?,TaFts H₂/8?2022 bp?and TaPBCP?969 bp?were got respectively.The homology alignment between wheat and Arabidopsis or Brochypodium distachyon were done,and results showed the homology between wheat and Brochypodium distachyon was higher.5.This reserach constructed the TaLCD and TaDCD overexpression vector,and transferred into Arabidopsis thaliana through Agrobacterium-mediated method.Finally the overexpression TaLCD and TaDCD transgenic Arabidopsis thaliana plants were obtained which provide a foundation for further research of the function of TaLCD and TaDCD.