Hydrogen Sulfide Enhances Nitric Oxide-induced Tolerance Of Hypoxia In Maize (Zea Mays L.) Tip Cells

Flooding is a major environmental stress that severely limits the growth and yields of crops that are sensitive to waterlogging or complete submergence.It is an effective way to enhance the plant's tolerance to flooding and to help promote yields of crops.Nitric oxide?NO?and hydrogen sulfide?H₂S?have emerged as key signaling molecules involving in response to many biotic or abiotic stresses in plants,while studies on understanding how these molecules involve in response to hypoxia in plants are quite less,especially H2 S.In this study,the pathway of NO and H2 S taking part in enhancing hypoxia tolerance of maize seedlings,which were used for experimental material,was studied by histochemical stain,fluorescence probes and relative key enzymes detection and qRT-PCR,results of this study were shown as following:?1?Pretreatment with SNP?sodium nitroprusside;NO donor?alleviated significantly maize root tip cells death measured by Evans Blue staining under hypoxic conditions.Using NO fluorescent probe DAF-FM DA,histochemical staining and relative biochemistry determination methods,it was found that exogenous NO pretreatment enhanced endogenous NO accumulation,and actived antioxidant defense to alleviate oxidative damage caused by excess reactive oxygen species?ROS?accumulation under hypoxia,meanwhile NO promoted cellular Ca²⁺ and enhanced activities of ADH?alcohol dehydrogenase?,ultimately enhanced survival rate of maize root tips.On the other hand,pretreatment with cPTIO resulted in a lower survival rate.?2?Root tip cells death was analyzed by Evans Blue staining during maize roots pretreated with exogenous 0.1 mM H₂S?NaHS as donor?after exposed to hypoxia,and the results displayed that survival rate was enhanced by pretreatment with H2 S.The work was performed using WSP-1 to measure intracellular H2 S and other methods to analyze relative physiological changes,and the results exhibited that H2 S was also involving in response to hypoxia like NO,and the oxidative damage caused by excess ROS accumulation were alleviated by enhancement antioxidant defense,and cellular Ca²⁺ content was also promoted,but not pretreatment with H₂S-synthesis inhibitor hydroxylamine?HA?and H₂S-scavenger hypotaurine?HT?.?3?The activities of the key enzymes in H2 S biosynthesis,namely L/D-CDs?L/D-cysteine desulfhydrases??O-acetyl-L-serine?thiol?lyase?OAS-TL?and ?-Cyanoalanine?CAS?,in maize roots pretreated with 0.2 mM NO,were enhanced in response to NO pretreatment,which resulted in H2 S accumulation.Although NO accumulation was not enhanced by pretreatment with H2 S under hypoxia conditions,H₂S-synthesis inhibitor hydroxylamine?HA?and H₂S-scavenger hypotaurine?HT?could eliminate NO induced hypoxia tolerance.Therefore,these results indicated that H2 S was involved in the NO-activated acquisition of hypoxia tolerance in plants and might act as a signal molecule downstream of NO in response to hypoxia.?4?Intracellular Ca²⁺ in root tips pretreated with 50 mM CaCl2,5 mM LaCl₃ or 3 mM EGTA was determined by Ca²⁺-sensitive fluorescent probe Fluo-8AM and relative physiological processes were measured,and the results showed that H2 S enhanced cytosolic Ca²⁺,and activities of ADH and PDC?pyruvate dehydrogenase?,which involve in anaerobic fermentation,were enhanced subsequently.At the same time,ethylene production was inhibited by H2 S which resulted in activating stationary growth strategy in plants.Together,H2 S served as a multi-faced transducer that enhanced hypoxia tolerance in maize?Zea mays L.??5?The expression of relative genes in maize root tips pretreated with 0.2 mM SNP and 0.1 mM NaHS were measured by qRT-PCR under hypoxia conditions,and the result displayed that NO and H2 S enhanced the expression of relative genes including rp3?rust resistance protein?and HRFP?Hypoxia responsive family protein?involving in stresses response,DBP?DNA-bing protein?and TA?transcroption activator?involving in transcriptional regulation,ADH?ethanol dehydrogenase?,PK?Pyruvate kinase?involving in energy metabolism,GS?glutamate synthase?,CYP51?cytochrome P450 14?-sterol demethylase?involving in redox balance regulation,IVD?Isovaleryl-CoA dehydrogenase?and SPI?serine protease inhibitor?involving in amino acid and protein metabolism,ME?NADP-dependent malic enzyme?involving in cytoplasmic pH control,CRT?calcium binding protein 1?involving in singnaling transduction,ORT?organic anion transporter?involving in ion transport and HIP?hypoxia induced protein?induced by hypoxia respectively,therefore,enhancement of hypoxia tolerance might be regulated by these genes whose expressions were promoted by NO and H2 S in plants.In conclusion,NO production induced by hypoxia activated H2 S accumulation,which enhanced antioxidant defense to alleviate oxidative damage caused by excess ROS accumulation.While the enhancement of H2 S also promoted cytosolic Ca²⁺,anaerobic fermentation and inhibition ethylene production to initiate stationary growth strategy,and plant then plant hypoxia tolerance was enhanced.