Research Of Differentially Expressed Proteins And Physiological Mechanisms Of Exogenous Spermidine On Alleviating NaCl Stress Of Cucumber

Salt stress is major enviromental stresses that limit agricultural production,increasing of saline soil areas become more and more severe problem.NaCl is the most common soluble salts in soils,when the concentration of 40 mM constitutes saline.High concentrations of salt ions around roots cause hypertonic stress,leaded plant water deficit,ionic imbalance,resulting in stunted plants and production.Cucumber(Cucumis sativus L.)is one of the worldwide vegetable crops,which are grown extensively and has good economic benefit.But cucumber roots distribut shallow,its renewable capacity is weak and very sensitive to salt stress.Polyamines are a class of low molecular weight aliphatic nitrogen,which have strong physiological activity.At physiological pH,polyamines have positively charged,could participate in the regulation of plant growth,mature,aging,and respond to abiotic stress.In recent years,increasing numbers of researchs about polyamine and plant stress tolerance,and explore the mechanisms from various ways.However,the mechanisms of polyamine improve salt stress tolerance are very complex,the specific molecular mechanisms remain to be further clarify.Use salt sensitive cucumber varieties 'Jinchun No.2' as test materials,nutrient solution culture to controllle rhizosphere,0.1 mM exogenous spermidine(Spd)apply in nutrient solution,under 75 mM NaCl and/or Spd treatment,measured the effects of exogenous Spd on plant growth,Na~+ and K~+ distribution,polyamine metabolism,differentially expressed proteins in leaves and roots,photosynthesis,glycolysis of cucumber seedlings;photosynthetic physiological,fruit yield and quality of fruiting period cucumber,to further reveal the mechanisms of exogenous spermidine improve cucumber salt tolerance in molecular and physiological levels.The main results were as follows:1.With the time prolonging,NaCl stress limited cucumber seedlings growth.The plant height,fresh weight of shoots and rooots were significantly reduced under NaCl treatment,those damage could be alleviated by exogenous Spd.Under NaCl stress,Na~+ accumulated and K~+ reduced in leaf and root,application with exogenous Spd controled distribution of K~+,Na~+ in organs level.X-ray microanalysis showed that,compared with NaCl stress,exogenous Spd limited the load of Na~+ in roots' epidermis,cortex,and reduced the lost of K~+ from mesophyll.2.Under NaCl stress,three forms of Put were increased significantly in roots amd leaves of cucumber seedlings,applied exogenous Spd significantly raised the content of the three forms of Spd and Spm,reducing free Put,and increased the conjuged and bounded forms.NaCl stress induced genes belong in Put biosynthesis expressed in cucumber seedling's leaves and roots,and applying exogenous Spd induced genes belong in Spd,Spm biosynthesis expressed leaves and roots,reduced catabolized related genes,maintained a higher content of Spd and Spm.That exogenous Spd through changed three kinds of polyamine content and form to relieved the injuries of cucumber seedlings under NaCl stress.3.Through the iTRAQ(isotope labeling relative and absolute quantitative)technology,221 differentially expressed proteins had been identified which responsed to NaCl stress or exogenous Spd in cucumber leaves.These proteins involved in photosynthesis,carbohydrate metabolism,amino acid metabolism,stress response,signal transduction antioxidant and some other metabolic pathways.Among them,applied exogenous Spd under NaCl stress regulated proteins on photosynthesis,photorespiration,tetrapyrrole synthesis,ROS removal,protein synthesis and degradation.These differentially expressed proteins may play an important role in exogenous Spd alleviated growth and photosynthesis inhibition of cucumber seedling under NaCl stress.4.To explore the relationship between photosynthetic performance and photosynthetic relevant differentially expressed proteins,the physiological and transcript changes were measured.NaCl stress decreased net photosynthetic rate(Pn),stomatal conductance(Gs)and intercellular CO2 concentration(Ci)and Rubisco carboxylation efficiency(CE)were significantly decreased in cucumber seedlnings,photorespiration rate(Pr)was significantly increased.Applyied exogenous Spd with NaCl increased Pn and CE,decreased Pr.Chlorophyll content and chlorophyll fluorescence parameters were significantly reduced after 6 d treatment.Exogenous Spd alleviated these damages.The transcription of differentially expressed proteins which related to photosynthetic carbon assimilation,Rubisco activase(RA)and phosphoglycerate kinase(PGK),were decreased under NaCl stress.Applied exogenous Spd treateatment increased RA,PGK and transcription level of a thioredoxin,which participated in positively regulated Calvin cycle.In addition,NaCl stress increased transcription of photorespiration related genes and a catalase(CAT)which was responsible for the removal ROS product of photorespiration.Exogenous Spd could downregulated photorespiration related gene transcription level and further increase the expression of CAT.Exogenous Spd could enhance exogenous Rubisco carboxylate efficiency,increase Calvin cycle flux,reduced photorespiration,to relieve the damage of photosynthesis and growth under NaCl stress.5.Based on iTRAQ technology appraisal for NaCl and/or Spd responsed proteins,a total of 172 differentially expressed proteins had been identified.These protein involved in small molecular carbohydrate metabolism,glycolysis,TCA and electron transport chain,nucleotide metabolism,lipid metabolism,amino acid metabolism and secondary metabolism,stress response,signal transduction,antioxidant metabolic pathways,exogenous Spd increased proteins belong to glycolysis,small molecular carbohydrate metabolism,osmoregulation substance metabolism,lipid metabolism,nucleotide metabolism,stress defense and signal transduction.Exogenous Spd may enhance NaCl stress tolerance of cucumber seedling root via promoting these proteins accumulated.6.Protein in glycolytic pathway were differentially expressed under NaCl and/or Spd.Determination of enzyme activity of glycolytic enzymes,compared with salt stress,Exogenous Spd increased enzyme activity of fructose-bisphosphate aldolase(FBA),enolase(ENO)and pyruvate kinase(PK).In addition,used the antibody of enolase for western blot experiments,the results show that relative quantitative of western blot results contrast with iTRAQ quantitative results.7.Determination of photosynthetic characteristics of cucumber fruiting period,showed that NaCl stress suppressed of Pn,Gs,Ci and transpiration rate(Tr)significantly.Exogenous Spd improve Pn,Gs and Ci.The chlorophyll fluorescence parameters determination showed that exogenous Spd could improve of maximal efficiency of PSII photochemistry(Fv/Fm),the actual photochemical efficiency of PSII(OPSII),photochemical quenching(qP)and non-photochemical quenching(NPQ)under NaCl stress.NaCl stress significantly reduced the biomass of cucumber,and exogenous Spd relieved the cucumber fruit deformity,increased fruit size,fruit numbers and yield per plant.Sugar/acid ratio was decreaseed,taste changed in cucumbers fruits under salt stresss.Cucumber fruits treated under applied exogenous Spd,had better fruit type,yield and quality.These results suggested that exogenous Spd could increase photosynthetic efficiency,improve fruits yield and quality under NaCl stress in fruiting period.