盐胁迫对大麦幼苗多胺生物合成的影响

盐胁迫对大麦幼苗多胺生物合成的影响

基本无病性植物（pas）非常小，亲水平台上钠，生命缓慢的异常行为，糖蜜（spd）和马蹄莲（spm）是生命的保障。其他uppi生命缓慢的行为是异常的，低于生命缓慢的水平地壳（c.c.2.13）。“公众发光”（卡姆纳利亚和苏门答腊，1994），“公共行动”（卡姆纳利亚、鲁姆、鲁姆、鲁姆等）（香，2008）（香，2008）（香，2008）（香，2008）（香，库恩，1994），以及“国家行动”（达顿，1989）。PAsarethoughttobeinvolvedinseveralkindsofabioticstressesinplants,suchasosmotic(Erdeietal,1996),saltstress(Azizetal,1998),acid(Velikovaetal,2000),heavymetals(Groppaetal,2001),aswellaschillinginducedstress(Serranoetal,1996).TherearemanyreportsonthechangesinPA(Put,Spd,Spm)contentsintheresponseofplantstosalinity.Duetothedifferenceinmaterialsandexperimentalconditionsused,theresultsshowedgreatinconsistance.Forexample,accordingtoPrakashandPrathapsenan(1988),levelsofendogenousPAsdecreasedinriceseedlingsunderNaCltreatment,whereasBasuetal(1988)reportedthatsalinityresultedinaccumulationofPAsinthesamematerial.KrishnamurthyandBhagnat(1984)reportedthatthecontentsofSpdandSpmincreasedsharplyinsalt_tolerantricecultivarsbysalttreatmentleadingenhancedleveloftotalPAs,whilePutcontentwasdecreasedrelatively.ItisofconsiderableinteresttonotethatPutaccumulatedexcessivelyinrootsofsalt_sensitivericecultivarswhenexposedtosalineconditions,whilecontentsofSpdandSpmincreasedslightly.Insorghum,SpdandSpmaccumulationoccurredduringsaltstressandwasconsideredasanadaptiveresponse(Erdeietal,1996).Santa_Cruzetal(1997)reportedthatthe(Spd+Spm)/Putratiosincreasedwithsalinityinsalt_toleranttomatospecies(Lycopersiconpennellii)butnotinsalt_sensitivespecies(L.esculentum).TheseresultssuggestedthathighlevelsofSpdandSpmaccumulatedinresponsetosaltstresswerebeneficialtoalleviationofsaltinjury,whiletheeffectsofPutwascontrarywithrespecttosalttoleranceofplants.However,transgenicexperimentdemonstratedthathighcontentsofPutcouldenhancesalttoleranceofrice(Oryzasativa)andpromoteditsbiomassundersalinity_stressconditions(RoyandWu,2001).SotherelationshipbetweenPAcontentsandsalttoleranceofplantsshouldbestudiedfurther.Inaddition,inourpreviousstudy(Zhaoetal,2000),boundPAs(bPAs)aswellasfreeones(fPAs)weredetectedinplasmamembraneandtonoplastvesiclesisolatedfromrootsofbarleyseedlings,andcontentsoffPutandbPut,fSpdandbSpdalteredgreatly,whileslightchangesinbothfSpmandbSpmlevelswereobserved,whichwasdifferenttosomeextentfrom!theresultsmentionedabove.Furthermore,anovelunknownPA(PAx)wasdetectedanditscontentwasfoundtobeofsomeimportancetosalttoleranceofbarleyseedlingsaswellasSpd.SoitisessentialtoreconsidertherelationshipbetweenendogenousPAlevelsandsalttoleranceofplants.Inexperimentsreportedhere,twobarleycultivarsofdifferentsaltsensitivitywereusedasexperimentalmaterials,endogenousfPAandbPAlevelsweredeterminedandactivitiesofsomekeyenzymesinvolvedinPAmetabolisminplantswerealsoassayed.Toourknowledge,thisisthefirstreporttoelucidatetherelationshipbetweensalttoleranceandPA(freeandbound)contentsintherootsofbarleyseedlings.1杏仁杏仁1.1htrafteriece自然生成Seedsofthetwobarley(HordeumvulgareL.)cultivarsJian4(salt_tolerant)andKepin7(salt_sensitive)weresurface_sterilized,imbibedandgerminated,andsowedinplasticpotswithsmallholesatbottomsandplacedingrowthchamber.Half_strengthHoaglandnutrientwassuppliedtoplantsthroughholes.Plantsweregrownat(20±1)℃with12hcool_fluorescentillumination(100μE·m-2·s-1).Barleyseedlingswithfirstleaffullyexpandedweretreatedwithhalf_strengthHoaglandsolutioncontaining0,50,100,200or300mmol/LNaCl.After7d,roottipsandyoungleavesoftheseedlingsweresampledandtested.Allexperimentswereperformedwith3replicates.1.2gr-pct反应GRwasassayedaccordingtothemethodofWangandZhao(1997).GRwasexpressedascm/d.1.3相关表1.1.1.1Na+andK+wereextractedin0.1mol/LHNO3andanalyzedwitha6400FlamePhotometer.1.4u2004范围TheADCandODCactivitieswereassayedaccordingtotheproceduredescribedbyBireckaetal(1985)withsomemodification,asdetailedearlier(ZhaoandLiu,2000a).TheADC(ODC)activitywasassayedin1.5mLofreactionmixturecontaining1mLoftheassaybufferwith100mmol/LTris_HCl,pH8.5,5mmol/LEDTA,40μmmol/Lpyridoxalphosphateand5mmol/LDTT,0.3mLoftheADCenzymeextract,and0.2mLof25mmol/LL_Arg(orOrn).Thereactionmixturewasincubatedat37℃for60min,andcentrifugedat3000gfor10min,0.5mLofthesupernatantwasmixedwith1mLof2mmol/LNaOH,then10μLbenzoylchloridewasaddedtothemixtureandstirredcontinuouslyfor20s.Afterconductedat25℃for60min,2mLofsaturatedNaCland2mLofetherwereaddedtothereactionmixtureandstirredthoroughly,thencentrifugedat1500gfor5min,1mLofetherphasewascollectedandevaporatedat50℃.Theremainderwasdissolvedin3mLofmethanol(HPLCgrade),anditsabsorptionvalueat254nmwasmeasuredwithaspectrophotometer(ModleU_3000,HITACHI,Tokyo,Japan).TheADCandODCactivitieswereexpressedasμmolAgm·g-1FW·h-1andμmolPut·g-1FW·h-1,respectively.TheTGaseactivitywasassayedaccordingtothemethoddescribedelsewhere(GrahamadnJames,1998)withsomemodification(ZhaoandLiu,2000a).ProteincontentwasdeterminedaccordingtothemethodofBradford(1976)usingBSAasthestandard.ThePAOactivitywasdeterminedasthemethodofZhaoandLiu(2000b).Roottipswerehomogenizedin50mmol/LNa2HPO4_KH2PO4buffer(pH7.0)onice.Thehomogenatewasfilteredthroughfourlayersofmuslinandthefiltratewasusedascrudeenzymeextract.TheTGaseactivitywasassayedin3.5mLofreactionmixturecontaining2mLofphosphatebuffer,0.5mLof3mmol/LSpd,0.5mLof30mmol/Lpyridoxalphosphateand0.5mLofenzymeextract.Thereactionmixturewasincubatedat37℃for60minandthereactionstoppedbyadding0.5mLof10%(W/V)TCA,andcentrifugedat5000gfor20min.Thesupernatantwascollectedandlettoreactwithbenzylaldehydefor20min,andabsorptionvaluewasmeasuredat435nm.TheenzymeactivitywasexpressedasΔA435·g-1FW·h-1,1/10ΔA435·g-1FW·h-1wasusedasoneenzymeactivityunit(1U).1.5PolyamineanalysisFreeandboundPAswereextractedinprechilledPCAaccordingtothemethodofScaramaglietal(1999)anddeterminedbythemethodofYangetal(1988).2产品系统2.1u3000ra联WithlowconcentrationsofNaCltreatment,GRofthetwobarleycultivarswerebothstimulatedtosomeextent.GRwasinhibitedwhenNaClconcentrationwasashighas200and300mmol/L.ItseemedthattherewasnosignificantdifferencesstatisticallyinGRbetweenJian4andKepin7at50,100,200and300mmol/LNaCl.LeafK+/Na+decreasedsharplywithNaClconcentrationincreasing,andK+/Na+inJian4washigherthanthatinKepin7(Fig.1).2.2tgaeindivi体制naclsing/agctivi坚持/envi体制/agctivi结构设计ADCisakeyenzymecatalyzingtheconversionofArgtofPut,anditsactivityroseverysignificantlywhenNaClconcentrationwasmorethan100mmol/L(P<0.01).TheconversionofOrntofPutiscatalyzedbyODC,whoseactivitywaslowerverysignificantlythanthatofADC(P<0.01).Inaddition,nosignificantchangeinODCactivitywasobservedwithNaCltreatment(P>0.05)(Fig.2).TGaseisakeyenzymecatalyzingtheconversionoffPAstobPAs.IncreasingNaClconcentrationscausedincreasesbothinTGaseandPAOactivities,anddecreasedtheactivitiesofTGaseandPAOat300mmol/LNaCl(Fig.2).2.3resp利益which/fpax/fpdswhich/salt排放/外部投资fspd+fpax/fpds/salttores/fpds/salttores/fpdswhich/salt/fpax/fpds/salttores/fpdswhich/salttores/fpds/fpds/fpax/fpds/salttority/salttority/fpds/fpds/salttority/fpdsfspd+bpo纳米statity/salttority/salttor3.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.4.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.4.3.5.3.3.4.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.AsaresultofincreasingNaClconcentrations,fPutcontentinbarleyseedlingrootsincreased.NaClof50-100mmol/LcausedaverysignificantincreaseintheleveloffSpdinKepin7,whileinJian4,allconcentrationscausedaverysignificantincreaseoffSpdcontent(P<0.01).NoverysignificantincreasesinfSpmlevelswereobservedundersaltstress(P>0.01).Inaddition,anabundantfreeunknownpolyamine(fPAx)withRf0.92wasdetected,whichlocatedbehindSpmandSpdonthin_layerchromatographicplate(RfofSpmis0.88andRfofSpdis0.78),anditscontentincreasedverysignificantlywithNaCltreatments(P<0.01),thatoffSpdalsoincreased(Table1).OfalldetectedfPAs,fSpdwasthemostabundantone,whichaccountedfor25%-45%proximately,whenNaClconcentrationwasmorethan200mmol/L,fPutwaspredominant.Sothevalueof(fSpd+fPAx)/fPutincreasedwithincreasingsalinityanddecreasedathighconcentrationsofNaCl.StatisticalanalysisshowedthattherewasasignificantpositivecorrelationbetweenGRand(fSpd+fPAx)/fPutratiowhateverinKepin7orJian4,andtheircorrelationcoefficientswere0.903**and0.901**(r0.01=0.798)respectively,suggestingGRofNaCl_treatedbarleyseedlingswascloselycorrelatedto(fSpd+fPAx)/fPut.Statisticalanalysisindicatedthat,with100mmol/LNaCltreatment,leafK+/Na+wasverysignificantlypositivelycorrelatedwithroot(fSpd+fPAx)/fPutratio(r=0.976**,r0.01=0.798).Theseresultsshowedthat(fSpd+fPAx)/fPutratiowascloselycorrelatedwithsalttoleranceofbarley,thestrongerthesalttolerance,thehigherofK+/Na+ratio,andviceversa.2.4与生物测定和重新级correlationwolbatchingwratching.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.5.4.3.4.3.4.3.4.3.4.5.4.3.4.5.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.5.4.3.4.5.4.5.4.3.4.5.4.3.4.5.4.5.4.5.4.5.4.5.4.3.4.5.4.5.4.5.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.3.3.3.3.3.4.3.4.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.5.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.Intheexperimentpresentedhere,bPAsaswellasfPAwerealsodetected,whilePAsconjugatedtosmallmolecules(suchashydroxycinnamicacid)inthesupernatantafterextractionwithperchloricacidwerenotfound.OfallkindsofbPAs,bPutwasthemostabundantone,whichaccountedfor30%-68%proximately.ThecontentofbSpmchangedalittlewithNaCltreatment,whilecontentsofbPut,bSpdandbPAxincreasedstrikinglyby50-200mmol/LNaCltreatmentandreducedsharplyat300mmol/LNaCl(Table2).AnanalysiswasperformedbetweenGRofbarleyseedlingsandrootbPAcontents,andtheresultindicatedthataverysignificantpositivelycorrelationexistedbetweenthembothinJian4andKepin7,withcorrelationcoefficientsof0.953**and0.923**(r0.01=0.798)respectively.Tables1,2showedthatcontentsofbPAswerehigherthanthoseoffPAsinrootsofbarleyseedlings,andinsalt_tolerantcultivarJian4bPAlevelswerehigherthanthatinsalt_sensitivecultivarKepin7.TheresultsofcorrelationanalysisbetweenGRandfPA/bPAratiosuggestedthattherewasaverysignificantlynegativecorrelationbetweenthem,astoJian4andKepin7,theircorrelativecoefficientswere-0.912**and-0.978**respectively.TheseresultsindicatedthatthebalanceoffPAsandbPAsinbarleyseedlingswasimportanttosalttolerance.bPutwasamainbPAinrootsofbarleyseedlings,soconversionformfPuttoconjugatedfromwasveryimportanttomaintainhighratioof(fSpd+fPAx)/fPutinbarleyseedlings.StatisticalanalysisshowedthatrootbPAlevelwaspositivelycorrelatedverysignificantlywiththeratioof(fSpd+fPAx)/fPut,astoJian4andKepin7,theircorrelativecoefficientswere0.914**and0.956**(r0.01=0.798)respectively.3苏putraftingwhich号,u2004,2001,2001,2002,2001,2002,2001,2002,2001,2001,2001,2001,2001,2001,2001,2001,2001,2001,2001,2001,2001,2001,2001,2001,2001,2001,2001,2001,2001,2001,2001,2001,2001,2001,2001,2001,2001,2001,2001,2001,2001,2019,2019,2019,2019,2019,2019,2019,2019,2019,2019,2019,2019,2019,2019,2019,2019,2019,2019,2019,2019,2019,2019,2019,9,26.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.3.4.3.4.3.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.3.3.3.3.3.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.3.4.4.3.4.3.4.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.4.3.3.3.3.3.3NaCl_treatedbarleyseedlingsinducedaconsiderableincreasinginADCactivityintheroots,whichresultedinanincreaseinfPutlevel.Inaddition,degradationofbPAscatalyzedbyPAOcouldalsoleadtotheaccumulationoffPut(FedericoandAngelini,1991).Inthepresentwork,PAOactivitywasactivatedat50-200mmol/LNaClanddecreasedat300mmol/LNaCl,however,itsactivityat300mmol/LNaClwasstillhigherthanthatofthecontrolplants(Fig.2),correspondingtothesechangesinPAO,thelevelsofbSpdandbPAxwerestilllowerthanthoseofthecontrolplants(Table2).TheaboveresultswereinagreementwiththeobservationsofFederico(FedericoandAngelini,1991).TGaseisakeyenzymecatalyzingtheconversionoffPAstobPAs(Serafini_FracssniandDelDuca,1995).TheactivityofTGasewaspromotedby50-200mmol/LNaClanddecreasedat300mmol/LNaCl,however,bPAlevelsdecreasedat200mmol/LNaCl(Fig.2;Table2),whichwasprobablyduetothedegradationofbPAscatalyzedbyPAO.TransgenicexperimentshaddemonstratedthatexcessiveaccumulationoffPutwastoxictoplantgrowthandledtoadecreaseinthegrowthofstemsandroots(RichardandAlexandra,1997),suggestingsharpaccumulationoffPutwasharmfultoplantgrowth.However,RoyandWu(2001)suggestedhighcontentsofPutcouldenhancethesalttoleranceofrice(Oryzasativa).Inouropinion,fPutaccumulationwasconsideredasanegativefactortosalttoleranceofbarleyseedlings,whichwasauthenticatedbythepreviousresult(KrishnamurthyandBhagnat,1984).Thus,alleviationofsaltinjuryanddetoxificationcouldbeachievedbyincreasingtheconversionoffPuttofSpm,fSpd,fPAxand/orboundones.Inaddition,Lietal(1999)hadreportedthattheconversionoffPuttofSpdorfSpmwasbeneficialtotheimprovementofsalttoleranceofrice.Inthepresentwork,nosignificantchangeinSpmlevelwasobservedwithNaCltreatment.Onthecontrary,fPAxcontentchangedverysignificantlyundersaltstress(P<0.01),50-200mmol/LNaCltreatmentsinducedaverysignificantincreaseinfPAxlevel,butwith300mmol/LNaCltreatment,fPAxcontentdecreasedmoreapparentlyinsalt_sensitivecultivarKepin7seedlingrootsthanthatinsalt_tolerantcultivarsJian4seedlingroots(Table1).ChangesinPAxcontentweresimilartothatinSpd(whateverinfreeorboundones).DependedonthefeaturesofdansylatedPAsonthin_layerchromatographyplate,PAxseemedtobealongchainPA.Basedontheabovefacts,theconversionoffPuttofSpdand/orfPAxmightplayanimportantroleinimprovingsalttoleranceof