ContentslistsavailableatScienceDirectPlantPhysiologyandBiochemistryjournalhomepage:www.elsevier.com/locate/plaphyResearcharticleChangesintheproteinpatternsinpea(PisumsativumL.)rootsundertheinfluenceoflong-andshort-termchillingstressandpost-stressrecoveryAnnaBadowieca,*,SylwiaSwigonskab,Stanis1awWeidneraDepartmentofBiochemistry,FacultyofBiologyandBiotechnology,UniversityofWarmiaandMazuryinOlsztyn,OczapowskiegoStreet1a,10-957Olsztyn,PolandbResearchandEducationCenter,LaboratoryofMolecularDiagnostics,FacultyofBiologyandBiotechnology,UniversityofWarmiaandMazuryinOlsztyn,PrawochenskiegoStreet5,10-720Olsztyn,PolandaarticleinfoArticlehistory:Received10June2013Accepted5August2013Availableonline16August2013Keywords:ChillingstressGerminationPearootsProteomicsRecoveryabstractAmongstmanyfactorsrestrictinggeographicaldistributionofplantsandcropproductivity,lowtem-peratureisoneofthemostimportant.Togainbetterunderstandingofthemolecularresponseofgerminatingpea(PisumsativumL.)tolowtemperature,weinvestigatedtheinfluenceoflongandshortchillingstressaswellaspost-stressrecoveryonthealterationsintherootproteomes.Theimpactoflongstresswasexaminedonthepeaseedsgerminatinginthecontinuouschillingconditionsof10Cfor8days(LS).Toexaminetheimpactofshortstress,peaseedsgerminatingfor72hintheoptimaltem-peratureof20Cweresubjectedto24-hchilling(SS).Additionally,bothstresstreatmentswerefollowedby24hofrecoveryintheoptimalconditions(accordinglyLSRandSR).Usingthe2DgelelectrophoresisandMALDI-TOFMSproteinidentification,itwasrevealed,thatmostoftheproteinsundergoingregu-lationundertheappliedconditionswereimplicatedinmetabolism,protectionagainststress,cellcycleregulation,cellstructuremaintenanceandhormonesynthesis,whichaltogethermayinfluencerootgrowthanddevelopmentintheearlystagesofplantlife.Theobtainedresultshaveshownthatmostofdetectedalterationsintheproteomepatternsofpearootsaredependentonstressduration.However,therearesomeanalogicalresponsepathwayswhicharetriggeredregardlessofstresslength.Thefunctionsofproteinswhichaccumulationhasbeenchangedbychillingstressandpost-stressrecoveryarediscussedhereinrelationtotheirimpactonpearootsdevelopment.Ó2013ElsevierMassonSAS.Allrightsreserved.1.IntroductionInthetimesofthealarmingincreaseofhumanpopulationandglobalclimaticchanges,understandingthemechanismsofplantresponsetoabioticstressbecameoneofthemostsignificantchallengesforbiologicalandagronomicalsciences.Plantsduringtheirlifecycleareexposedtonumerousstrainsofbioticandabioticorigin.Oneofthemajorfactorslimitingplantproductivityandtheirgeographicaldistributionislowtemperature.Non-freezingAbbreviations:2D,PAGEtwo-dimensionalelectrophoresisinpolyacrylamidegels;C1,C2,controlsamplesoftheseedsgerminatingintheoptimaltemperature;LS,long,continuouschillingstress;LSR,recoveryafterlongchillingstress;MALDI-TOFMS,matrix-assistedlaseddesorption/ionizationtime-of-flightmassspec-trometer;ROS,reactiveoxygenspecies;SS,short,rapidchillingstress;SR,recoveryaftershortstress.*Correspondingauthor.Tel.:þ48895233520.E-mailaddress:anna.badowiec@uwm.edu.pl(A.Badowiec).0981-9428/$eseefrontmatterÓ2013ElsevierMassonSAS.Allrightsreserved.http://dx.doi.org/10.1016/j.plaphy.2013.08.001temperaturesrangingfrom0to15C,calledchillingstress,frequentlyoccurintemperateregionsduringearlygrowingseasonandmaycauseasubstantialcroploss[1].Althoughlowtemperatureshaveanegativeimpactofongrowth,developmentandvitalityofplants[2],manyofthemhaveevolvedvariousadaptivestrategiestominimizecellulardamageandwithstandtheadverseenvironmentalconditions.Plantsareabletoadjusttheirgrowthanddevelopment,aswellastheirphysiologicalandmolecularfunctionstoestablishanewlevelofcellularhomeostasis.Numerousreportsindicatethattheregulationofgeneexpressionisoneofthemainstrategiesofplantresponsetoachangeableenvironment.Ithasbeenprovedthatstressresponseoccursnotonlyonatranscriptomebutalsoonaproteomeandmetabolomelevelinplantcells[3].Theinvestigationoftheproteinpatternalterationsduringexposuretochillingmayhelptounravelarelationshipbetweenproteinabundanceandtheprocesseswhichundergoregulationinresponsetothestress.Understandingthenaturalmechanisms316A.Badowiecetal./PlantPhysiologyandBiochemistry71(2013)315e324
lowtemperaturestresshavefocusedonthemodelplantsArabi-dopsisthalianaandOryzasativa[4].However,theresultsobtainedforthesespeciescannotalwaysbeappliedtootherplantfamiliessuchaslegumes.ThemostrecentproteomicstudiesconcerningtheinfluenceoflowtemperaturestressonpeahasbeenperformedbyDumontetal.[5](roots,stemsandleaves)andGrimaudetal.[6](chloroplasts).However,noextensivestudyofpearootpro-teomes,coveringtheexaminationofchillingstressofdifferentdurationtogetherwiththerecoveryafterstressexemptionhasbeendoneyet.Thisstudyisapartofabiggerwork,aimedatcomparingtheplantswithdifferentsusceptibilitytolowtemper-atureandtheirproteomicresponsetothechillingstressofdifferentduration,occurringintheearlystagesofplantdevelopment(BadowiecandWeidner,unpublishedresults).Fig.1.Schemeoftheexperiment.C1econtrolsampleofpeaseedsgerminatingintheoptimaltemperatureconditions(20C)for3days;C2econtrolsampleofpeaseedsgerminatinginoptimaltemperature(20C)for4days;SSeseedssubjectedtorapid24-hchillingstress(10C)after3daysofgerminationin20C;SReseedssubjectedtorecoveryaftershortstress:3daysin20C,1dayin10C,1dayin20C;LSepeaseedsgerminatingfor8daysinlongcontinuouschillingconditions(10C);LSReseedssubjectedto24-hrecoveryin20Cafter8daysofgerminationinlongchillingcon-ditionsThesolidlineindicatestheexperimentalpartsconductedintheoptimaltemperature(20C),brokenlineindicatespartsconductedinthelowtemperature(10C).
2.Results2.1.MorphologicalchangesThegeneralschemeoftheexperimentsisshowninFig.1.Theseedswhichconstitutedthecontrolweregerminatingin20Cfor72h(controlC1)and96h(controlC2).Toinducethelongcontinuouschillingstress(sampleLS),seedsweregerminatingin10CuntiltheirrootsachievedthemorphologicalsimilaritytotherootsfromthecontrolC1(8days).Inordertoexaminetheimpactoftherecoveryafterlongchillingstress,peasgerminatingfor8daysin10Cweretransferredfor24hto20C(sampleLSR).Shortrapidstresswasinducedbytransferringtheseedsgerminatingfor72hin20Ctolowtemperature(10C)foradditional24h(sampleSS).Therecoveryaftershortstresswasinducedbyincreasingthetemperatureto20Cforanother24h(sampleSR).ThemorphologyofpeaseedsgerminatingunderdifferentexperimentalconditionsisdepictedinFig.2.Thecontinuouslowtemperature(LS)duringgerminationhasanegativeinfluenceontheelongationofpearoots.ThelengthofrootsfromLSachievedsimilaritytothelengthofthosefromC1after8daysofgerminationin10C.ChillingstressofshortdurationalsocausedinhibitionofrootdevelopmentincomparisontotherootsfromcontrolC2.The24-hrecoveryafterbothlong(LSR)andshortstress(SR)enhancedtheelongationofroots.Thecontentoffreshanddryweightintherootsfromgermi-natingpeaseedsisshowninFig.3.Theaveragecontentoffreshanddrymassin72-hrootsgerminatinginthecontrolconditionsC1andthemassofrootsfrompeagerminatingfor8daysin10C(LS)didnotdiffersignificantly.Therecoveryafterlongchilling(LSR)causedasignificantincreaseinbothfreshanddryweightofpearootsindeterminingplantstresstolerancewouldallowthebreederstointroducenew,chillingtolerantcultivarsofvaluablecropplants.Takingintoconsiderationthecomplexityandtissue-specificityofstressresponsepathways,determiningstress-relatedbiomarkerswhichcouldsimplifytheselectionofcultivarsmoretoleranttochillingisparticularlydifficult.Inthepresentedworkweexaminedthegerminatingseedsofpea(PisumsativumL.),whichbelongstothefamilyoflegumes,asecondmostimportantgroupofcropplantsafterthegrasses.Theirseedsconstituteanimportantsourceofvaluableproteinsinhumanandanimalnutrition.Itisasignificantforagecropwidelybredintemperateregions.Theglobalproductionofthepeaseedsin2011wasnearly17Mtons(dataaccordingtohttp://faostat.fao.org).Theearlyround-seededvarieties,towhichbelongtheexaminedherecultivar‘Szesciotygodniowy’,exhibithightolerancetolowtem-peraturesduringsowingtime.Toseparateandidentifytheproteinsfrompearoots,2Dgelelectrophoresisfollowedbymassspectrometrywereemployedinthiswork.Thismethodhasalreadybeensuccessfullyutilizedtoanalyzetheexpressionofplantproteinsunderdifferentformsoftheabioticstress.Todate,mostoftheproteomicreportsconcerningFig.2.Themorphologicalchangesoccurringintherootsofpeaseedsgerminatingfor72hintheoptimaltemperature(C1),germinatingfor8daysunderlongchillingstress(LS),subjectedto24hrecoveryafterlongchillingstress(LSR),aswellasgerminatingfor96hintheoptimaltemperature(C2),subjectedtorapid24hchillingafter72hofgermination(SS)andto24hofrecoveryaftershortchilling(SR).
A.Badowiecetal./PlantPhysiologyandBiochemistry71(2013)315e324317
Fig.3.Changesinfresh(FW)anddryweight(DW)ofpearootsisolatedfromtheseeds:A)germinatingfor72hintheoptimaltemperature(C1),germinatingfor8daysunderlongchillingstress(LS),subjectedto24hofrecoveryafterlongchillingstress(LSR);B)germinatingfor96hintheoptimaltemperature(C2),subjectedtorapid24-hchillingafter72hofgermination(SS)andsubjectedto24hofrecoveryaftershortchilling(SR).ResultsarepresentedasmeansÆSD.Valuesfollowedbythesamesu-perscriptarenotsignificantlydifferentbetweenthecontrolandstresssamplesaswellasbetweenstressandrecoverysamples(p<0.05).
comparisontothesampleLS.Subjectingthegerminatingseedstoshortchilling(SS)resultedinasignificantinhibitionofthefreshanddrymatterincorporationincomparisontorootsofpeaseedsgerminatingincontrolconditionsC2.Thesubsequentrecovery(SR)causedthesignificantincreaseofbothvalues,comparingtothesampleSS.2.2.ProteinidentificationThemaingoalofthepresentstudywastoinvestigatethein-fluenceofthechillingstressofdifferentdurationandpost-stressrecoveryontheproteomicchangesoccurringintherootsofgerminatingpeaseeds.Theappliedinvestigationmethodenabledustoidentifyproteinsalteredinabundanceintheappliedcondi-tions.Morethan1300proteinspotswerereproduciblydetectedonCoomassiestainedgels.Seventy-fourproteinswithatleast1.75-folddifferenceinabundanceafterchillingstressorrecoveryweresubjectedtothetrypticdigestionandanalyzedwithMALDI-TOFMS.Sixty-oneoftheseproteinsweresuccessfullyidentified.Weidentified5proteinswhichamountincreasedand5whichamountdecreasedduringthelongexposuretochilling(LS).Thesubsequent24hofrecoveryintheoptimaltemperature(LSR)resultedintheincreaseofthequantityof22proteinsandthedecreaseof13proteinsingerminatingpearoots.Proteinidentifi-cationresultsareshowninTable1.ProteinswhichabundancealteredinLSandLSRarealsomarkedinFig.4.Theappliedshortrapidchillingstress(SS)causedanincreaseinthequantityof3proteinsandadecreaseinthequantityof6other.Weidentifiedalso4proteinsup-and3down-regulatedduringthefollowingrecovery.TheidentificationresultsareshowninTable2.ProteinswhichexpressionchangedinSSandSRarealsomarkedinFig.5.Itisworthnoticingthatweidentifiedmanyproteinswhichaccumulationchangedundermorethanoneoftheappliedcondi-tions.ThenumbersofproteinscommontoseveraltreatmentsareshownontheVenndiagramsinFig.6.Theabundanceofoneofthementionedproteins(adenosylhomocysteinase)wasincreasedinbothSSandLS.Anotherprotein(2-phosphoglyceratedehydratase)exhibitedhigheraccumulationinLSandthenlowersaccumulationduringthefollowingrecoveryLSR.Thequantityofotherprotein(S-adenosylmethioninesynthase)decreasedinbothSRandLSR.Thequantityofaproteinidentifiedas1-aminocyclopropane-1-carboxylateoxidaseincreasedinSSanddecreasedafterwarddur-ingthefollowingrecoverySR.Therelativeamountofoneprotein(methylenetetrahydrofolatereductase)wasfoundtoberaisedinSSandreducedinLSR.Theaccumulationof3proteins(phosphoglyceratekinaseandtwoalphatubulins)waslowerinLSthaninC1andthenincreasedduringthefollowingrecoveryLSR.Thequantitiesofthreeotherproteins(galactokinase,glutathioneS-transferaseandputativepectinesterase)decreasedduringtheshortstressSSandthenincreasedduringthepost-stressrecoverySR.Oneofthem(putativepectinesterase)wasalsoup-regulatedduringrecoveryafterlongstress(LSR).Therewerenocommonproteinswhichrevealedadecreasedabundanceduringbothshortandlongstressandonlyoneprotein(putativepectinesterase)commonfortherecoveryafterbothshortandlongchillingtreatment.3.DiscussionInthisworkweperformedaproteomicanalysisofpearootssubjectedtolongcontinuous(LS)andshortrapidchilling(SS)duringgermination,aswellassubjectedtopost-stressrecoveryafterbothstresstreatments(accordinglyLSRandSR).Ourinves-tigationrevealedacomplexityofnetworksthatmayactcoopera-tivelyinordertoestablishanewcellularhomeostasisandinfluencethegrowthanddevelopmentofpearootsinresponsetolowtemperatureofdifferentlength.Sixty-oneoutofseventy-fivedifferentlyexpressedproteinsweresuccessfullyidentifiedbyMALDI-TOFMSandthesubsequentdatabasesearch.Besidethealreadyknowncold-responsivepro-teinssuchasenolase,L-ascorbateperoxidaseorheatshockpro-teins,weidentifiedseveralotherswhichprobablyplayapivotalroleincarbohydrate,energyandaminoacidsmetabolismaswellascellwallcomponentsbiosynthesis.Theireffectonthemetabolicpathwaysinconjunctionwitharesponsetotheappliedconditionswillbediscussedfurther.3.1.ProteinsregulatedduringthelongchillingstressTheproteinwhichaccumulationchangedthemostamongtheproteinsalteringinabundanceafterlongchilling(LS)wasadeno-sylhomocysteinase,knowntoindirectlycontrolDNAmethylationandinfluencegenestranscription.Itsaccumulationwasalsostim-ulatedinAcerplatanoidesafterabscisicacidapplication[7]whatindicatesthattheremightbeasignificantrelationshipbetweenadenosylhomocysteinaseabundanceandthesuppressionofgermination.Itisworthnoticingthatitsabundancewaspositivelyregulatedunderbothchillingtreatments,suggestingthestronginfluenceoftheappliedstressonregulationofgeneexpression,possiblyaffiliatedwithrootgrowthinhibition.TheotherproteinmoreabundantinLSthaninC1wasdetectedasoneofthekeyenzymesofglycolysise2-phosphoglyceratedehydratase,knownasenolase.Overexpressionofthisenzymeundercoldstresshasalreadybeendescribedpreviously[8,9].Theaccumulationofenolasewassuggestedtobeanindicatorofcoldstressacclimation[5].Itthisstudythequantityofenolasenotonlystronglyincreasedduringlongchillingbutalsodecreasedalmosttwiceduringpost-stressrecovery,implyingthatintherootsofpeagerminatinginlowtemperatureoccurprocessesconnectedtoacclimation.Otherpositivelyregulatedproteins:GDP-mannose3,5-epimeraseandputative12-oxophytodienoatereductaseareinvolvedinpro-tectionagainststress.GDP-mannose3,5-epimeraseisaninterme-diateforascorbicacidsynthesisandaffectstheleveloftheantioxidantglutathione,playingthiswayadetoxifyingroleincells[10].Oxophytodienoatereductase,ontheotherhand,takespartin318A.Badowiecetal./PlantPhysiologyandBiochemistry71(2013)315e324
Table1MALDITOFidentificationoftheproteinswhichweredifferentlyaccumulatedinresponsetolongcontinuouschillingstress(LS)andpost-stressrecovery(LSR)intherootsofgerminatingpeaseeds.IDProteinnameFCaAccessionnumberSC/PbExp.pI/MWcCalc.pI/MWdProteinswhichquantitychangedunderlongchillingconditions(LS)Increasedquantity1Adenosylhomocysteinase[EC:3.3.1.1]22-Phosphoglyceratedehydratase[EC:4.2.1.11]4Putative12-oxophytodienoatereductase[EC:1.3.1.42]5GDP-mannose3,5-epimerase[EC:5.1.3.18]6Aspartateaminotransferase1[EC:2.6.1.1]Decreasedquantity8Acetyl-CoAcarboxylasecarboxyltransferasesubunitbeta[EC:6.4.1.2]9Alphatubulin10Alphatubulin11Phosphoglyceratekinase,cytosolic[EC:2.7.2.3]13AlphatubulinProteinswhichquantitychangedduringrecoveryafterlongchilling(LSR)Increasedquantity15L-Ascorbateperoxidase,cytosolic[EC:1.11.1.11]18Luminal-bindingprotein/HSP7019Triosephosphateisomerase[EC:5.3.1.1]20MediatorofRNApolymeraseIItranscriptionsubunit21Caffeicacid3-O-methyltransferase3[EC:2.1.1.68]9Alphatubulin22L-Ascorbateperoxidase,cytosolic[EC:1.11.1.11]23Fructose-1,6-bisphosphatase,cytosolic[EC:3.1.3.11]24Glyceraldehyde-3-phosphatedehydrogenase,cytosolic[EC:1.2.1.12]27L-Ascorbateperoxidase,cytosolic[EC:1.11.1.11]28NADP-dependentisocitratedehydrogenase[EC:1.1.1.42]29Heatshockprotein90-130Putativepectinesterase[EC:3.1.1.11]31Alphatubulin32ATPsynthasesubunitbeta34Caffeoyl-CoAO-methyltransferase[EC:2.1.1.104]35ATPsynthasesubunitalpha,mitochondrial11Phosphoglyceratekinase[EC:2.7.2.3]37PsHSC71.038Heatshockprotein81-339Alphatubulin13AlphatubulinDecreasedquantity42Fructose-bisphosphatealdolase,cytoplasmic[EC:4.1.2.13]43F-Box/Kelch-repeatproteinAt3g1753044HypotheticalproteinZEAMMB73_053553452-Phosphoglyceratedehydratase[EC:4.2.1.11]46Eukaryoticinitiationfactor4A/DEADboxRNAhelicase47Glyceraldehyde-3-phosphatedehydrogenase,cytosolic48Translationallycontrolledtumorproteinhomolog49Malatedehydrogenase,cytoplasmic[EC:1.1.1.37]50Methylenetetrahydrofolatereductase1[EC:1.5.1.20]51Alphatubulin52S-Adenosylmethioninesynthase[EC:2.5.1.6]22-Phosphoglyceratedehydratase[EC:4.2.1.11]54Predictedbetaamylase[EC:3.2.1.2]abcd2.52.32.11.91.83.12.51.91.81.8XP_003524926P42896B9FFD3Q93VR3P28011A4QLB5P46259P46259AF275639ACU1877531/1433/1427/737/1620/526/928/1120/739/1238/55.4/753.7/325.1/306.1/694.5/314.7/545.2/464.3/524.9/475.8/205.7/545.3/485.2/425.7/438.9/466.6/564.9/504.9/505.7/425.4/223.52.62.42.42.42.32.32.22.22.12.02.02.02.01.91.91.91.91.81.81.81.82.62.62.32.22.22.12.02.02.01.91.91.91.8P48534Q42434AFK34912Q9LKR3Q43047P46259P48534Q8RW99P34922P48534AAS49171XP_002273244O64479P46259BAF40515Q40313P05493AF275639CAA83548XP_002864424XP_003629736ACU18775P46257Q9LUP5AFW86695NP_001237329AAN74635P34922P50906O48905Q9SE60P46259BAC81655NP_001237329XP_00363202566/1117/745/1221/1333/928/1138/738/664/1757/818/614/927/627/831/846/832/1339/1217/926/1736/1038/560/1220/647/737/1457/1830/759/1132/825/630/1341/1333/1438/114.2/233.8/484.4/213.8/476.0/365.2/344.2/244.2/155.7/365.5/345.2/293.9/405.4/275.1/333.8/554.5/304.9/294.9/354.0/464.3/425.6/355.8/206.6/424.9/574.5/235.1/565.2/516.4/373.9/196.2/395.9/324.8/545.4/513.7/306.1/545.5/275.0/745.7/275.1/745.6/404.9/505.5/274.5/386.6/375.5/276.2/465.0/818.7/384.9/505.2/535.5/286.0/555.7/425.0/715,0/784.9/505.4/226.8/397.4/458.9/265.3/485.4/476.6/374.7/196.4/365.6/674.9/506.3/405.3/486.3/56Expressionfoldchange.Sequencecoverage(%)/numberofpeptidesmatched.Experimentalvaluesofisoelectricpointandmolecularmassoftheprotein.Theoreticalvaluesofisoelectricpointandmolecularmassoftheprotein.biosynthesisofjasmonicacid,signalmoleculeaffiliatedwithplantdefenseagainstvariousstressesincludinglowtemperature[11].Thelastproteinwhichabundancechangedpositivelyinthisgroupwasidentifiedasaspartateaminotransferase,whichaltersaminoacidsandnitrogenmetabolisminplants[12].Theroleofthisenzymeinresponsetocoldisnotclear.TheproteinswhichamountwasnegativelyregulatedinLSareimplicatedinbothmetabolismandcellstructuremaintenance.Itisnotsurprisingthattheabundanceofacetyl-CoAcarboxylasecarboxyltransferasebetasubunitandphosphoglyceratekinase,enzymesrelevantforcarbohydratemetabolism,significantlydecreasedunderlongchillingconditions.AloweramountoftheformerundercoldstresshasalreadybeennoticedinAbelmoschusmoschatus[13].Alsophosphoglyceratekinasewasfoundtobeearly-responsivetobothcoldandsaltstressinriceleaves[14].Itisnoteworthythatitsquantityroseafterstressrelease,provingthatlowtemperaturereducedtherateofcellmetabolismduringgermination.Themultiplealphatubulinspotsfoundinthisworkmayindi-catethepresenceofthenumerousproteinisoformsortheproductsofpost-translationalmodificationsanddegradation.Ithasbeenprovedbeforethatthecompositionoftubulinisotypesinryerootsdeterminesthestabilityofmicrotubulesunderlowtemperaturestress,whatseemstobecriticalforplantcoldtolerance[15].A.Badowiecetal./PlantPhysiologyandBiochemistry71(2013)315e324319
Fig.4.2Dgelmapsofdifferentlyaccumulatedproteins,extractedfromtherootsofpeaseeds,germinatinginthecontrolconditions(C1),inlongchillingconditions(LS)andsubjectedtorecoveryafterlongchillingstress(LSR).TheproteinspotswerevisualizedbyCoomassieG-250staining.ThenumbersofspotscorrespondtothoseshowingquantitativedifferencesbetweenC1andLSaswellasbetweenLSandLSR.
3.2.ProteinsregulatedduringtherecoveryafterlongchillingstressMostoftheproteinswhichquantityincreasedinLSRareinvolvedinstressresponseandcellstructuremaintenance;othersareengagedincarbohydrateandenergymetabolism.Temperaturechangefromlow(10C)toambient(20C)apparentlytriggersROSoverproductionwhichconsequentlyforcestheincreaseintheac-tivityofantioxidantdefensesystem.ItisalreadyknownthatplantswithhigherROSscavengingabilityexhibitanincreasedtolerancetoenvironmentalstresses[16].AmongtheproteinsinvolvedincelldetoxificationinLSRwereL-ascorbateperoxidaseandNADP-dependentisocitratedehydrogenase.TheincreasedabundanceofL-ascorbateperoxidasehasbeenlinkedbyDumontetal.[5]tothecoldacclimationinpea.Ithasalsobeendetectedinourotherreport,wheretherelativeabundanceofthisproteinincreasedduringtherecoveryaftershortstressintherootsofgerminatingbean(BadowiecandWeidner,unpublishedresults).Anotherpro-teininvolvedintheantioxidativemachinery,NADP-dependentisocitratedehydrogenase,hasacapacitytogeneratethereducingpowerintheformofNADPHinresponsetothecausedbytheabioticstressorsoxidativestress[17].Manyproteinsengagedinstressresponsebelongtoaheatshockproteinfamily.Membersofthislargeanddiversifiedgroupareresponsibleforproperproteinfolding,refolding,assemblyanddegradation.Theyplayalsoanimportantroleinthestabilizationofmembranesandhavevariousotherfunctionswhichleadtorees-tablishingthecellularhomeostasisinstressfulconditions[18].ThemediatorofRNApolymeraseIItranscriptionsubunithasbeenpreviouslyfoundtobeup-regulatedduringgerminationandexhibitedinductioninresponsetoheatshockinArabidopsis[19].Mediatorcomplexisasignificantcontrollerofgeneexpressionsotheregulationofthisprotein,togetherwiththeincreaseintheabundanceofheatshockproteins,mayindicatetheoccurrenceofthenovelproteinssynthesisandtheneedfortheenhancedqualitycontroloftheproteinprocessingaftertemperatureshift.Anothersignificantfunctionalgroupoftheproteinsaccumu-latedduringLSRincludestheproteinsinvolvedinmetabolismofcellwallcomponents:caffeicacid3-O-methyltransferase,caffeoyl-CoAO-methyltransferaseandputativepectinesterasewhicharetheenzymesofligninandpectinmetabolismpathways.Theinductionofgenesinvolvedinligninsynthesishasbeenlinkedwithcellwallstrengtheninginresponsetothelowtemperaturestress[20],thereforetheincreaseinarelativequantityofcaffeicacid3-O-methyltransferaseandcaffeoyl-CoAO-methyltransferaseinthisworkisintriguing.PossiblyasubstantialriseintheabundanceoftheseenzymesinLSRdoesnotindicatetheenhancedprotectionagainsttheadverseconditionsbutratherconstitutesaresponsetoanincreasingdemandofnewbuildingmaterialfortherapidlyextendingcells.Thishypothesisseemstobeconfirmedbytheincreasedamountofpectinesterase,whichisknowntomodifyadegreeofgalacturonylresiduesmethylationincellwallpectin.Suchmodificationmakesthecellwallcomponentsmoresuscep-tibletodegradationbypolygalacturonasesandconsequentlyleadstocellwalllooseningandaffectsitsextensibility[21].Moreover,pectinesteraseaccumulationwasalsostimulatedduringrecoveryaftershortchillinginthiswork.ThequantityofmanyenzymestakingpartinbothglycolysisandgluconeogenesiswasfoundtobestronglyincreasedinLSR.Amongthemwereglyceraldehyde-3-phosphatedehydrogenase,phos-phoglyceratekinaseandtriosephosphateisomerase.Sinceglycol-ysisandgluconeogenesissharemostoftheenzymesanddonotoccurwiththesameefficiencyincells,thestimulationoftheaccumulationoffructose-1,6-bisphosphatase,theenzymespecificonlytogluconeogenesis,suggeststhereversalofglycolyticflowanddominationofcatabolicreactionsresultinginglucosesynthesisduringrecoveryafterlongstress.Highamountofsugarsisrequiredforgerminationandthefurtherseedlingestablishmentaswellasprovidesbuildingblocksforothercellmolecules[22].Additionally,weobservedanincreaseinabundanceofalphaandbetasubunitsofATPsynthase.Regulationofbothproteins,togetherwiththeproteinsinvolvedincarbohydratemetabolism,320A.Badowiecetal./PlantPhysiologyandBiochemistry71(2013)315e324
Table2MALDITOFidentificationoftheproteinswhichweredifferentlyaccumulatedinresponsetoshortrapidchillingstress(SS)andpost-stressrecovery(SR)intherootsofgerminatingpeaseeds.IDProteinnameFCaAccessionnumberSC/PbExp.pI/MWcCalc.pI/MWdProteinswhichquantitychangedinresponsetoshortchilling(SS)Increasedquantity1Adenosylhomocysteinase[EC:3.3.1.1]551-Aminocyclopropane-1-carboxylateoxidase[1.14.17.4]50Methylenetetrahydrofolatereductase[EC:1.5.1.20]Decreasedquantity30Putativepectinesterase[EC:3.1.1.11]56Galactokinase[EC:2.7.1.6]57Heatshockprotein70,mitochondrial58Cyclin-P1-159GlutathioneS-transferase[EC:2.5.1.18]60HypotheticalproteinZEAMMB73_3616502.11.71.73.22.01.91.91.81.8XP_003524926P31239Q9SE60O64479CAF34022P37900Q0J9W0BAC81649AFW8899031/1439/1025/627/626/615/1031/849/1254/75.4/624.4/375.8/335.4/275.0/604.5/665.2/574.2/265.0/145.7/545.1/365.6/678.7/385.4/555.8/727.1/284.9/274.4/11Proteinswhichquantitychangedduringrecoveryaftershortchilling(SR)Increasedquantity56Galactokinase[EC:2.7.1.6]1.861Alpha-1,4-glucan-proteinsynthase(UDPforming)[2.4.1.-]1.830Putativepectinesterase[EC:3.1.1.11]1.859GlutathioneS-transferase[EC:2.5.1.18]1.8Decreasedquantity62S-Adenosylmethioninesynthase[EC:2.5.1.6]3.463Heatshockprotein71.21.9551-Aminocyclopropane-1-carboxylateoxidase[1.14.17.4]1.7abcdCAF34022O04300O64479BAC81649P49612AAA82975P3123926/624/827/649/1241/1332/1720/65.0/604.9/405.4/274.2/265.8/514.2/514.4/375.4/555.7/428.7/384.9/276.4/405.2/725.1/36Expressionfoldchange.Sequencecoverage(%)/numberofpeptidesmatched.Experimentalvaluesofisoelectricpointandmolecularmassoftheprotein.Theoreticalvaluesofisoelectricpointandmolecularmassoftheprotein.supportstheideaoftheenergy-consumingcatabolicreactionsinitiatedafterlowtemperaturestressrelease.ThereweremanyreportsofATPasesubunitsresponsivenesstotemperaturefluctu-ations[8,14].Theincreaseinabundanceofbothproteinshasalsobeennoticedduringtherecoveryaftershortstressinbeanroots(BadowiecandWeidner,unpublishedresults).Theaccumulationof13proteins,includingsomeglycolyticen-zymes,decreasedduringrecoveryaftercontinuouschillingtreat-ment.Membersofaldolasesfamilyplaythemultiplefunctionsingrowthanddevelopmentaswellasinsugar,ABAandstresssignalinginArabidopsisinresponsetodiverseabioticstresses[23].Therehavealsobeenreportsoftheroleofcytosolicfructose-bisphosphatealdolaseinprotectionagainstabioticstressinrice[14].Morethantwo-folddecreaseinquantityexhibitedeukaryoticinitiationfactor4A,knownalsoasDEADboxRNAhelicase.Itwasobservedthattheaccumulationofthisproteindecreasesintheconditionsofoxidativestressinriceseedlingleaves[24].AlthoughtheexactmechanismofDEADboxproteinsactioninresponsetoachangingtemperatureisnotfullyknownyet,VashishtandTuteja[25]considerthemasatargetforthestresstoleranceengineering.S-Adenosylmethioninesynthasecatalyzestheformationofaprecursorforethyleneandpolyamines[26].Theregulationofitsexpressionhasalreadybeenpairedseveraltimeswithaprotectionagainstlowtemperaturestressandaslowdownofplantgrowth[8,26].ItisworthnoticingthattherelativeamountofS-adeno-sylmethioninesynthasedroppedduringthepost-stressrecoveryalsointherootsofbean(BadowiecandWeidner,unpublishedre-sults),sothestressexemptionappearstoinhibittheaccumulationofthisenzyme.ThereleaseofahormonalblockademayinfluencerootdevelopmentaswellastheincreaseinfreshanddryweightcontentinLSR.TheobservedaccelerationofrootgrowthinLSRmayrelyoncellelongationratherthancelldivisionswhichissuggestedbydown-regulationoftranslationallycontrolledtumorprotein.Itscellularfunctionsinplantsincludeapositivecontrolofcellcycledurationbutnotpost-mitoticcellgrowth[27].Anotherenzymewithadecreasedabundancewasmalatede-hydrogenase,whichissuggestedtobeinvolvedintheacquisitionofcoldandsaltstresstoleranceinplants[28].InthestudyofDumontetal.[5]ithasbeenrelatedtothecoldacclimationofpearoots.AlsomethylenetetrahydrofolatereductasequantitywasreducedinLSR.Sinceitservesasamethyldonorforsynthesisofmethioninefromhomocysteine,itiscrucialformaintainingintracellularconcen-trationofmethionineandS-adenosyl-L-methionine.TransferofmethylgroupsfromS-adenosyl-L-methionineontooxygenorcar-bonatomsmayresultinnucleicacidsandproteinsmodification[29].Thestimulationofstarchdegradation,whichresultsinanincreasedleveloftheosmoprotectantsandmaltose,mayalsoplayaprotectiveroleagainstlowtemperatureinplantcells[30].Relativeabundanceofbetaamylasedecreasedduring24hafterchillingstressrelease,probablydiminishingthelevelofalreadyunnec-essaryosmoprotecivemoleculesinrootcells.ThestrongdecreaseinabundanceexhibitedalsoahypotheticalproteinAt3g17530,containingF-boxdomainand2Kelch-repeats.Itmaybeinvolvedinaproteolyticpathwayinplants.ThefunctionofotherproteinidentifiedinthisgroupehypotheticalproteinZEAMMB73_053553isnotknown.3.3.ProteinsregulatedduringtheshortchillingstressThesearchforcold-responsiveproteinsintherootsofpeasubjectedtotheshortrapidchillingduringgermination(SS)resultedindetectionofthreeproteinswhichquantityincreasedand6proteinswhichquantitydecreased.Adenosylhomocysteinaseandmethylenetetrahydrofolatereductaseaccumulationwasalsopositivelyregulatedunderotherconditionsinthisexperiment(LSandLSR).Theyareinvolvedinregulationofgeneexpressionandaminoacidsmetabolism.Thethirdproteinwhichaccumula-tionincreasede1-aminocyclopropane-1-carboxylateoxidaseisdirectlyimplicatedintheproductionofethylene.ThoughethyleneaffectsmanyaspectsofanormalplantgrowthandA.Badowiecetal./PlantPhysiologyandBiochemistry71(2013)315e324321
Fig.5.2Dgelmapsofdifferentlyaccumulatedproteinsextractedfromtherootsofpeaseedsgerminatinginthecontrolconditions(C2),subjectedtoshortchilling(SS)andrecoveryaftershortchilling(SR).TheproteinspotswerevisualizedbyCoomassieG-250staining.Thenumbersofspotscorrespondedtothoseshowingquantitativedif-ferencesbetweenC2andSSaswellasbetweenSSandSR.
developmentwhileinlowconcentration,expositiontoadverseconditionsincreasestheproductionof“stressethylene”whichresultsinaninhibitionofcellgrowthandproliferation[31].Theabundancesof6proteinswerediminishedduringshortstress.Threeofthemeputativepectinesterase,galactokinaseandglutathioneS-transferaseincreasedinabundanceduringthesubsequentrecovery.Asithasbeenalreadydiscussedabove,pectinesteraseisinvolvedinthemodificationofcellwallcompo-nentsandgalactokinaseprotectsplantcellsagainstthecytotoxicityofgalactose.Thenegativeeffectofgalactoseonplantorgangrowthhasbeenwelldocumented[32].Thedecreasedamountofthisenzymecouldbeoneofthereasonsofrootelongationslowdownduringtheexposurecoldstress.Takingintoconsiderationthatoneoftheproteinswhichquantitywasreducedwasidentifiedascyclin-P1-1,theretardationofrootgrowthmaybealsoaresultofcellcyclearrest[21].TheinfluenceofcyclinsoncelldivisionunderstressconditionshasalreadybeenobservedinArabidopsisroots[33].Theabundanceoftwoproteinsinvolvedinprotectionagainststress:mitochondrialheatshockprotein70andglutathioneS-transferasediminishedinresponsetotherapidchilling.Thefirstonechaperonesthebiosynthesisofmitochondrialproteins[34],theotherisnotonlyakeycomponentoftheROSscavengingmechanismbyperformingadetoxificationofhydroxyperoxide,butalsoinfluenceshormonehomeostasis,regulationofapoptosisandplantresponsetobioticandabioticstress[35].WeobservedasimilarpatternofglutathioneS-transferaseaccumulationintherootsofbeanseedssubjectedtoshortchilling(BadowiecandWeidner,unpublishedresults).3.4.ProteinsregulatedduringtherecoveryaftershortchillingstressAmongtheproteinswhichquantityincreasedduringthere-coveryaftershortchillingstress(SR)weregalactokinase,putativepectinesteraseandglutathioneS-transferase,whichwereprevi-ouslydown-regulatedundershortchillingconditionsanddis-cussedabove.Alpha-1,4-glucan-proteinsynthasewasanotherproteinwhichamountincreasedduringtherecoveryafterexposi-tiontoshortstress.Itwasanotherprotein,alongsidepectines-terase,possiblyinvolvedinthemetabolismofcellwallcomponentsinSR[36].TothegroupofproteinswhichamountwasreducedduringtherecoverybelongS-adenosylmethioninesynthase,1-amino-cyclopropane-1-carboxylateoxidaseandheatshockprotein71.2.Theyareimplicatedinethylenesynthesisandprotectionagainststress.TakingintoconsiderationthattheaccumulationofS-adeno-sylmethioninesynthasewasnegativelyregulatedduringrecoveryafterbothlongandshortchillingand1-aminocyclopropane-1-carboxylateoxidasewaspositivelyregulatedinresponsetoshortstress,ethyleneisoneofthemostimportantplayersimplicatedintemperaturestressresponseinpea,butthemechanismoftriggeringitsbiosynthesismaybedifferentforthestressofdifferentnatureandlength.Thethirddetectedproteininthisgroup,heatshockprotein71.2,actsasamolecularchaperoneandassistsproteinfoldingduringtranslation.ItsexpressionisusuallytriggeredbyROSproduction[37].Summarizingtheresultsobtainedinthisstudywemayconcludethatgerminationintheprolongedlowtemperatureconditionsaffectstheproteinsinvolvedinmetabolismofcarbo-hydrates,aminoacids,nitrogenandcellstructurecomponentsofpearoots.Shortrapidchilling,ontheotherhand,inducesstresshormonebiosynthesis,influencesthecellwallmetabolismandcellcycleregulation.Bothlongandshortchillingstimulimayaffectgeneexpressionandtriggerthemechanismsofprotectionagainststress,thoughitcanbeachievedinavariousway.Whenitcomestoproteinalterationsoccurringduringrecoveryafterlongandshortchilling,itisapparentthatchangingthetemperaturefromlowtoambientduringgerminationconstitutesaheatshockforpearootsandentailslaunchingthediversemechanismsofanti-stresspro-tection.CommonforbothLSRandSRistheincreasedamountoftheproteinsinvolvedincellwallmetabolismandthediminished322A.Badowiecetal./PlantPhysiologyandBiochemistry71(2013)315e324
Fig.6.Venndiagramanalysisoftheproteinsdifferentiallyaccumulatedinthevariousgerminationconditions.Thenumberofproteinsup-ordown-regulatedunderlong(LS)andshort(SS)chillingstressandrecoveryafterlong(LSR)andshort(SR)stressareshownintherespectivesegments:A)numberofproteinsdown-regulatedbylongandshortchillingaswellasup-regulatedduringrecoveryafterlongandshortchilling;B)numberofproteinsup-regulatedbylongandshortchillingaswellasdown-regulatedduringrecoveryafterbothchillingconditions.
quantityoftheenzymesimplicatedintheproductionofgrowthinhibitors.CharacteristicforLSRaredisturbancesincarbohydratemetabolismandtheaccumulationofproteinsinvolvedinROSscavenging.4.Materialsandmethods4.1.GerminationconditionsSeedsofpea(P.sativumL.)cultivar‘Szesciotygodniowy’,sup-,Poland),wereusedintheexperiment.pliedbyTorseedS.A.(TorunPriortosowing,theseedsweresurfacesterilizedfor10minwith0.5%solutionofsodiumhypochloriteandwashedthoroughlywithdistilledwater.Seedswereplacedbetweentherolledsheetsoftissue-paperandinsertedintoglasscylinderscontainingenoughwatertoensureconstanthumidityofthepaper.Theseedswhichconstitutedthecontrolwereplacedinagrowthchambersetfor20Candgerminatedfor72h(controlC1)and96h(controlC2).Toinducethelongcontinuouschillingstress(sampleLS),seedswereplacedin10CdirectlyaftersowingandgerminateduntiltheirrootsachievedthemorphologicalsimilaritytotherootsfromthecontrolC1(8days).Inordertoexaminetheimpactoftherecoveryafterlongchillingstress,theseedsgerminatingfor8daysin10Cweretransferredfor24hto20C(sampleLSR).Shortrapidstresswasinducedbytransferringtheseedsgerminatingfor72hin20Ctolowtemperature(10C)foradditional24h(sampleSS).Therecoveryaftershortstresswasinducedbyshiftingtheseedsforanother24hto20C(sampleSR).ThegerminationstagewasperformedintheSANYOMir-154cooledincubator.Aftertheappointedtimetherootswerecutandimmediatelyfrozeninliquidnitrogen.4.2.FreshanddryweightcontentmeasurementForthemeasurementofthefreshanddryweightcontent,minimum30rootsfromatleast3independentbiologicalreplicatesfromeachofexaminedtreatmentswereweighedimmediatelyaftercutting.Rootswerethendriedat105Cfor24handweightedagaininordertomeasurethedryweightcontent.DatawereanalyzedwithStatistica10.0(StatSoft,Poland),usingStudent’st-testandreportedasmeansÆSD.Differencesbetweenthecontrolsandstresssamplesaswellasbetweenstressandrecoverysampleswereconsideredstatisticallysignificantwithasignificancelevelof95%.4.3.ProteomeextractionInordertoextracttherootproteomes,300mgoffrozenfreshtissuewasgroundinliquidnitrogenandincubatedwithshakingfor1hinice-coldextractionbuffercontaining7.4Murea,2.1Mthiourea,62mMCHAPS,8mMdithiothreitol,0.2%TritonX-100,16.5mMTrizmabase,21mMTrizmahydrochloride,1%IPGbufferpH4e7(GEHealthcare),60U/mlDNaseI,5.8Kunitz/mlRNaseA;1tab./10mlproteaseinhibitorsCocktailCompleteMini(Roche).Aftertheincubationtimethesampleswerecentrifuged(12000Âg,10min,4C)andtheproteinextractsfromthesupernatantswerepurifiedandconcentratedwithReadyPrepÔ2-DCleanupKit(Bio-Rad).4.4.Two-dimensionalelectrophoresisExtractedproteinswereseparatedusing2D-PAGE(two-dimensionalelectrophoresisinpolyacrylamidegels)accordingtoGörgetal.[38].Priortotheisoelectrofocusingtheproteinextractsweredissolvedinthebuffercontaining7Murea,2Mthiourea,2%CHAPS,0.5%IPGbufferpH4e7(GEHealthcare),80mMdithio-threitol,0.002%bromophenolblue.Totalof650mgproteinwasloadedonto24cmImmobilineDryStripGels(GEHealthcare)withthelinearpHgradient4e7.IsoelectrofocusingwasperformedintheEttanIPGphor3system(GEHealthcare),withavoltageroutineasfollows:30V/10h(activerehydrationat20C),500V/1h(stepandhold),1000V/1h(gradient),8000V/3h(gradient),8000V/3:30h(stepandhold).Directlybeforetheanotherstepof2D-PAGE,stripswereequilibratedfor15minwith65mMdithiothreitolforproteinreductionandforanother15minwith135mMiodoace-tamideforproteinalkylation.Theequilibrationbuffercontained6Murea,75mMTriseHCl(pH8.8),29.3%glycerol,2%sodiumdodecylsulfate,0.002%bromophenolblue.Theequilibratedstripswereplacedonto12.5%polyacrylamidegelsandsealedwith0.5%moltenagarosesolution.TheelectrophoresisofdenaturedproteinswascarriedoutintheEttanDALTsixelectrophoreticunit(GEHealthcare)asdescribedbyLaemmli[39].Electrophoresiswasperformedat25C(2.5W/gelfor30min,17W/gelfor3:30h).Threeindependentbiologicalreplicatesforeachsamplewereperformed.A.Badowiecetal./PlantPhysiologyandBiochemistry71(2013)315e324323
ProteinsseparatedingelswerevisualizedwithCoomassieBrilliantBlueG-250accordingtotheprotocoldescribedbyNeuhoffetal.[40].Gelimagesweredigitalizedat300dpiusingImage-ScannerIII(GEHealthcare).GelanalysiswasperformedwithImageMasterÔ2DPlatinum6.0software(GEHealthcare).Proteinspotsweredetectedandquantifiedonthebasisoftheirrelativevolumeinrelationtothetotalvolumeofthewholesetofmatchedspots.Proteinsexhibitingthehighest,reproduciblealterations(atleast1.75-fold)weresubjectedtostatisticalanalysis.TheManneWhit-neyUtestwaschosentoevaluatestatisticalsignificanceofthedifferencesbetweenthecontrolandstresssamplesaswellasbe-tweenthestressandrecoverysamples(atp<0.05).Theproteinswhichabundancechangedsignificantlyweredesignatedtomassspectrometryforidentification.4.5.ProteindigestionandMALDI-TOFanalysisTheproteinsofinterestweremanuallycutoutfromthegelswithascalpelblade,washedwith100mlofamixtureacetonitrile/50mMammoniumbicarbonate(1/1,v/v)andthendehydratedwithacetonitrile.Drygelplugswererehydratedwith100mlof50mMammoniumbicarbonate.After5minanequalvolumeofacetonitrilewasaddedandincubatedtogetherfor15min.After-ward,spotsweredehydratedwith100mlofacetonitrileanddriedinavacuumcentrifuge.Aminimumvolume(5ml)ofdigestionsolutioncontaining15ng/mltrypsin(Promega),reconstitutedin25mMammoniumbicarbonate,wasaddedtothedriedgelfrag-ments.Thereactionwascarriedoutovernightat37C.Afterthefirst30minofincubation,anequalvolumeof25mMammoniumbicarbonatewasaddedinordertoavoiddryingofthegelplugsduringthedigestionstage.Peptideswereextractedfromthegelfragmentsafteradditionof0.5mlofacetonitriletoeachsampleand5minofsonication.Peptidesamplesweremixedwithsatu-ratedsolutionofamatrix(a-cyano-4-hydroxycinnamicacid),dis-solvedinasolutionof50%acetonitrile,0.1%trifluoroaceticacid.PeptidemixtureswereanalyzedusingMALDI-TOFAutoflexIIISmartBeammassspectrometer(BrukerDaltonics).ThesearchthroughthedatabasesofNationalCenterforBiotechnologyInfor-mationandSwissProtwasperformedusingMASCOTsearchengine(http://www.matrixscience.com).ForPeptideMassFingerprintsearch,thefollowingparameterswereapplied:taxonomyVir-idiplantae,monoisotopicmassesMHþ,0.2Damasstolerance,onemissedcleavagesiteallowed,carbamidomethylationofcysteineasfixedmodificationandmethylationofmethionineasvariablemodification.AcknowledgmentsWearegratefultoM.Stobieckiandhisco-workers:q.Marczak,K.ChmielewskaandP.RodziewiczfromTheInstituteofBioorganicChemistry(IBCHPAS,Poland)forsharingtheirknowledgeandexperienceonmassspectrometry.AnnaBadowiecwassupportedbyascholarshipprogramforPhDstudents“DrINNO2ebuildingasocialpotentialofhighlyqualifiedspecialistsintheWarmia-Mazury”.References[1]D.J.Allen,D.R.Ort,Impactofchillingtemperaturesonphotosynthesisinwarm-climateplants,TrendsPlantSci.6(2001)36e42.[2]B.Rymen,F.Fiorani,F.Kartal,K.Vandepoele,D.Inz,G.T.S.Beemster,Coldnightsimpairleafgrowthandcellcycleprogressioninmaizethroughtran-scriptionalchangesofcellcyclegenes,PlantPhysiol.143(2007)1429e1438.[3]R.Kooke,J.J.B.Keurentjes,Multi-dimensionalregulationofmetabolicnet-worksshapingplantdevelopmentandperformance,J.Exp.Bot.63(2012)3321e3323.[4]M.Rossignol,J.B.Peltier,H.P.Mock,A.Matros,A.M.Maldonado,J.V.Jorrin,Plantproteomeanalysis:a2004e2006update,Proteomics6(2006)5529e5548.[5]E.Dumont,N.Bahrman,E.Goulas,B.Valot,H.Sellier,J.Hilbert,C.Vuylsteker,I.Lejeune-Henaut,B.Delbreil,Aproteomicapproachtodecipherchillingresponsefromcoldacclimationinpea(PisumsativumL.),PlantSci.180(2011)86e98.[6]F.Grimaud,J.Renaut,E.Dumont,K.Sergeant,A.Lucau-Danila,A.S.Blervacq,H.Sellier,N.Bahrman,I.Lejeune-Hénaut,B.Delbreil,E.Goulas,Exploringchloroplasticchangesrelatedtochillingandfreezingtoleranceduringcoldacclimationofpea(PisumsativumL.),J.Proteomics80(2013)145e159.[7]T.A.Paw1owski,ProteomeanalysisofNorwaymaple(AcerplatanoidesL.)seedsdormancybreakingandgermination:influenceofabscisicandgibberellicacids,BMCPlantBiol.9(2009)48.[8]S.P.Yan,Q.Y.Zhang,Z.C.Tang,W.A.Su,W.N.Sun,Comparativeproteomicanalysisprovidesnewinsightsintochillingstressresponsesinrice,Mol.Cell.Proteomics5(2006)484e496.[9]S.Swigonska,S.M.Weidner,Proteomicanalysisofresponsetolong-termcontinuousstressinrootsofgerminatingsoybeanseeds,J.PlantPhysiol.170(2013)470e479.[10]S.Cui,F.Huang,J.Wang,Y.Cheng,J.Liu,Aproteomicanalysisofcoldstressresponsesinriceseedlings,Proteomics5(2005)3162e3172.[11]H.Liu,B.Ouyang,J.Zhang,T.Wang,H.Li,Differentialmodulationofphoto-synthesis,signaling,andtranscriptionalregulationbetweentolerantandsensitivetomatogenotypesundercoldstress,PLoSONE7(2012)e50785.[12]Y.Zhou,H.Cai,J.Xiao,X.Li,Q.Zhang,X.Lian,Over-expressionofaspartateaminotransferasegenesinriceresultedinalterednitrogenmetabolismandincreasedaminoacidcontentinseeds,Theor.Appl.Genet.118(2009)1381e1390.[13]R.Li,C.Wang,T.Chen,P.Chen,Quantitativeproteomicanalysisofcold-responsiveproteinsinAbelmoschusmoschatus,J.Anim.PlantSci.14(2012)2006e2023.[14]M.Hashimoto,S.Komatsu,Proteomicanalysisofriceseedlingsduringcoldstress,Proteomics7(2007)1293e1302.[15]A.Abdrakhamanova,Q.Y.Wang,L.Khokhlova,NickPIsmicrotubuledisas-semblyatriggerforcoldacclimation?Pl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