Molecular responses of genetically modified maize to abiotic stresses as determined through

23 Pages·2017·1.41 MB·English

RESEARCHARTICLE
Molecular responses of genetically modified
maize to abiotic stresses as determined
through proteomic and metabolomic
analyses
RafaelFonsecaBenevenuto1,2,3*,SarahZanonAgapito-Tenfen2,ViniciusVilperte1¤,Odd-
GunnarWikmark2,4,PeetJansenvanRensburg5,RubensOnofreNodari1
a1111111111 1 DepartmentofCropScience,FederalUniversityofSantaCatarina,Floriano´polis,Brazil,2 GenøkCenter
forBiosafety,Tromsø,Norway,3 DepartmentofEngineering&NaturalSciences,WesternNorwayUniversity
a1111111111
ofAppliedSiences,Sogndal,Norway,4 UnitforEnvironmentalScienceandManagement,North-West
a1111111111
University,Potchefstroom,SouthAfrica,5 HumanMetabolomics,North-WestUniversity,Potchefstroom,
a1111111111
SouthAfrica
a1111111111
¤ Currentaddress:InstituteforPlantGenetics,FacultyofNaturalSciences,LeibnizUniversityofHannover,
Hannover,Germany
*[email protected]
OPENACCESS
Abstract
Citation:BenevenutoRF,Agapito-TenfenSZ,
VilperteV,WikmarkO-G,vanRensburgPJ,Nodari
Somegeneticallymodified(GM)plantshavetransgenesthatconfertolerancetoabiotic
RO(2017)Molecularresponsesofgenetically
modifiedmaizetoabioticstressesasdetermined stressors.Meanwhile,othertransgenesmayinteractwithabioticstressors,causingpleiotro-
throughproteomicandmetabolomicanalyses. piceffectsthatwillaffecttheplantphysiology.Thus,physiologicalalterationmighthavean
PLoSONE12(2):e0173069.doi:10.1371/journal.
impactontheproductsafety.However,routineriskassessment(RA)analysesdonotevalu-
pone.0173069
atetheresponseofGMplantsexposedtodifferentenvironmentalconditions.Therefore,
Editor:WeiWang,HenanAgriculturalUniversity,
weherepresentaproteomeprofileofherbicide-tolerantmaize,includingthelevelsofphyto-
CHINA
hormonesandrelatedcompounds,comparedtoitsnear-isogenicnon-GMvarietyunder
Received:November3,2016
droughtandherbicidestresses.Twentydifferentiallyabundantproteinsweredetected
Accepted:February14,2017 betweenGMandnon-GMhybridsunderdifferentwaterdeficiencyconditionsandherbicide
Published:February28,2017 sprays.Pathwayenrichmentanalysisshowedthatmostoftheseproteinsareassignedto
energetic/carbohydratemetabolicprocesses.Amongphytohormonesandrelatedcom-
Copyright:©2017Benevenutoetal.Thisisan
openaccessarticledistributedunderthetermsof pounds,differentlevelsofABA,CA,JA,MeJAandSAweredetectedinthemaizevarieties
theCreativeCommonsAttributionLicense,which andstressconditionsanalysed.Inpathwayandproteomeanalyses,environmentwasfound
permitsunrestricteduse,distribution,and
tobethemajorsourceofvariationfollowedbythegenetictransformationfactor.Nonethe-
reproductioninanymedium,providedtheoriginal
less,differencesweredetectedinthelevelsofJA,MeJAandCAandintheabundanceof
authorandsourcearecredited.
11proteinswhencomparingtheGMplantanditsnon-GMnear-isogenicvarietyunderthe
DataAvailabilityStatement:Allrelevantdataare
sameenvironmentalconditions.Thus,thesefindingsdosupportmolecularstudiesinGM
withinthepaperanditsSupportingInformation
files. plantsRiskAssessmentanalyses.
Funding:TheauthorswouldliketothankCAPES
forscholarshipsprovidedtoRBandCNPqforthe
scholarshiptoVVandRON.Financialsupportwas
providedbyTheNorwegianAgencyfor
DevelopmentCooperation(MinistryofForeign
Affairs,Norway)undertheGenØkSouth-America
ResearchHubgrantFAPEU077/2012.Theauthors
PLOSONE|DOI:10.1371/journal.pone.0173069 February28,2017 1/23
MolecularanalysisofGMmaizeunderstressconditions
alsothanktheMetabolomicCentreofthe 1.Introduction
DepartmentofBiochemistry(North-West
Recurrentadvancesinmoleculartechniquesandscientificknowledgehaveresultedintheabil-
University,SouthAfrica)forquantitativeanalysisof
phytohormonesandrelatedcompounds.Thiswas itytogeneticallymanipulatemanyorganisms,especiallythedevelopmentofgeneticallymodi-
ajointprojectbetweenUFSCandGenØk–Center fied(GM)plants.AndbecauseGMplantsareregulatedinmostcountries,riskassessment
forBiosafety. (RA)andequivalencecriteriatoassessthesafetyofGMcropshavebeenconductedduringthe
Competinginterests:Theauthorshavedeclared pastfewyears[1–7].Theconceptof‘substantialequivalence’canbedefinedastheclosenutri-
thatnocompetinginterestsexist. tionalandelementalsimilaritybetweenaGMcropandanon-GMnear-isogenicvariety.Ithas
oftenbeenusedtoclaimthatGMcropsareassafeandnutritiousascurrentlyconsumed
plant-derivedfoods[8].However,theparameters,criteriaandanalysesusedtodeclareaGMO
substantiallyequivalentareunclearandreducedtoalimitedsetofvariables,suchasthetotal
amountsofcarbohydrates,proteinsandminerals.Inaddition,pesticideresiduescanbecome
partofthecompositionofaherbicide-tolerantGMplant,andtheymayaddtoxicpropertiesto
thefinalplantproduct,eitheraloneorbyaffectingplantmetabolism[9].Nonetheless,the
identificationofsuchpesticideresiduesisoverlookedincompositionalanalyses[10].There-
fore,oneofthemostreliablewaystodetectunintendedconsequencesofgeneticmodification
isthroughamoredetailedsurveyofmolecularcomponents[11].
Herbicideusehasbeensteadilyincreasing,particularlyafterthereleaseofherbicide-toler-
antGMcrops[12]whichencompassapproximately80%ofGMcrops,andareabletoaccu-
mulateresidueswithoutdyinginordertofacilitateweedmanagement[13].Bydefinition,
herbicidesarechemicalsthatimpairplantdevelopmentand,therefore,areusedtoeliminate
manydifferentweeds[14].Herbicideapplicationisconsideredoneofthemajorabioticstress-
orsofbothweedandherbicide-tolerantGMplants,leadingtostressresponsepathwaysin
exposedplants,thusaffectinggeneexpressionandphysiology[15–17].Stressisdefinedasany
externalabioticorbioticconstraintthatreducesaplant’sabilitytoconvertenergyintobio-
mass[18].Inmostcases,lowcropproductivitycanbeattributedtovariousabioticstresses,
includinghighsalinity,drought,cold,andheat,allofwhichnegativelyinfluencethesurvival,
biomassproduction,andyieldofmajorcrops[19–21].Specifically,droughtstresscanbe
definedasanyreductionintheamountofwateravailableforthecrop,regardlessofirrigation,
whichreducesitsproductivitybelowexpectedyieldsunderadequatewatersupply.Droughtis
acommonrecurringphenomenonanditseffectsareextremelycomplex[22,23].Watershort-
ageleadstoreducedgrowthfortheentireplant,especiallyintheshoots,aswellasalterations
ontheregulationofgeneexpressionproducts,suchashormones,transcriptsandproteins
[24–27].
Changesattranscriptlevelarenotnecessarilyreflectedbycorrespondingchangesinplant
proteome[28,29].Thus,thoroughinvestigationsregardingchangesinplantproteomesubmit-
tedtostressconditionsarecrucial,sincetheydirectlyindicateplantstressresponses.Unlike
thegenome,whichisexpectedtobeconstantforanorganism,theproteomeishighlydepen-
dentonenvironmentalinfluences,suchasabioticstresses[4].Profilingtools(e.g.proteomics)
appliedtothemolecularcharacterizationstepcanassistontheidentificationofpotentially
hazardousproducts,suchastoxins,allergensandantinutrients.Inaddition,withtheemer-
genceofnewGMOtypes,broaderuseofmolecularprofilingtoolsarerequiredforreliable
pre-marketRA[30].Moreover,planthormonesplaycentralrolesintheabilityofplantsto
adapttochangingenvironmentsandtheresultantabioticstress.Phytohormonescanalsorap-
idlyaltergeneexpressionbyinducingorpreventingthedegradationoftranscriptionalregula-
torsviatheubiquitin-proteasomesystem[31].Furthermore,someoftheseplanthormonesare
themainmediatorsofplantdefensemechanismsagainststress,suchasplantgrowthregula-
tors(e.g.abscisicacid(ABA),ethylene,cytokinins(CK),auxin(IAA),gibberellin(GA),jasmo-
nate(JA),salicylicacid(SA),amongothers)[31].
PLOSONE|DOI:10.1371/journal.pone.0173069 February28,2017 2/23
MolecularanalysisofGMmaizeunderstressconditions
Generally,RAanalysesdonotincludeinformationontheresponseofGMplantsexposed
todifferentenvironmentalconditions.Waterdeficithasbeencommoninmajorgrowing
regionswhere,atthesametime,theapplicationofherbicidesisacommonoccurrence.Thus,
itseemsreasonabletoassesstheresponseofGMplantstoadverseconditions,especiallyin
lightofclimatechangetimes.Therefore,totestourhypothesisthatabioticstressconditions
leadtooff-targeteffectsinproteinandmetabolitelevelsinGMplants,wehaveperformeda
proteomicandmetabolomiccharacterizationoftheherbicide-tolerantNK603GMmaizeand
itsnon-GMnear-isogenicline,underdistinctstressconditions,includingdroughtandherbi-
cideapplication.
2.Materialandmethods
2.1.Plantmaterialandexperimentaldesign
Inordertoelucidatetheresponsesofageneticallymodifiedmaizeplanttowaterstressand
herbicideapplication,twoindependentexperimentswereperformed.Bothexperimentswere
carriedoutunderidenticalgrowingconditions,exceptfortheapplicationofRoundupherbi-
cideinthesecondexperiment.
Experiment1aimedatcomparingtheherbicide-tolerantGMmaizeDKB245RR2(trans-
formationeventNK603;uniqueidentifierMON-ØØ6Ø3-6fromMonsantoCompany,
glyphosate-basedherbicidetolerance,DekalbBrandCorn)anditsnear-isogenicnon-GM
counterpartDKB245underwaterstress.Seedsofthesehybridswerepreviouslygerminated
inagrowthchamber(Eletrolab™Model202/3)setto16hlightperiodand25˚C(±2˚C).
Seedlingswereplacedin8LplasticpotsfilledwithPlantmaxHTsubstrate(Buschle&Lep-
perS.A),transferredtoagreenhouseandwatereddaily,keepingsubstratemoistureat125%
(1.25gH O/gofsoil)controlledbyasoiltensiometer(WaterMeterMODWS-76).The
2
plantswerekeptintheseconditionsuntiltheyreachedfiveleavesfullyemerged(stageV5),
thenasubsetofbothvarieties(GMandnon-GM)werestressedbywithholdingirrigation,
accordingtoVicentetal.[16]withmodifications.Thedroughtstressperiodwasmaintained
untilsubstratemoisturereached25%(0.25gH 0/gofsoil),approximately15daysafter
2
waterrestriction.Atthistime,leafsampleswerecollected.Studiesonplantresponseto
waterdeprivationreportasignificantdecreaseinleafconductancefromtheseventhor
eighthdayofwaterstress,indicatingthatthestressisbeingperceivedbytheplant[32,33].
Forthisreason,plantsinExperiment1weresubjectedto15daysofwaterstress,allowing
theopportunityfordirectresponsetothestresscondition.
Experiment2aimedattestingtheinfluenceofherbicideapplicationontheherbicide-toler-
antGMmaizehybrid(NK603).ThisexperimentwascarriedoutsimilarlytoExperiment1,
exceptthattwosequentialapplicationsofthecommercialherbicideRoundupOriginal(Mon-
santodoBrasilLTDA)weredoneinasubsetoftwelveplants.Herbicideapplicationwasper-
formedfollowingthemanufacturer’srecommendation.Thefirstapplicationtookplace15
daysafterplantemergence,andthesecondapplicationwasperformed30daysafteremer-
gence,bothatdosesof2.0L/hainaconcentrationof960gofglyphosate(isopropylamine
salt)perha.SimilartoExperiment1,thedroughtstressperiodwasmaintaineduntilsubstrate
moisturereached25%.
Forbothexperiments,12plantspertreatmentwererandomlysampledandseparatedinto
threegroupsoffourplantseach.Thefourplantsofeachgroupwerepooledandwereconsid-
eredasonebiologicalreplicate(sample)ofeachtreatmentfortheproteomicandplanthor-
moneanalysis.Then,leafpieceswerecutfromeachbiologicalreplicate,weighed,immediately
placedin3.8mlcryogenictubes,andfrozeninliquidnitrogen.Thesampleswerekeptat
-80˚Cuntilproteinandhormoneextraction.
PLOSONE|DOI:10.1371/journal.pone.0173069 February28,2017 3/23
MolecularanalysisofGMmaizeunderstressconditions
2.2.Proteomicanalysis
2.2.1.Proteinextractionandfluorescencehybridization. Fortwo-dimensional(2D)
Differencegelelectrophoresis(DIGE)proteomicanalysis,wefollowedthemethodologyof
Agapito-Tenfenetal.[1].Briefly,approximately100mgofeachsampleweregroundinamor-
tarwithliquidnitrogenandsubsequentlyextractedandprecipitatedusingphenolandmetha-
nol/ammoniumacetate.Pelletswereresuspendedinaurea/thioureabuffercompatiblewith
furtherfluorescentlabelling(4%CHAPS(w/v),5mMPMSF,7Murea,2Mthioureaand30
mMTris-base).Proteinquantificationwasperformedwiththe2-DQuantKit(GEHealthcare
Bio-SciencesAB,Uppsala,Sweden).Eightyμgofeachproteinsamplepoolwerelabelledwith
400ρmol/μlofCyDyeDIGEfluors(Cy3andCy5;GEHealthcare)andsubsequentlystoredat
-80˚C.AninternalstandardlabelledwithCy2wasusedineveryrunfornormalization.The
internalstandardwasamixtureofequalamountsofeachplantvarietysample.Afterprotein-
fluorhybridization,lysine(10mM)wasusedtostopthereactionandthenmixedtogetherfor
2D-DIGE.Samplepairswererandomlyselectedforbi-dimensionalelectrophoresisruns.
2.2.2.2-DDIGEconditions. Afterproteinlabelling,sampleswerepreparedfortheiso-
electricfocusing(IEF)step.Stripgelsof24cmandalinearpHrangeof4–7(GEHealthcare)
wereused.Stripswereinitiallyrehydratedwithlabelledproteinsamples(7Murea,2Mthio-
urea,2%CHAPS(w/v),0.5%IPGbuffer(v/v)(GEHealthcare),2%DTT).Stripswerethen
processedusinganEttanIPGPhorIEFsystem(GEHealthcare)inatotalof35,000Volts.h-1
and,subsequently,reducedandalkylatedfor30minunderslowagitationinTris-HClsolution
(75mM),pH8.8,containing2%SDS(w/v),29.3%glycerol(v/v),6Murea,1%DTT(w/v)and
2.5%iodocetamide(w/v).The2D-DIGEconditionswereperformedasdescribedbyWeiss
andGo¨rg[32].GelswereimmediatelyscannedwithaFLA-9000modularimagescanner(Fuji-
filmLifescience,Dusseldorf,Germany).Toensuremaximumpixelintensitybetween60,000
and90,000pixelsforthethreedyes,allgelswerescannedata100μmresolutionandthepho-
tomultipliertube(PMT)voltagewassetbetween500and700V.AsdescribedbyAgapito-
Tenfenetal.[2],weperformedpreparativegelsforeachplantvarietyinordertoextractrele-
vantspots.Thesewereperformedwitha450μgloadoftotalproteinpoolsin24cmgelsfrom
eachvariety,separately,andstainedwithcolloidalCoomassieBrilliantBlueG-250(MS/MS
compatible).
2.2.3.Imageanalysis. ThescannedgelimagesweretransferredtotheImageQuantV8.1
softwarepackage(GEHealthcare)andappropriatelycropped.Afterwards,theimageswere
exportedtoImageMasterTM2DPlatinum7.0,version7.06(GEHealthcare),forcomparative
analysis.Each2D-DIGEgelisloadedwithtreatmentandcontrolsamplesplusaninternal
standardcontrol(equalmixofallsamplesoftheexperiment)andleabeledwithadifferent
fluorescencefluor.Therefore,theDIGEsoftwareusesaco-detectionalgorithm,whichiscapa-
bleofrealizingsimultaneousdetectionofproteinspotslabelledindifferentimages,butpresent
onthesamegelandnormalizeitwiththeinternalcontrol.Thus,thisco-detectionsignificantly
increasestheaccuracyofacomparativeproteomicstudy,especiallycomparedtotraditional
2D-PAGEusingComassieBrilliantBlue.Thus,thesimultaneouselectropheresisoftreatment
andcontrolsamples,followedbynormalizationwiththecorrespondinginternalstandard
greatlyminimizestechnicalvariationandthusdoesnotrequiretechnicalreplicategels.Infact,
technicalreplicatesarerepresentedbytheinternalstandardcontrolwhichispresentinevery
DIGEgel.
2.2.4.In-geldigestionandproteinidentificationbyMS/MS. Spotsfrompreparativegels
wereexcisedandsenttotheProteomicPlatformLaboratoryattheUniversityofTromsø(Nor-
way)forprocessingandanalysis.Theyweresubjectedtoin-gelreduction,alkylation,andtryp-
ticdigestionusing2–10ng/μltrypsin(V511A;Promega)[34].Peptidemixturescontaining
PLOSONE|DOI:10.1371/journal.pone.0173069 February28,2017 4/23
MolecularanalysisofGMmaizeunderstressconditions
0.5%formicacidwereloadedontoananoACQUITYUltraPerformanceLCSystem(Waters,
Milford,MA,USA),containinga5-μmSymmetryC18Trapcolumn(180μm×20mm;
Waters)infrontofa1.7-μmBEH130C18analyticalcolumn(100μm×100mm;Waters).Pep-
tideswereseparatedwithagradientof5–95%acetonitrile,0.1%formicacid,withaflowrate
of0.4μl/minelutedtoaQuadrupoleTime-of-Flight(Q-TOF)Ultimamassspectrometer
(Micromass;Waters).Thesampleswererunindata-dependenttandemMSmode.Peaklists
weregeneratedfromMS/MSbyProteinLynxGlobalserversoftware(version2.2;Waters).
Theresulting‘pkl’filesweresearchedagainsttheNCBIproteinsequencedatabasesusing
MascotDistiller,ver.2.5.1.0(MatrixSciences;http://matrixscience.com).Dataweresearched
againsttheNCBInrdatabase,ver.20151109,withViridiplantaetaxonomy(GreenPlants;
3334509sequences)andagainst‘allentries’and‘contaminants’(76068736sequences;
27658295194residues)forcontaminationverification.Thefollowingparameterswereadopted
fordatabasesearches:completecarbamidomethylationofcysteinesandpartialoxidationof
methionines;peptidemasstolerance±100ppm;fragmentmasstolerance±0.1Da;missed
cleavages1;andsignificancethresholdlevel(P<0.05)forMascotscores(-10Log(P)).Even
thoughhighMascotscorescouldbeobtainedwithsignificantvalues,acombinationofauto-
mateddatabasesearchesandmanualinterpretationofpeptidefragmentationspectrawasused
tovalidateproteinassignments.Molecularfunctionsandcellularcomponentsofproteinswere
searchedagainsttheEncyclopediaofGenesandGenomes(KEGG)Orthologysystemdata-
base,release69.02014(http://kegg.jp/kegg/ko.html).
2.2.5.Pathwayenrichmentanalysis. Differentiallyabundantproteinsweresubmittedto
enrichmentanalysisbytheonlinetoolagriGO,v1.2[35],usingtheSingleEnrichmentAnalysis
(SEA)category,withthefollowingparameters:1)Selectedspecies:Zeamaysssp;2)Statistical
testmethod:Hypergeometric;3)Multi-testadjustmentmethod:Hochberg(FDR);4)Signifi-
cancelevelof0.05;5)Minimumnumberof5mappingentries;and6)Geneontologytype:
PlantGOSlim.Next,theonlinetoolREVIGO[34]wasusedtoremovetheredundantGene
Ontology(GO)terms.OnlysignificantGOterms(FalseDiscoveryRate(FDR)values<0.05)
wereusedwiththefollowingparameters:1)Allowedsimilarity:medium(0.7);2)Database
withGOtermsizes:Zeamays;and3)Semanticsimilaritymeasure:SimRel.
2.3.Planthormoneandrelatedcompounds
2.3.1.Compoundextraction. Sampleswerepolledupto40mgoffreeze-driedleaftissue
(biologicalreplicates)fortheextractionofphytohormonesandrelatedcompounds.Theleaf
tissuewasgroundinliquidnitrogenuntiltotalspraying,accordingtoPanetal.[36].Subse-
quently,500μlofextractionsolutionconsistingofisopropanol,H OandHCl(2:1:0.002,v/v/
2
v)wereaddedineachtube.Tubeswerethenplacedonashakerat100rpmfor30minat4˚C.
Afterstirring,1mlofdichloromethanesolventand10μlof2-acetamidophenol(10ppm)
(internalstandard)wereaddedtoeachtubeandstirredat100rpmfor30minat4˚C.Samples
werecentrifugedat13,000gfor5minat4˚C.Aftercentrifugation,900μlofsolventpresentin
thelowerphaseweretransferredintoanewtubeusingaPasteurpipette.Thesolventmixture
wasconcentrated(notcompletelydried)usinganitrogenevaporatorwithnitrogenflowand
thendissolvedin100μlofmethanol.Sampleswerekeptat-20˚Cuntilanalysis.
2.3.2.Liquidchromatography—Tandemmassspectrometry. Thequantitativeanalysis
ofphytohormonesandrelatedcompoundswasconductedattheMetabolomicCentreof
theDepartmentofBiochemistry(North-WestUniversity,SouthAfrica).Quantificationof
phytohormonesandrelatedcompounds,whichbelongtothemainclassesinvolvedinplant
defense-relatedmetabolicpathwaysduringstress,wasperformedforSalicylicAcid(SA),Jas-
monicacid(JA),MethylJasmonate(MeJA),Abscisicacid(ABA),Indoleaceticacid(IAA)and
PLOSONE|DOI:10.1371/journal.pone.0173069 February28,2017 5/23
MolecularanalysisofGMmaizeunderstressconditions
Cinnamicacid(CA).Quantificationwasdeterminedusingreverse-phaseliquidchromatogra-
phy-tandemmassspectrometrywithmultiplereactionmonitoring[36].AnAgilent1290Liq-
uidchromatographcoupledtoanAgilent6460triplequadrupolemassspectrometerwasused.
ChromatographywasperformedwithanAgilentZorbaxEclipseplusC18(2.1x100mm,
1.8μm).Thechromatographicseparationstartedwith5%solventB(methanol)and95%sol-
ventA(waterand0.1%formicacid).ThepercentageofsolventBwasincreasedto90%over7
min.From7to9min,solventBwasmaintainedat90%andthendecreasedafter9minto
reach5%solventBat12min.Apost-runof4minwasusedtoensurecolumnre-equilibration.
Aconstantflowrateof0.2ml/minandacolumntemperatureof50˚Cwereused.
TheESIsourcewithAgilentJetStreamtechnologywassetineitherpositiveornegativeion-
izationforeachofthetwomethodsused.Agastemperatureof325˚C,gasflowof10L/min
andnebulizerpressureof45psiwereusedinbothmethods.Multiplereactionsmonitoring
(MRM)transitionofeachanalytewasoptimized(Additionalfile1).Threeruns(technicalrep-
licates)wereconductedforeachbiologicalpool,toinformthetechnicalvariation.
2.4.Statisticalanalysis
MultipleCo-InertiaAnalysis(MCIA)wasperformedinordertoexploretheexperimental
qualityandthemainsourcesofvariationinproteomicandplanthormonedatasetssimulta-
neously.Fortheproteomicsexperiment,one-wayANOVAwasusedtoinvestigatedifferences
atindividualproteinlevels.TukeytestatP<0.05wasusedtocomparethemultiplemeansin
thedataset.Thecalculationswereperformedonnormalizedspotvolumeratiosbasedonthe
totalintensityofvalidspotsinasinglegel.DifferencesatP<0.05wereconsideredstatistically
significant.StatisticalanalyseswereperformedusingImageMasterTM2DPlatinumver.7.06
(GEHealthcare).Forplanthormonequantitativeanalysis,one-wayANOVAandTukeytest
werealsousedatstatisticalsignificanceofP<0.05.Becauseofnon-normaldistribution,the
datawerenaturallogarithm(LN)transformed.Foldchangesarealsopresentedinlogarithm
base2(Log2FC).Log2FCisthemostwidelyusedalternativetransformationoftheratiosince
ithastheadvantageofproducingacontinuousspectrumofvaluesandrepresentingup-and
downregulatedcompoundvaluesinasimilarandreaderfriendlyfashion[37].
3.Resultsanddiscussion
Proteomicprofileandkeymetaboliccompoundswereanalysedincomparisontothenon-GM
near-isogenicvarietyunderthesameexperimentalconditionsaimingatexploringmolecular
responsesofGMmaizevarietytodroughtandherbicidestressconditions.Tofacilitatethe
presentationofresults,wehavenamedeachplantvarietyanditscorrespondinggrowingcon-
ditionsasfollows:Experiment1—non-GMD(conventionalhybridunderdroughtstress);
non-GM(Conventionalhybridundernormalgrowthconditions);GMD(GMhybridunder
droughtstress);GM(GMhybridundernormalgrowthconditions).Experiment2—GMDH
(GMunderdroughtstressandherbicideapplication);GMD(GMhybridunderdrought
stress)(Table1).
Table1. Commercialmaizevarietiesandtreatmentsusedinthisstudy.
Varietycommercialname GMeventname Transgenes Stresscondition Studydesignation
DKB245 non-GM - Drought non-GMD
DKB245 non-GM - Control non-GM
DKB245RR2 NK603 CP4EPSPS Drought GMD
DKB245RR2 NK603 CP4EPSPS Control GM
DKB245RR2 NK603 CP4EPSPS Drought+Herbicide GMDH
doi:10.1371/journal.pone.0173069.t001
PLOSONE|DOI:10.1371/journal.pone.0173069 February28,2017 6/23
MolecularanalysisofGMmaizeunderstressconditions
3.1.ExperimentalqualityassessmentusingMCIA
MCIAhasbeenrecognizedasanexcellenttoolforintegratingtheresultsofdifferent"omics"
techniques.Itisanexploratorydataanalysismethodthatisabletoprovideasimplegraphical
representationthatidentifiestheconcordancebetweenthesemultipledatasets[38].Itisnote-
worthythatonecomponentofMCIAcanbeusedtohighlighttheabsenceorpresenceofco-
structurebetweenthedatasets,therebypermittingthechoiceofthestrongestelementsforthe
followinganalysis[38],andbasedonthesemerits,MCIAwasappliedtothisexploratoryanaly-
sisofproteomicandmetabolicstudiesinbothexperiments.
Thecoordinatesofeachpoolofbiologicalreplicatesforeachtreatmentareconnectedby
lines,thelengthofwhichindicatesthedivergence(theshortertheline,thehigherthelevelof
concordance)betweenthehormone(andrelatedcompounds)andproteinrelativeabundance
levelsforaparticularreplicate.FortheExperiment1dataset(Fig1A),theMCIAplotshows
thetwoprincipalcomponents(PC1xPC2).PC1separatedthebiologicalreplicatesofmaize
varietiessubmittedtodroughtstressfromthoseofnon-stressedvarieties,accountingfor
41.35%ofthetotalvariationinthedataset.PC2separatedGMfromnon-GMvarieties,which
accountedfor19.73%ofthetotalvariation.Fig1BshowstheMCIAplotforExperiment2.In
thiscase,theMCIAplotshowssimilartrendsinproteomeandmetabolicprofiles,indicating
thatthesourcesofbiologicalinformationformostvariantsweresimilar.TheMCIAplotalso
Fig1.MCIAprojectionplot.(A)MCIAprojectionplotforExperiment1,representingtheproteomicandmetabolomicdatasets:PC1=41.35%and
PC2=19.73%.(B)MCIAprojectionplotforExperiment2,representingtheproteomicandmetabolomicdatasets:PC1=47.29%andPC2=27.7%.PC1is
representedbythefirstaxis(horizontal),andPC2isrepresentedbythesecondaxis(vertical).EigenvalueandPercentagegraphicsshowtheamountof
variationinthedatasetcorrespondingtoeachPC.Differentsymbolsrepresenttherespective“omics”analysisandareconnectedbylineswherethelengthis
proportionaltothedivergencebetweenthedatafromasamereplicate.Linesarejoinedbyacommonpoint,representingthereferencestructure,which
maximizescovariancederivedfromtheMCIAsyntheticanalysis.Colorsrepresentthebiologicalreplicates.
doi:10.1371/journal.pone.0173069.g001
PLOSONE|DOI:10.1371/journal.pone.0173069 February28,2017 7/23
MolecularanalysisofGMmaizeunderstressconditions
showedthatbothPC1andPC2separatedsampleswithherbicideapplicationfromthose
whithoutapplication.PC1accountedfor47.29%ofthevariationinthedataset,whilePC2
accountedfor27.70%ofthevariation.ThecombinationofbothPCscorrespondto~75%of
thevariationintheexperiment,representingahighpercentageofthetotalvariation.Basedon
theseresults,weconcludethatthemainsourceofquantitativevariationinourstudyorigi-
natedfromtheenvironment(factorstress),followedbythehybrids(geneticmodificationfac-
tor).Asexpectedfordifferentgrowingconditions,theseresultsagreewiththoseofprevious
studies[2,4,39,40].Mesnageetal.[13]usedthesameMCIAapproachtodeterminetherela-
tionshipofproteomeandmetabolomeprofilesofnon-GMandGM(NK603)maizeseeds.
Overall,theauthorsshowedthattheGMtransformationprocesswasthemajorcontributorto
variationintheproteinandmetaboliteprofiles,followedbythesprayingofherbicide[13].
Othermultivariateanalyses,suchasPrincipalComponentAnalysis(PCA),havebeenusedto
demonstratesimilaritiesinthequantityofproteinsandmetabolitesbetweendifferentcondi-
tionsinmulti-omicsstudies.However,unlikeMCIAanalysis,PCAhastobedoneseparately
foreachdifferent“omic”approach[1–3].Therefore,whenconductinggene,proteinand
metaboliteexpressionanalysesinthedifferentmaizehybridsMCIAcouldbeamorereliable
integrativetoolinthiscasethanPCA.
3.2.Differentialproteomicanalysis
Totalproteincontentmeanwas4.50±0.14mg.g-1(freshweight)ofleafmaterial,whichis
consideredasufficientquantityfor2D-DIGEanalysis.Nosignificantdifferencewasobserved
intheone-wayANOVA(P<0.05)fortheproteincontentmeanbetweendifferentgrowing
conditionsinExperiments1and2.Thenumberofspotsinatwo-dimensionalproteingelis
oneofthemostimportantcomponentsforacomparativeproteomicsanalysis.Essentially,the
numberofspotspresentinthegelsofeachtreatmentcorrelateswithcomparativeefficiency.
Here,theaveragenumberofspotsdetectedwas643and,nostatisticallysignificantdifference
wasfoundbetweenorwithintreatments(plantvarietiesandgrowingconditions)ineither
experiment(one-wayANOVA,P<0.05;datanotshown).Comparativeproteomicstudies
using2D-DIGEanalysisinplantleaveshaveshownanaveragenumberofdetectedspotsrang-
ingfrom500to900,whichagreeswithourresults[41–43].
Overallcomparativeanalysisrevealedatotalof20differentproteinsthatwerepresent,
absent,orup/downregulatedinoneofthetreatmentsatstatisticallysignificantlevel(P<0.05)
(Tables2and3).Proteinsthatwerenotdetectedinthisstudywerebelowthedetectionlimit
of±1ngandwerethereforeconsideredabsentinthesample.All20differentproteinswere
identifiedusingQuadrupoleTime-of-Flight(Q-TOF)tandemmassspectrometryanalysis
(MS/MS)(P<0.05).Table2showstheLog2fold-changevariationofidentifiedproteinfor
eachcomparison.Table3showsadescriptionofeachoftheseproteins,includingtheassoci-
atedbiologicalprocessandmolecularfunctionaccordingtotheUniprotdatabase.
Weperformedapathwayenrichmentanalysisinordertorankassociationsbetweentheset
ofdifferentiallyregulatedproteinsrepresentingmetabolicpathwaysandtherespectivestatisti-
calprobability.Thisanalysisallowsustoidentifytherelevantresultsofpotentiallyaltered
pathways.AllsignificantlyenrichedGOterms(FDR<0.05)areshowninFig2.Thealtered
biologicalprocesseswiththehighestnumberofassignedproteinsweremetabolicprocess
(GO:0008152),catalyticactivity(GO:0003824)andcellularmetabolicprocess(GO:0044237).
Thedifferentiallyabundantproteinsrelatedtoeachdifferentbiologicalprocesswillbedis-
cussedseparatelyinthefollowingsections.
3.2.1.Experiment1—Effectsofdroughtstress. Twoseparatepairwiseanalyseswerecar-
riedouttoassesstheeffectsofdroughtstressinGMandnon-GMplants:GMDxGMand
PLOSONE|DOI:10.1371/journal.pone.0173069 February28,2017 8/23
MolecularanalysisofGMmaizeunderstressconditions
Table2. Fold-change(Log2FC)ofdifferentiallyabundantproteinsinherbicide-tolerantGMmaizeNK603anditsnon-GMnear-isogenicvariety
grownundersuboptimalconditionsinExperiment1and2. Apositivevalueindicatesthattheproteinshowedup-regulationforthefirsttreatmentofthe
comparison.Anegativevaluemeansthattheproteinshoweddownregulationforthefirsttreatmentofthecomparison.‘ns’indicatesthatnosignificantdiffer-
ence(ANOVAP<0.05)wasfoundfortherelativeabundanceofthisproteinonthatspecificcomparison.
Protein UniProtID Experiment1 Experiment2
Factor1(Stress) Factor2(Genetic Unifactorial
Modification)
non-GMDx GMDx GMxnon- GMDxnon- GMDHxGM
non-GM GM GM GMD D
ATPsynthasesubunitgamma,chloroplasticprecursor(Zea P0C1M0 -1.34 -1.19 ns ns 1.33
mays)
Ferredoxin-NADPreductase(Zeamays) B6T9S5 0.57 -0.75 ns -0.93 0.72
PhotosystemIreactioncentersubunitII(Zeamays) B6SKI1 0.71 ns ns -0.80 ns
PhotosystemIreactioncentersubunitIVA(Zeamays) B6SPC1 1.21 ns ns ns ns
ATPsynthaseCF1alphasubunit(SaccharumhybridcultivarSP- A0A0U2U1F5 ns -0.95 ns ns ns
80-3280)
ATPsynthaseCF1betasubunit(chloroplast)(Zeamays) P00827 ns 0.76 ns ns ns
NAD-dependentepimerase/dehydratase(Zeamays) B4FH62 ns -0.77 ns ns ns
PhotosystemIsubunitVII(chloroplast)(Zeamays) P11601 ns -0.81 ns -0.84 ns
Oxygen-evolvingenhancerprotein2,chloroplastic(Zeamays) A0A096QKN7 ns -0.85 ns -0.67 -0.44
Phosphoglyceratekinase(Zeamays) C0PDB0 ns -0.96 ns 1.42 ns
Cytochromeb6-fcomplexiron-sulfursubunit(Zeamays) B4FTU7 ns 0.63 ns ns ns
Inorganicpyrophosphatase(Zeamays) B6SQQ0 ns -0.65 ns ns 0.57
psbP-likeprotein2,chloroplastic(Zeamays) A0A096TZ12 ns ns 0.88 ns ns
3-phosphoshikimate-1-carboxyvinyltransferase(Glycinemax) Q71LY8 ns ns 1.26 1.17 ExclusiveGM
D
Ribulosebisphosphatecarboxylase/oxygenaseactivase, Q9ZT00 ns ns -0.80 ns ns
chloroplasticprecursor(Zeamays)
Ribulosebisphosphatecarboxylaselargesubunit(Zeamays) A0A059Q6R9 ns ns -0.86 ns ns
ChainA,TheCrystalStructureofPsbp(Zeamays) A0A096Q0E5 ns ns 0.49 ns ns
Malatedehydrogenase(Zeamays) B4FZU8 1.15 ns ns ns ns
Fructose-bisphosphatealdolase(Zeamays) C0PD30 ns ns 0.64 ns ns
UncharacterizedproteinLOC100273394(Zeamays) B4FV82 ns -1.06 ns ns ns
doi:10.1371/journal.pone.0173069.t002
non-GMDxnon-GM.Atotalof14differentiallyabundantproteinswereobservedinthetwo
comparisons.Fiveoftheseproteinsweredifferentiallyabundantincontrolonly,withlog2
fold-changevalues(log2FC)rangingfrom-1.34to1.15(Table2).Nevertheless,4ofthese5
proteinswereupregulatedinthestresstreatment,exceptforATPsynthasesubunitgamma
(chloroplasticprecursor),whichwasdownregulatedinstress.These5proteinsareinvolvedin
molecularfunctionsbelongingtoenergymetabolismandstressresponsebiologicalprocesses
(Table3).ThecomparisonbetweenGMDxGMrevealed11differentiallyabundantproteins
withlog2FCrangingfrom-1.19to0.76,andmostoftheproteinsweredownregulatedinthe
GMDsamples.These11proteinsarenotonlyinvolvedinenergymetabolism,butalsoinpri-
maryreactionsofcarbohydratemetabolism,suchasglycolysisandphosphorylation(Table3).
Asexpected,someoftheproteinswerefounddifferentiallyabundantinbothcomparisons,
butwithdistinctfold-changes.Forinstance,ATPsynthasesubunitgamma(chloroplasticpre-
cursor)wasdownregulatedintheGMDsamples(log2FC=-1.19)andinnon-GMDsamples
(log2FC=-1.34),whencomparedtothecontrolunderoptimalconditions.ATPsynthaseisan
enzymethatmodulatestheelectrochemicalgradientacrossthethylakoidmembraneand
directlyinfluencestherateofphotosyntheticelectrontransport,thusrepresentingakeyfactor
intheregulationofphotosyntheticenergyconversioninchloroplasts[44].Kohzumaetal.[45]
PLOSONE|DOI:10.1371/journal.pone.0173069 February28,2017 9/23
MolecularanalysisofGMmaizeunderstressconditions
Table3. Descriptionofdifferentiallyexpressedproteinsfoundinthisstudy. Proteinswereconsidereddifferentiallymodulatedatstatisticallysignificant
differenceinnormalizedvolumeineachcomparisonbetweenthetreatments(GM;non-GM;GMD;non-GMD;GMDH)atANOVAP<0.05accordingto
Table2.ProteinswereclassifiedinfunctionalcategoriesbasedontheKEGGOrthologydatabasesandoncarefulliteratureevaluation.Thetablereportsspot
number(MatchID),accessionnumberandproteinname,togetherwithMascotscore,sequencecoverage,numberofmatchedpeptides,theoreticaland
experimentalmolecularweight(MW)andisoelectricpoint(pI).
Spot Accession ProteinName Mascot Sequence Peptides Theor. Theor. Exp. Exp. BiologicalProcess/
ID Score Coverage Mass pI(pH) Mass pI MolecularFunction
(%) (kDa) (kDa) (pH)
1689 NP_001150872 ATPsynthasesubunitgamma, 520 31 12 54 5.31 52 5.4 Energymetabolism/
chloroplasticprecursor(Zea Stressresponse(ATP
mays) synthesiscoupledproton
transport)
1576 ACG33858 Ferredoxin-NADPreductase, 536 44 19 40 8.56 44 8.2 Energymetabolism
(Zeamays) (oxidoreductase;
nucleotidebinding)
1364 ACG25364 PhotosystemIreactioncenter 220 24 5 21 9.77 20 9.1 Energymetabolism
subunitII(Zeamays) (photosynthesis)
1329 NP_001149700 PhotosystemIreactioncenter 204 47 6 14 9.79 15 9.1 Energymetabolism
subunitIVA(Zeamays) (photosynthesis)
1881 YP_024377 ATPsynthaseCF1alphasubunit 842 40 17 55 5.87 55 5.8 Energymetabolism(ATP
(SaccharumhybridcultivarSP- hydrolysis-coupledproton
80-3280) transport)
1826 NP_043032 ATPsynthaseCF1betasubunit 237 16 5 54 5.31 55 5.4 Energymetabolism(ATP
(chloroplast)(Zeamays) hydrolysis-coupledproton
transport)
1511 ACG31416 NAD-dependentepimerase/ 264 35 7 31 9.11 30 9.1 Carbohydratemetabolism
dehydratase(Zeamays) (catalyticactivity)
1261 NP_039445 PhotosystemIsubunitVII 110 46 3 8 6.51 8 6.2 Energymetabolism
(chloroplast)(Zeamays) (photosyntheticelectron
transportinphotosystemI;
oxidoreductase)
1429 XP_008652004 Oxygen-evolvingenhancer 345 37 8 31 9.50 33 9.1 Energymetabolism
protein2,chloroplastic(Zea (photosynthesis;calcium
mays) ionbinding;photosystem
I)
1756 NP_001147628 Phosphoglyceratekinase(Zea 413 23 10 50 6.07 48 5.8 Carbohydratemetabolism
mays) (glycolyticprocess;
phosphorylation)
1365 ACG28186 Cytochromeb6-fcomplexiron- 204 28 5 24 8.52 21 8.4 Energymetabolism
sulfursubunit(Zeamays) (photosynthesis;
oxidoreductaseactivity;
electrontransport)
1529 ACG27183 Inorganicpyrophosphatase(Zea 744 49 19 31 5.79 30 5.9 Carbohydratemetabolism
mays) (phosphate-containing
compoundmetabolic
process;magnesiumion
binding)
1341 XP_008654301 psbP-likeprotein2,chloroplastic 301 36 8 25 8.34 25 7.5 Energymetabolism
(Zeamays) (photosynthesis;calcium
ionbinding;photosystem
II)
1763 AAL67577 3-phosphoshikimate- 944 50 19 47 5.13 49 5.2 Energymetabolism
1-carboxyvinyltransferase (chorismatebiosynthetic
(Glycinemax) process;shikimate
pathway);
1743 NP_001104921 Ribulosebisphosphate 487 31 12 47 6.29 49 6.4 Energymetabolism
carboxylase/oxygenaseactivase, (Calvincycle;ATP
chloroplasticprecursor(Zea binding;activationof
mays) RuBisCO)
1848 NP_043033 Ribulosebisphosphate 521 27 12 52 6.33 52 6.3 Energymetabolism
carboxylaselargesubunit(Zea (photosynthesis;carbon
mays) fixation)
(Continued)
PLOSONE|DOI:10.1371/journal.pone.0173069 February28,2017 10/23