Mulberry Transcription Factor MnDREB4A Confers Tolerance to Multiple Abiotic Stresses in ...

17 Pages·2015·11.3 MB·English

RESEARCHARTICLE
Mulberry Transcription Factor MnDREB4A
Confers Tolerance to Multiple Abiotic Stresses
in Transgenic Tobacco
Xue-QinLiu☯,Chang-YingLiu☯,QingGuo,MengZhang,Bo-NingCao,Zhong-HuaiXiang,
Ai-ChunZhao*
StateKeyLaboratoryofSilkwormGenomeBiology,KeyLaboratoryforSericultureFunctionalGenomicsand
BiotechnologyofAgriculturalMinistry,SouthwestUniversity,Chongqing,400716,China
☯Theseauthorscontributedequallytothiswork.
*[email protected]
Abstract
Thedehydrationresponsiveelementbinding(DREB)transcriptionfactorshavebeen
reportedtobeinvolvedinstressresponses.MoststudieshavefocusedonDREBgenesin
subgroupsA-1andA-2inherbaceousplants,buttherehavebeenfewreportsonthefunc-
tionsofDREBsfromtheA-3–A-6subgroupsandinwoodyplants.Moreover,mulberry
OPENACCESS treesareecologicallyandeconomicallyimportantperennialwoodyplants,buttherehas
beenlittleresearchonitsstressphysiology,biochemistryandmolecularbiology.Inthis
Citation:LiuX-Q,LiuC-Y,GuoQ,ZhangM,CaoB-
N,XiangZ-H,etal.(2015)MulberryTranscription study,aDREBgenefromthemulberrytree,designatedasMnDREB4A,classifiedintothe
FactorMnDREB4AConfersTolerancetoMultiple A-4subgroupbyourpreviousstudy,wasselectedforfurthercharacterization.Ourresults
AbioticStressesinTransgenicTobacco.PLoSONE
showedthattheMnDREB4Aproteinwaslocalizedtothenucleuswhereitactivatedtran-
10(12):e0145619.doi:10.1371/journal.pone.0145619
scription.ThepromoterofMnDREB4Acandirectprominentexpressiondownstreamofthe
Editor:ZhulongChan,ChineseAcademyof β-glucuronidase(GUS)geneunderheat,cold,droughtandsaltstress,andGUSstaining
Sciences,CHINA
wasdeepestafter12hofstresstreatment.TheMnDREB4A-overexpressiontransgenic
Received:July3,2015
tobaccoshowedtheimprovedgrowthphenotypeunderuntreatedconditions,suchas
Accepted:December7,2015 greenerleaves,longerroots,andlowerwaterlossandsenescencerates.Overexpression
Published:December22,2015 ofMnDREB4Aintobaccocansignificantlyenhancetolerancetoheat,cold,drought,and
saltstressesintransgenicplants.Theleafdiscsandseedlingsoftransgenicplants
Copyright:©2015Liuetal.Thisisanopenaccess
articledistributedunderthetermsoftheCreative reducedleafwiltingandsenescenceratescomparedtothewildtypeplantsunderthedif-
CommonsAttributionLicense,whichpermits ferentstressconditions.Furtherinvestigationshowedthattransgenicplantsalsohad
unrestricteduse,distribution,andreproductioninany
higherwatercontentsandprolinecontents,andlowermalondialdehydecontentsunder
medium,providedtheoriginalauthorandsourceare
untreatedconditionandstressconditions.OurresultsindicatethattheMnDREB4Aprotein
credited.
playsanimportantroleinplantstresstolerance.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformationfiles.
Funding:TheNationalHi-TechResearchand
DevelopmentProgramofChina(No.2013AA100605-
3)andtheChinaAgricultureResearchSystem(No.
Introduction
CARS-22-ZJ0102).
Abioticstresses,suchasheat,cold,high-saltlevels,anddrought,presentmajorchallengesto
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. sustainablefoodproductionbecausetheyreducepotentialyieldsbyupto50%incropplants
PLOSONE|DOI:10.1371/journal.pone.0145619 December22,2015 1/17
MnDREB4AImprovetheToleranceofTobaccotoAbioticStresses
[1]Therefore,developingcropsthatarebetteradaptedtoabioticstressesisimportantforfood
productioninmanypartsoftheworld.Majoreffortshavebeenmadetoenhancestress-toler-
ancethroughtheuseofconventionalplantbreeding,andgeneratedmanycropvarietieswith
improvedstresstolerance.Inthisprocedure,thestressresistantgeneticvariabilityisidentified
byscreeninggermplasmcollectionsandselections.Beneficialtraitsaresubsequentlyintro-
ducedintocultivarsusingcomplexmatingdesigns[2].However,thisapproachistime-con-
suming,andlabor-andcost-intensive.Furthermore,unwanted,linkedtraitscanalsobe
transferred,whichmeansthatthemethodcansufferfrompoorselectivity[3].Contrarytothe
conventionalplantbreedingmethods,geneticengineeringcandirectlyintroducespecified
genesintoplants,whichoffersthepossibilityofimprovingstresstolerancebyamoreattractive
andquickmethod[4,5].Todate,geneticengineeringhasbeensuccessfullyusedtoimprove
plantyieldandquality[6],increasepestresistance[7],reduceweedcompetition[8]and
increaseabioticstresstolerance[9],andithasbeenwidelyappliedtomanydifferentspecies,
suchasrice[9,10],wheat[8],maize[11],potato,[12]andtomato[13].Forabioticstress,the
induciblegenesareclassifiedintofunctionalandregulatorygenesaccordingtothefunctionsof
theirencodingproteins[14].Introducingasingle,functionalgenehasnotbeenalwayssuccess-
fulinconferringtolerancebecausetherearemanydifferentpathwaysinvolvedincontrolling
plantabioticstress.Conversely,theexpressionofaregulatorygene,especiallyastressrelated
transcriptionfactor,canimproveplantabioticstresstolerancemoreeffectively[15]becauseit
isexpectedtomodulatetheexpressionofalargenumberofrelevantdownstreamgenes[16].
Thus,transcriptionfactorgeneswillprobablybecomeapowerfultoolinthegeneticengineer-
ingofplantabioticstresstolerance[17,18].
Recently,researchershaveidentifiedseveralkeytranscriptionfactors(TFs)involvedinthe
regulationnetworksactivatedduringabioticstress[19,20],includingethyleneresponsiveele-
mentbindingfactors(ERF),basic-domainleucine-zipper(bZIP),myeloblastosis(MYB),and
WRKYbindingtranscriptionfactors[21,22].ProbablythebeststudiedgroupofTFsinvolved
inabioticstressisthedehydrationresponsiveelementbindingproteins(DREB)genesbelong-
ingtotheAP2/ERFfamily.ThefirstDREBfamilymemberwasisolatedfromArabidopsisand
itrespondstolowtemperatureandwaterdeficit[23].AllDREBscontainaconservedAP2
(Apetala2)domainofapproximately60aminoacidsanditcanbindtothedehydrationrespon-
siveelement(DRE)/c-repeatsequence(CRT)coresequences,whicharepresentinnumerous
abioticstressresponsivegenespromoters[24].Inthisdomain,therearesevenkeyresidues
thatareessentialforhighlyspecificinteractionswiththeDRE/CRTelement.Therearefour
args,twotrpsandonevalresidues[25].IntheN-terminaloftheAP2domain,theYRGele-
ment,containingapproximately20aminoacids,playsanimportantroleinidentifyingand
bindingtotheDRE/CRTelement,especiallytheVal(V14)atposition14andGlu(E19)at
position19[26,27].TheN-terminaloftheAP2domaincontainstheRAYDelement,which
hasapproximately42aminoacidsandtheassociatedWLGmotif.Bothregulatethespecial
bindingactivityofTFsbyinfluencingtheconformationoftheYRGelementorbyinteracting
withotherproteins[28].
PlantDREBproteinsareclassifiedintosixsubfamiliesaccordingtotheirfunctionandchar-
acterization:A-1,A-2,A-3,A-4,A-5,andA-6[26].Manyofthemhavebeenidentifiedand
functionallycharacterizedindifferentspecies.MoststudieshavefocusedontheDREBgenesin
theA-1andA-2subgroups,overexpressionofA-1andA-2familiesDREBgenescaninduce
theexpressionofstressresponsivedownstreamgenesandenhanceabioticstresstolerance[29–
35].However,therehavebeenfewreportsonthefunctionsofDREBsfromtheA-3~A-6
subgroups.
Mulberry(MorusalbaL.)treesareecologicallyandeconomicallyimportantperennial
woodyplants[36].Ithadbeenreportedthatmulberrycanadapttomanydifferentsituations,
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MnDREB4AImprovetheToleranceofTobaccotoAbioticStresses
includingcold,waterlogged,droughtandsalineenvironments[37].However,therehasbeen
littleresearchonitsstressphysiology,biochemistryandmolecularbiology[38],andthereis
verylittleinformationavailableaboutthemulberryDBREfunctions.Inourpreviousstudy,we
identifiedandcharacterized30DREBgenesfromtheM.notabilisGenomeDatabase[39],and
theirfunctionsduringabioticstresswerepreliminarilypredicted.
Basedonourpreviousresearch,MnDREB4AwasclassifiedintoA-4subgroupanditcanbe
inducedbymultipleabioticstresses[39].Furthermore,thepromoterofthisgenecontained
severalcis-elementsthatwererelatedtoadversestresses.Therefore,weselectedMnDREB4A
forfurtherinvestigation.Inthisstudy,wecharacterizeditssubcellularlocalization,promoter
activityandfunction.ThetoleranceofthetransgenictobaccoharboringMnDREB4Atomulti-
pleabioticstresseswasalsoevaluated.
MaterialsandMethods
CloningandbioinformationanalysisoftheMnDREB4Agene
Thecodingsequence(CDS)oftheMnDREB4AwasamplifiedandclonedformM.notabilisas
describedinourpreviousstudy[39].AlloftheloggedDREBproteinsequencesforotherspe-
ciesweredownloadedfromthefollowingwebsites:theNationalCenterforBiotechnology
Information(NCBI,http://www.ncbi.nlm.nih.gov/);theArabidopsisinformationresource
(TAIR,http://www.arabidopsis.org/);EnsemblPlants(http://plants.ensembl.org/index.html);
thedatabaseofricetranscriptionfactors(DRTF,http://drtf.cbi.pku.edu.cn/index.php)and
theplanttranscriptionfactordatabase(PlantTFDB,http://planttfdb.cbi.pku.edu.cn/).The
informationaboutthesesequencesisshowninS1Table.Thesequenceswerealignedusing
ClustalX2.0[40]andillustratedusingtheGenedocprogram[41].Theconservedmotifsin
MnDREB4AproteinswereidentifiedusingamotifbasedsequenceanalysistoolcalledMEME
[42].Thethree-dimensionalstructureoftheMnDREB4AproteinwaspredictedusingSWISS--
MODEL(http://swissmodel.expasy.org/).ThesubcellularlocalizationofMnDREB4Aproteins
waspredictedusingtwowebsitetools:TargetP1.1Server(http://www.cbs.dtu.dk/services/
TargetP/)andEuk-mPLoc2.0(http://www.csbio.sjtu.edu.cn/bioinf/euk-multi-2/).
SubcellularlocalizationoftheMnDREB4Aprotein
MnDREB4AwascombinedwiththeN-terminalorC-terminalofgreenfluorescentprotein
(EGFP)toconstructafusionproteinexpressioncassette.TheMnDREB4AandEGFPwere
insertedintotheKpnIandEcoRIsiteoftheplantexpressionvectorpLGNL,whichhadbeen
preservedinourlaboratory.Thegenespecificprimersthatcontainedrestrictionenzyme
sitesareshowninS2Table.ThevectorsthatcontainedoneoftheMnDREB4A::EGFPfusion,
EGFP::MnDREB4AfusiongenesortheEGFPexpressioncassettewereconfirmedbysequenc-
ingandthentransientlyintroducedintoonion(AlliumcepaL.)epidermalcellsbyAgrobacter-
iumtumefaciensLBA4404,asdescribedbyLiuetal.[43].ThenucleiwerestainedbyDAPI(4',
6-diamidino-2-phenylindole)andimageswerecapturedforeachcolorchannelwithafluores-
cencemicroscope(Nikon,Tokyo,Japan).
ConstructionoftheMnDREB4Apromoter-GUSfusionexpression
plasmidandArabidopsistransformation
0
The1500bp5 upstreamregionofMnDREB4AandtheGUSsequencewereamplifiedby
PCRusingthespecificprimerswithrestrictionenzymesitesshowninS2Table.Thepromot-
erswerefusedwiththeGUSgeneandthenthefusionconstructwasinsertedintotheKpnI
andEcoRIsitesofthepCAMBIA2300vector.Thereconstructedplasmid(MnDREB4A
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MnDREB4AImprovetheToleranceofTobaccotoAbioticStresses
pro::GUS)wasconfirmedbysequencingandtransformedintoAgrobacteriumtumefaciens
EHA105bythefreeze—thawmethod[44].ThepositiveAgrobacteriumtumefacienswas
transformedintoArabidopsisthalianabythefloraldipmethod[45].TransgenicArabidopsis
wereselectedon1/2MSmediumcontainingafinalconcentrationof50mg/Lkanamycinand
confirmedbyPCRanalysis.TheplasmidMnDREB4Apro::GUSwasusedasapositivecontrol
andthegenomicDNAofwildtype(WT)ArabidopsiswasusedasnegativecontrolinthePCR
analysis.
SouthernblottingandGUSstainingoftransgenicArabidopsis
GenomicDNAsfromtheWTandthedifferenttransgenicArabidopsislineswereprepared
fromleavesusingthecetyltrimethylammoniumbromidemethod(CTAB)[46].Exactly20μg
ofDNApersamplewasdigestedwithEcoRIovernightat37°C.TheWTArabidopsisgenomic
DNAwasusedasthenegativecontrol.ThedigestedDNAswereseparatedon0.8%(w/v)
agarosegelusingelectrophoresisandthentransferredtoapositivelychargedHybond-N
membrane(RocheDiagnosticsGmbH,Mannheim,Germany).TheGUSprobe,labeledwith
digoxigenin(DIG),wassynthesizedbyPCRfollowedthemanufacturer’sinstructions(Roche
DiagnosticsGmbH,Mannheim,Germany).TheSouthernblotwasfinallyperformedfollowing
thepre-hybridization,washingandchemiluminescentdetectionprocedure[47].
GUSactivitywasassessedhistochemicallyusingkanamycin-resistantT1andT2generation
transgenicArabidopsisthatcontainedMnDEEB4Apro::GUS.Thestainingsolutionforthe
GUSassaywaspreparedasdescribedbyRaeetal.[48].Theplantswereincubatedwiththe
stainingsolutionat37°Cforabout12h.The7-day-oldseedlingsineachsamplewereexposed
to40°C(heat),4°C(cold),150mMNaClor20%PEG6000(drought)treatment.Thesamples
weresubjectedtoGUSstainingafter0,1,3,6,9,12,and24hofexposure.TheGUSstaining
wasquantifiedbyImageJ(NationalInstitutesofHealth,Maryland,USA).Allresultsarerepre-
sentativeofthreeindependentexperiments.
Constructionandtransformationofthevectorfortobaccotransformation
TheMnDREB4Asequence,flankedbyaKpnIandaSpeIrestrictionsite,wereamplifiedby
PCRusingthespecificprimersshowninS2Table.TheobtainedDNAfragmentsweredigested
bycorrespondingrestrictionenzymesandtheninsertedbetweentheKpnIandSpeIsitesof
theplantexpressionvectorpLGNLunderthecontrolofthecauliflowermosaicvirus(CaMV)
35Spromoter.Therecombinantplasmid(CaMV35S::MnDREB4A)wasconfirmedbysequenc-
ingandtransformedintoAgrobacteriumtumefaciensEHA105usingthefreeze—thawmethod
[44].ThenthepositiveAgrobacteriumtumefacienswastransformedintotobacco(Nicotiana
tabacumL.)plantsusingtheleafdiscmethod[49].Transgenictobaccowasselectedforon1/2
MSmediumcontainingafinalconcentrationof50mg/LkanamycinandconfirmedbyGUS
staining,andgenomicPCR.TodeterminetheexpressionlevelsoftheMnDREB4Aintrans-
genictobaccoplants,totalRNAwasextractedwithRNAisoplus(Takara,Dalian,China),the
firststrandcDNAwassynthesizedusingthePerfectRealTimeversionofthePrimerScript™
RTreagentKitwithgDNAEraser(PerfectRealTime)(TaKaRa,Dalian,China).TheqRT-PCR
procedurewasthesameastheonedescribedinourpreviousstudy[38].TheNtActingene
(GenBankAccessionnumber:U60489)wasusedasaninternalreference,theforwardand
reverseprimersforamplificationwereNtActin-F(5'-TCACAGAAGCTCCTCCTAATCCA
-3')andNtActin-R(5'-GAGGGAAAGAACAGCCTGAATG-3').Besides,theseedsofthe
confirmedtransgeniclineswereculturedinMSmediumcontaining1mg/L6-BAforfurther
research.
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MnDREB4AImprovetheToleranceofTobaccotoAbioticStresses
DifferenceanalysisbetweentransgeniclinesandWTtobaccounder
untreatedconditions
Theleaves(takenfromthesamepositiononeachsample),rootsandwaterlossratesforthe
transgeniclineandWTtobaccowerecomparedunderthesamegrowthconditions.Inaddi-
tion,theleaveswerecutinto1cmdiameterdiscs,whichwerethenculturedwith1/2MSnutri-
entsolutioninaclimatechamber(NingboSoutheastInstrumentCorporation,Ningbo,China)
(25°C,16hday/8hnight).Therootswerepulledoutandcleanedcarefullysothattheydidnot
break.Todeterminethewaterlossrate,theleaveswereweighed(FW)immediatelyandincu-
batedat30°Cwith40%relativehumidityandthenweighedatdesignatedtimes(Wt).The
waterlossratewascalculatedasfollows:
Waterlossrate ¼ðFW (cid:2) WtÞ=FW(cid:3)100%:
Eachmeasurementwasrepeatedthreetimes.
Physiologicalandabioticstresstoleranceanalysisoftransgenic
tobaccos
ThetransgeniclinesandWTtobaccosamplesweregrownonMSmediumuntilrootformation
andthentransferredintosoilinaclimatechamber(25°C,16hday/8hnight).Afterabout8
weeks,thetobaccoplantswereapproximately15cmhigh.Thentheyweresubjectedtoheat
(40°C),cold(4°C),drought(20%PEG6000),andsalt(400mMNaCl)stressesuntilsignificant
differencesappearedbetweenthetransgeniclineandWTtobacco.Whenthisoccurred,the
leavesfromthesamepositionsonthesamplewerecollectedforrelativewatercontent(RWC),
freeprolineandmalonaldehyde(MDA)contentmeasurements.Eachtreatmentwasreplicated
threetimes.Toexploretheeffectsofthevarioustreatmentsonleaves,1cmdiameterleafdiscs
werecutfromhealthyandfullyexpandedleavesoftransgenicandWTtobaccoplants.Then
theleafdiscsweretreatedwithheat,cold,drought,andsaltstresses,respectively.
Aftertheabioticstresstreatments,thefreshweights(FW)ofthetransgeniclineandWT
tobaccoleaveswereimmediatelyrecorded.Theleavesweresoakedfor12hindistilledwaterat
roomtemperatureunderaconstantlightandtheturgidweight(TW)wasrecorded.Totaldry
weight(DW)wasrecordedafterdryingfor24hat80°C.RWCwascalculatedasfollows:
RWCð%Þ¼½ðFW (cid:2) DWÞ=ðTW (cid:2)DWÞ(cid:4)(cid:3)100%
[50].
Fortheprolinecontentanalysis,exactly~0.5goffreshleaveswashomogenizedin10mlof
3%sulfosalicylicacidandcentrifugedat2,000×gfor15min.Theresulting2mlextractwas
incubatedwith2mlofninhydrinreagent,whichcontained2.5%(w/v)ninhydrin,60%(v/v)
glacialaceticacidand40%6Mphosphoricacid,and2mlofglacialaceticacid,at100°Cfor40
min.Thereactionwasterminatedinanicebathand5mloftoluenewasadded.Thesamples
werethenvortexed.Thereactionmixturewasincubatedat23°Cfor24handtheabsorbance
ofthesupernatantat520nmwasdeterminedbyaspectrometer(TECHCOMP,Shanghai,
China).Thentheprolinecontentwascalculatedasdescribedpreviously[51].
TheMDAcontentwasdeterminedasdescribedbyHodgesetal.[52].Around0.5goffresh
leaveswashomogenizedin10ml10%trichloroaceticacid(TCA)andcentrifugedat12,000×g
for10min.Then2ml0.6%thiobarbituricacid(TBA)dissolvedin10%TCAwasaddedtoa2
mlaliquotofthesupernatant.Themixturewasheatedinboilingwaterfor30minandthen
quicklycooledinanicebath.Aftercentrifugationat2,500×gfor10min,theabsorbanceofthe
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MnDREB4AImprovetheToleranceofTobaccotoAbioticStresses
supernatantat450,532,and600nmwasdeterminedwithaspectrometer(TECHCOMP,
Shanghai,China).ThentheMDAcontentwascalculatedasdescribedpreviously[52].
Statisticalanalysis
TheresultswereorganizedandanalyzedbyExcel2013(Microsoft,Redmond,USA),Origin
7.0(OriginLab,MA,USA)andSPSSStatistics17.0(SPSSInc.,Chicago,IL,USA)software.The
meanswerecomparedbyStudent’st-testwerecalculatedtofindthesignificantdifference
betweendifferentlines.Meanvaluesthatweresignificantlydifferentwithintreatmentfrom
eachotherwereindicatedbyasterisks.
Results
BioinformationanalysisoftheMnDREB4Agene
Multiplesequenceanalysisshowedthatalldeducedproteinsexhibitedthetypicalfeaturesof
DREBproteins,includinganAP2domain,andYRG,WLG,andRAYDmotifs(Fig1a).More-
over,theseproteinsalsohadtheconservedvaline(V14)atthe14thpositionandglutamicacid
(E19)atthe19thpositionintheAP2domain.TheAP2domaindisplayedahigherdegreeof
aminoacididentitythantheN-terminiandC-terminioftheproteins.
TogaininsightintothenatureoftheAP2domainofmulberryDREBproteinanditshomo-
loguesfromotherspecies,includingPopulustrichocarpa,Vitisvinifera,Malusdomestic,
Gossypiumhirsutum,Arabidposisthaliana,Zeamays,andOryzasativa,sequencelogoswere
producedtoexaminethelevelofconservationateachresidueposition.Theresultsrevealed
that46(70.8%)oftheconservedaminoacidswereidenticalintheAP2domain,andtheresi-
dueslocatedinotherpositionsdisplayedvaryinglevelsofconservation(Fig1b).Inadditionto
V14andE19,therewerefourarginine(R),twotryptophan(W)andavaline(V)participating
intheinteractionbetweentheDREBproteinsandspecificDNAsequences(Fig1b).Apart
fromtheAP2domain,twootherconservedmotifswerealsofound(Fig1b),butthefunctions
ofthesemotifsrequirefurtherinvestigation.
BasedontheSWISS-MODELonlineprediction,theMnDREB4Aencodingaproteincon-
tainingthreeβ-foldedsheetsandoneα-helix(S1aFig).Theα-helixpackedapproximatelypar-
alleltotheβ-foldedsheets.TheV14andE19residues,whichallowtheproteintobindDNA,
werelocatedinthesecondβ-foldedsheet(β-2)(S1bFig).Whenthemodelwasrotatedatcer-
tainangles,thesecondβ-foldedsheet(β-2)wasinthemostfavorablepositiontobindthe
DNA(S1cFig),whichwasconsistentwiththatofotherreportedDREBgenes[25].
SubcellularlocalizationoftheMnDREB4Aprotein
AlthoughtheMnDREB4Adidn’tcontainanobviousnuclearlocalizationsignal(NLS),itwas
predictedtobenuclearproteinaccordingtotheTargetP1.1ServerandEuk-mPLoc2.0.Toval-
idatetheprediction,theMnDREB4A::EGFPvectorandtheEGFP::MnDREB4Afusiongene,
underthecontroloftheCaMV35Spromoter(S2Fig),weretransfectedintotheonionepider-
malcells.TheEGFPvectorwasusedasacontrolandwastransfectedseparately(S2Fig).The
greenfluorescencesignalsoftheMnDREB4A::EGFPandEGFP::MnDREB4Afusionproteins
wereobservedonlyinthenucleus(Fig2),whichimpliedthattheMnDREB4Aproteinswere
nucleus-localizedproteins.Thisresultwasconsistentwiththeproteinstructureprediction.
Promoteractivityanalysis
ToexaminewhethertheabioticstressresponsivenessoftheMnDREB4Agenewastranscrip-
0
tionallycontrolledby5 upstreamregions,transgenicArabidopsisweregeneratedby
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MnDREB4AImprovetheToleranceofTobaccotoAbioticStresses
Fig1.Proteinsequencemulti-alignmentandsequenceslogosforDREBsfromdifferentspecies.(a)AlignmentwasperformedusingtheGeneDoc
program.TheconservedV14,E19,YRG,RAYDandWLGmotifsarehighlightedbyasterisksandlines.Conservativesequencesarehighlightedbyblack
andgreyshading.(b)Theoverallheightofeachstackindicatestheconservationoftheproteinsequenceattheaminoacidposition,whereastheheightsof
theletterswithineachstackrepresenttherelativefrequencyofthecorrespondingaminoacid.TheYRG,RAYDandWLGmotifsarehighlightedbyasterisks.
ThekeyresiduesthatareessentialforthehighlyspecificinteractionswithandforbindingtotheDRE/CRTelementarehighlightedbycolorfulcirclesand
squares.
doi:10.1371/journal.pone.0145619.g001
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MnDREB4AImprovetheToleranceofTobaccotoAbioticStresses
Fig2.SubcellularlocalizationoftheMnDREB4Atranscriptionfactorinonionepidermalcells.TheMnDREB4A::EGFPorEGFP::MnDREB4Afusion
genesunderthecontrolofCaMV35Spromoterweretransfectedintoonionepidermalcells.ThepositivecontrolwasatransfectedplasmidcontainingEGFP
alone.DAPIandarrowswereusedtoindicatethenucleus.Imageswerecapturedforeachcolorchannelwithafluorescencemicroscope(Nikon,Tokyo,
Japan).
doi:10.1371/journal.pone.0145619.g002
introducingaMnDREB4Apro::GUSfusedgene(S3aFig).Kanamycin-resistantprogeny(T1
generation)wereconfirmedbygenomicPCR(S3bFig).ThePCRresultshowedthatthe
GUS-nosandMnDREB4AprogenescouldbedetectedinArabidopsistransgenicstrains1,2,
6,9and10(designatedasTS1,TS2,TS6,TS9,andTS10).TheSouthernblottingshowedthat
therewasonlyonecopyofMnDREB4Apro::GUSinTS1,TS2,andTS9,andmultiplecopies
inTS6andTS10(S3cFig).
One-week-oldT2seedlingsweretreatedwithheat(40°C),cold(4°C),drought(20%
PEG6000),andsalt(150mMNaCl).Afterthedifferenttreatments,thestrongestGUSexpres-
sionswereobservedintheleavesandrootsofTS2(Fig3),TS1,TS9,andTS10(S4a–S4cFig),
andlowerinTS6(datanotshown).TheGUSactivitywasthehighestafter12htreatmentand
thendeclined.AlthoughtheGUSaccumulationlevelsinthedifferenttransgeniclinesweredif-
ferent,theirresponsetrendsunderabioticstresseswereidentical.WealsofoundthattheGUS
reporterwasmainlyexpressedintheleavesandrootsanditpreferentiallylocalizedtothevas-
culatureofvarioustissues.
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MnDREB4AImprovetheToleranceofTobaccotoAbioticStresses
Fig3.ExpressionpatternsfortheGUSreportergeneunderthecontroloftheMnDREB4ApromoterintransgenicArabidopsis.(a)Histochemical
GUSstaininginTS2wasperformedafterabioticstresstreatmentatdesignatedtimepoints.Heat:40°C,Cold:4°C,Salt:150mMNaCl,Drought:20%
PEG6000.(b)TheGUSstainingwasquantifiedbyImageJ(NationalInstitutesofHealth,Maryland,USA).
doi:10.1371/journal.pone.0145619.g003
ThedifferencebetweentransgeniclineandWTtobaccoundernormal
conditions
ToinvestigateMnDREB4Afunction,therecombinantplasmid(CaMV35S::MnDREB4A)(Fig
4a)wastransformedintotobaccoandproducedtransgenicplants.Thesetransgeniclines
wereselectedonselectionmediumcontainingkanamycin.Thetransgenicplantswereana-
lyzedfurtherbyPCRusinggenomicDNAasthetemplate,and35Sforwardandgene-specific
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MnDREB4AImprovetheToleranceofTobaccotoAbioticStresses
Fig4.Therecombinantplasmid(CaMV35S::MnDREB4A)usedinthetransgenictobacco,andthePCRandqRT-PCRanalysisresults.(a)The
recombinantplasmid(CaMV35S::MnDREB4A).(b)PCRanalysisusinggenomicDNAasthetemplate,and35Sforwardandgene-specificreverseprimers.
(c)TheexpressionleveloftheMnDREB4AgeneinthetransgeniclinesandthecontrolplantswasexaminedbyqRT-PCRanalysis.Thedatawerecalculated
usingthe2–ΔΔCtformula.ThesignificantlydifferentvaluesamongWTandtransgenictobaccoplantsareindicatedwithasterisks(n=3).Twoasterisks
indicateasignificantdifferenceatP<0.01.
doi:10.1371/journal.pone.0145619.g004
reverseprimers(Fig4b).TheexpressionlevelsoftheMnDREB4Ageneinthetransgeniclines
andthecontrolplants(WT)wereexaminedbyqRT-PCRanalyses(Fig4c).Expressionof
MnDREB4Aintransgeniclines1,5and7(designatedasOE1,OE5,andOE7)wasover
300-foldgreaterthaninthecontrolplants(Fig4c).TheOE1transgeniclinewaschosenfor
furtherexperiments.
ThetransgenicOE1tobaccoandtheWTwerecultivatedunderthesameconditions.How-
ever,whencomparedtotheWTundernormalconditions,theOE1tobaccoplantsshowed
obviousphenotypicleafandrootdifferences.Forexample,theOE1leavesweresmallerand
greenerthantheWTleaves(Fig5a)andtheOE1rootswerelongerthantheWTroots(Fig5b).
Furthermore,theOE1plantsexhibitedlowerratesofwaterlossateachtimepointthanthe
WTafterleafdetachment(Fig5c).Whentheleafdiscshadbeenculturedin1/2MSnutrient
solutionforabout1month,thediscsfromtheWTplantsbecameyellowandsenescencewas
moreobviousthanforthetransgenictobaccoOE1disks(Fig5d).Alltheseresultsindicated
thattheOE1transgenictobaccoshouldhaveastrongertolerancetoadverseconditionsthan
theWT.
OverexpressionofMnDREB4Aenhancedtolerancetomultipleabiotic
stressesintransgenictobacco
TheOE1andWTleafdiscsandseedlingsweretestedwithvariousabioticstressestocharacter-
izethefunctionofMnDREB4Atransgeniclineunderheat,cold,drought,andsaltstress.Fig6a
showsthatthelargestareasofdamagewereobservedinWTtobaccoleafdiscsafter4daysof
heat(40°C)treatment,whileOE1discssufferedonlyslightdamage.After1monthofcold
(4°C)ordrought(20%PEG6000)treatment,theWTdiscsexhibitedmoreobviouschlorosis
PLOSONE|DOI:10.1371/journal.pone.0145619 December22,2015 10/17