Triticum aestivum WRAB18 functions in plastids and confers abiotic stress tolerance when

19 Pages·2017·3.66 MB·English

RESEARCHARTICLE
Triticum aestivum WRAB18 functions in
plastids and confers abiotic stress tolerance
when overexpressed in Escherichia coli and
Nicotiania benthamiana
XiaoyuWang,LinshengZhang*,YaneZhang,ZhenqingBai,HaoLiu,DapengZhang
CollegeofLifeSciences/StateKeyLaboratoryofCropStressBiologyforAridAreas,NorthwestA&F
University,Yangling,China
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*[email protected]
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Abstract
WRAB18,anABA-inducibleproteinbelongstothethirdfamilyoflateembryogenesisabun-
dant(LEA)proteinswhichcanbeinducedbydifferentbioticorabioticstresses.Inthepres-
OPENACCESS entstudy,WRAB18wasclonedfromtheZhengyin1cultivarofTriticumaestivumand
Citation:WangX,ZhangL,ZhangY,BaiZ,LiuH, overexpressedinEscherichiacolitoexploreitseffectsonthegrowthofE.coliunderdiffer-
ZhangD(2017)TriticumaestivumWRAB18 entabioticstresses.ResultssuggestedtheenhancedexhibitionoftoleranceofE.colito
functionsinplastidsandconfersabioticstress
thesestresses.Meanwhile,theWRAB18-transgenictobaccoplantswereobtainedtoana-
tolerancewhenoverexpressedinEscherichiacoli
lyzethestress-relatedenzymaticactivitiesofascorbateperoxidase(APX),peroxidase
andNicotianiabenthamiana.PLoSONE12(2):
e0171340.doi:10.1371/journal.pone.0171340 (POD)andsuperoxidedismutase(SOD),andtoquantifythecontentofmalonaldehyde
(MDA)underosmoticstress,highsalinity,andlowandhightemperaturestress.Theactivi-
Editor:GuangxiaoYang,HuazhongUniversityof
ScienceandTechnology,CHINA tiesofAPX,PODandSODinthetransgenictobaccolineswerehigherwhilethecontentof
MDAwaslowerthanthoseofWTlines.Moreover,plastidlocalizationofWRAB18inNicoti-
Received:November7,2016
anabenthamianaplasmacellswerefoundfusingwithGFP.Inaddition,purifiedWRAB18
Accepted:January19,2017
proteinprotectedLDH(Lactatedehydrogenase)enzymeactivityinvitrofromvariousstress
Published:February16,2017
conditions.Inbrief,WRAB18proteinshowsprotectiveactionbehavingasa“molecular
Copyright:©2017Wangetal.Thisisanopen shield”inbothprokaryoticandeukaryoticcellsundervariousabioticstresses,notonlydur-
accessarticledistributedunderthetermsofthe
ingABAstress.
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformation
Introduction
files.
Funding:Thisstudywassupportedbyfunding Abioticstresseswerekeyrestrictionfactorsduringthegrowthandproductionofcrops.Plants
fromtheStateKeyLaboratoryofCropStress havedevelopednumerousdefensemechanismstoprotectthemselvesundercomplexgrowth
BiologyforAridAreas(CSBA2015007)andthe conditionsthroughlong-termevolutionaryprocesses.Thesemechanismsarefoundatthecel-
SpecializedResearchFundfortheDoctoral
lularandmolecularlevels,suchaschangesinmembranelipidcomposition,productionofnew
ProgramofHigherEducation(20120204110033).
proteinpolymers,increasedcontentsofsugars,organicacids,solubleproteins,proline,and
Thefundershadnoroleinstudydesign,data
ABA,andothercorrespondingchangesnecessarytoresistoravoidadverseconditions[1].
collectionandanalysis,decisiontopublish,or
preparationofthemanuscript. LEAproteinsrepresentoneoftheseadaptionsandarebelievedtoplayanimportantrolein
PLOSONE|DOI:10.1371/journal.pone.0171340 February16,2017 1/19
WRAB18enhancesstresstoleranceincells
Competinginterests:Theauthorshavedeclared adversephysiologicalregulatoryprocesses,althoughtheseproteinsarecommoninplants
thatnocompetinginterestsexist. underabioticstressconditions[2].However,theirspecificphysiologicalandbiochemical
functionsremainunclear.Therefore,furtherstudiesonthemechanismsofLEAproteinsare
required[3].
Lateembryogenesisabundant(LEA)proteinswerefirstidentifiedinlate-stagematurecot-
tonseedsbyDureandCroudhin1981[4].Sincethen,theyhavebeenidentifiedinmanyother
speciesincludinghigherplants,suchaswheat[5],rice[6],maize[7],barley[8],andbean[9],
andinprimaryspecies,suchasArtemiafranciscana[10],nematodes[11],andbacteria[12].
LEAproteinsarewidelydistributedinseeds,roots,stems,andleavesofplants[13].Theyare
classifiedintosevengroupsaccordingtotheaminoacidsequencesandconservedmotifs[14].
Group3LEAproteinshavebeencharacterizedbasedonan11-aminoacidmotif,TAQAA-
KEKAGE[15,16].Theyexistinarandomlycoiledstructureinplantcellsandhaveahighper-
centageofhydrophilicaminoacids,suchasglycine,serine,andthreonine,whichforma
reversibleα-helixunderdehydratedconditionsandsubsequentlyrevertbacktotherandomly
coiledstructureuponrehydration[17,18].Thehydrophilicα-helixmayberelatedtoprotein-
proteinandprotein-lipidinteractionsduringstresssituations[19],thatpotentiallymaintains
membranestabilityandintegrationunderstress[20,21].However,thefunctionsdiscussed
aboveareinferredbasedonexistingobservations,andnodirectevidenceisavailableinvivo
tosupporttheseconclusions.Therefore,furtherstudiesarerequiredtoexplorethespecific
functions.
Atthispoint,numerousofnovelgroup3LEAproteinshavebeenisolatedandidentifiedin
differentspecies.Twenty-threeLEAgeneswereidentifiedfromthePinus.tabuliformisbyGao
etal.(2016),sixofthembelongtothethirdgroupofLEAfamily,whichcanimprovetheresis-
tanceofE.colistrainsagainstheatandsaltstresseswhentheyareexpressedabundantlyinE.
coli[22].FournovelgenesidentifiedfromthesorghumgenomewereclassifiedintoLEA3A
andLEA3Bsubgroupsaccordingtotheconservativespecificmotifsofgroup3LEAproteins
[23].SinceLEAproteinshavebeendiscovered,andsequencefeatureshavebeenanalyzed,
theirspecificfunctionswereexplored.SeveralLEAproteinswereisolated,analyzed,and
detected.LEAproteinshavebeentransferredintodifferentcelltypestoexaminethecontribu-
tionstheymaketobiologicalcellsunderstress.ZmLEA3,locatedinboththecytosoland
nucleus,bindstometalssuchasFe3+,Mn2+,Zn2+,andMn2+underosmoticandoxidative
stress.FurtherexperimentalinvestigationsshowedthatoverexpressionofZmLEA3inyeast
(GS115)andtobacco(Nicotianabenthamiana)improvedthetolerancetobioticstress[7].The
protectiveeffectofOsLEA4onthegrowthofE.coliduringstresswasconfirmed,anditwas
showedthatOsLEA4functionsasaprotectivefactorinprokaryoticcells[24].Furthermore,
HVA1,agroup3LEAgene,wasclonedfrombarleyandexpressedinrice,datashowedthat
transgenicricehadastrongtolerancetosaltstressanddehydration[25,26].Moreover,after
insertingHVA1intothewheatgenome,thetransgenicwheatshowedtheincreaseinbiomass
andwaterusageefficiencyunderhydratedconditions[27].CellsexpressingMg3-GFPwere
foundtoshowreducedcellshrinkageeffectsduringdehydration[28].Overall,numerousstud-
ieshaveshownthatLEAproteinsplayanimportantroleinprotectingplantsfromdamage
causedbydrought,loworhightemperature,orsalinitystress.
Inpreviousreports,thefull-lengthsequenceofWRAB18(accessionno.AB115914)was
obtained,anditscorrespondingaminoacidsequencewasanalyzedusingtheBLASTalgo-
rithm.Itbelongstothe3Asubgroupofgroup3LEAproteinfamilyharboringtheconserved
domain[14,29].WRAB18isahydrophilicproteinwithhighaminoacidquiteidenticalto
WRAB19inwheat[30]andHVA1inbarley[31].IthasbeenreportedthatWRAB18is
inducedbyABAtreatmentandrespondstoABAstress,butitsotherrolesinabioticstress
remainunclear[32].
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WRAB18enhancesstresstoleranceincells
TofurtherexplorethefunctionofWRAB18,wecloneditscDNAsequencefromthewheat
cultivarZhengyin1.Inbothprokaryoticandeukaryoticspecies,invivoandinvitroexperi-
mentswereperformedtoverifytheresponseofWRAB18tootherstressfactors(excluding
ABA-relatedinductivity),andthesubcellularlocalizationandprotectiveeffectstowardsother
enzymeactivityundervariousstresseswereexamined.
Materialsandmethods
Plantmaterialsandstresstreatments
Winterwheat(TriticumaestivumcultivarZhengyin1)wasusedthroughoutthisstudy.Seeds
werepreservedbytheStateKeyLaboratoryofCropStressBiologyforAridAreas,Northwest
A&FUniversity,Yangling,China.Seedswerefirstsoakedindistilledwaterovernightand
treatedwith70%alcoholfor5min,washedsixtimeswithsterilewater,andthenplacedoncul-
turedishesovertwolayersofwettedfilterpaperunder200μEm-2s-1lightwiththelight/dark
cycleconditionsof16/8hat28˚Cfor2weeks.Shootswerewatered,andthedouble-layeredfil-
terpaperwasdrenchedtoavoidanaerobicstresscausedbyafloodingofwater.
TheNicotianabenthamianaseedsusedinthesubcellularlocalizationexperimentwere
obtainedfromCollegeofLifeSciences,NorthwestA&FUniversity,Yangling,Chinaand
sowninsoilmixedwithvermiculiteata1:1ratiofor4weeksinagreenhouseunder16-hpho-
toperiodat25˚Candwerewateredwithdeionizeddistilledwater(ddH O).
2
AmplificationoftheWRAB18geneandexpressionoftheWRAB18
protein
TotalRNAwasextractedfromwheat(TriticumaestivumcultivarZhengyin1)usingTrizol
reagent(Invitrogen).First-strandcDNAsynthesiswasperformedusing500ngtotalRNA,and
oligodTandrandomprimerswereusedaccordingtothemanufacturer’sinstructionsofPri-
meScriptRTreagentKitwithgDNAEraser(TaKaRa).TheopenreadingframeofWRAB18
wasamplifiedfromcDNAusinggene-specificprimers(S1Table)basedonthesequencefrom
NCBI(accessionno.AB115914).TheproductswereclonedintopMD18-Tvectors(TaKaRa)
andsequenced.PhysicochemicalpropertyanalysisofWRAB18wasdonebasedonthefollow-
ingwebsitehttp://web.expasy.org/protparam/.
TheopenreadingframeofWRAB18wasclonedfromcDNAusinggene-specificprimers
withtheappropriaterestrictionenzymedigestionsitesforEcoRIintheforwardprimerand
HindIIIinthereverseprimer(S1Table).TheproductswereligatedintopET28a(Novagen),
andsequencinganalysisverifiedtransformationoftheWRAB18-pET28arecombinantplasmid
intoEscherichiacolistrainBL21(DE3)competentcells(TransGenBiotech).WRAB18was
expressedwithanN-terminus6×Histag,athrombincleavagesiteandaT7tag,sincethestop
codonwasdesignedinthereverseprimertoavoidappendingaC-terminal6×Histag.
Thepositivestrainsweregrownat37˚CinLuria-Bertani(LB)agarmediumsupplemented
withkanamycin(50μg/mL).After3–4hofgrowthuntiltheOD reached0.4–0.5,isopro-
600
pylthio-β-D-galactoside(IPTG)wasaddedtotheculturestoafinalconcentrationof1.0mM
andgrownat37˚Cfor12htoinduceexpressionofthetargetprotein.Afterboilingthebacte-
rialsuspensionat95˚Cfor3mins,thesupernatantwasexaminedby12%SDS-PAGE,and
proteinbandswerestainedwithCoomassieBrilliantBlueR-250[33].
ImmunoblottinganalysiswasperformedtoconfirmtheexpressionofWRAB18.Proteins
weretransferredtopolyvinylidenefluoride(PVDF)microporousmembraneat80mAfor50
min.Afterwashingthreetimesfor10mineachtimewith20mlof1×TBSTbuffer(20mM
Tris-Hcl,200mMNaCl,0.1%Tween20,pH7.5),themembranewasblockedina5%nonfat
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WRAB18enhancesstresstoleranceincells
driedmilksolutionfor1h.Incubationwithanti-His-tagrabbitpolyclonalantibody(CWBIO-
TECH)raisedagainstrecombinantproteinwasperformedovernightat4˚C.Washedthree
timesfor10mineachtimewith20mlof1×TBSTbuffer,themembranewereincubatedfor1
hatroomtemperaturewiththesecondaryantibody(goatanti-rabbitIgG;Beyotime)and
washedasabove,onetimemorefor10minwith20mLof1×TBSbuffer(20mMTris-Hcl,200
mMNaCl,pH7.5).Thenmembranewasstainedwithfreshlyprepareddevelopingbuffer
(200μlNBTsolutionand200μlBCIPsolutionin5mLalkalinephosphatasebuffer)untilsig-
nalswereclearlyvisible.Thereactionwasstoppedbywashingthemembraneindeionizeddis-
tilledwater(ddH O).
2
AssaysforabioticstresstoleranceofE.colitransformants
StresstoleranceassayswereperformedtodetecttheeffectofWRAB18onE.coliviability
underadverseconditions.ThetransformantharboringemptypET28aplasmidwasusedas
control.BL21/WRAB18andBL21/pET28acultures,aswellasIPTGinduction,wereprepared
asdescribedabove.TheconcentrationofinducedculturesinliquidLBwasadjustedtoan
OD of1.0,afterwhich1mLwasinoculatedinto50mLfreshliquidLBwithkanamycin
600
(50μg/mL)andIPTG(1.0mM)in250mLflasks,andtheinitialconcentrationwasadjustedto
thesameOD foreachstress.InordertomeasurethesalinitytoleranceoftransformedE.
600
colicells,theliquidLBwassupplementedwithanadditional500mMNaCl.Ontheother
hand,800mMmannitolwasaddedtotheliquidLBtodetectthedroughttoleranceofE.coli
cells.NaClandmannitolwasdissolvedintheliquidLBmediumwithoutchangingthebacte-
rialculturevolume.Bothofthetwobacterialsuspensionsweredevelopedat37˚Cand200
rpm.Forthethermophylacticexperiments,samplesweretransferredto45˚C,whilecellswere
culturedat28˚Cand200rpmforthecryophylacticassays.RecombinantBL21/WRAB18and
BL21/pET28aintheinducedculturesinliquidLBsupplementedwithkanamycin(50μg/mL)
andIPTG(1.0mM)wereculturedwithoutanystressorat37˚Cand200rpm.Ateachtime,3
mLofculturewereusedtomeasuretheOD usingaspectrophotometer.Eachexperiment
600
wasperformedintriplicate.
Transgenictobaccooverexpressionassay
TheWRAB18genewasamplifiedusingprimerscontainingXbaIandSacIrestrictionenzyme
sites(S1Table).ThecDNAwasthendigestedwithXbaIandSacI,purified,andligatedtoline-
arizedpBI121binaryvector(Clontech)containingthepowerfulCauliflowermosaicvirus
(CaMV)35Spromoterandthekanamycinresistancegene.10ngoftherecombinantplasmid
wasaddedinto50μLAgrobacteriumtumefaciens(GV3101)competentcellsin0.5mLeppen-
dorftubeandmixedbytappingthetube.Themixturewastransferredtoapre-chilled(onice)
electroporationcuvettewith2mmgapsizes.Electroporationcuvettewasputintheholderof
MicroPulserElectroporator(Bio-Rad)adjustingthegenepulserunit“SetVolts”settingto2.5
kVandthe“CAP”settingto25μFD.Theresistancewassetto400Oonthepulsecontroller
unit,andthetimeconstantwas~9msec.Afterbeingincubatedonrollerdrumat28˚Cfor~2
hours,200μLofculturewasplatedonselectivemedium.Positivestrainswereusedtoinfect
tobaccoleaves,whichwerecutintosmallpieces(2×2cm).Weobtainedcallusbytissuecul-
tureonsolidMSmedium.ThetranformantionofNicotianiabenthamianawasperformed
usingtheleafdiscmethod[34].BudswereplacedinMSmediumcontaining100mg/Lkana-
mycintoselectpositiveplants.Aftergrowingfor2months,theyweretransferredtosoiluntil
theseedswereharvested.
Semi-quantitativeRT-PCRwasperformedusing4-week-oldtransgenictobaccoleavesto
identifyoverexpressingtransgenictobaccoplants.Theexpressionofβ-actingenewasusedasa
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WRAB18enhancesstresstoleranceincells
control.Atotalof10leavesfrom10lineswereanalyzedfortheexpressionoftheWRAB18
genefromgenomicDNAandcomparedwithlevelsinWTplants.
Experimentsassessingthegrowthandphysiologicalparametersof
transgenictobacco
Weobservedphenotypicdifferencesbetweentransgenicandwild-type(WT)plants.We
soakedtransgenicandWTtobaccoseedsindistilledwaterfor2daysandgrewthemonspe-
cificMurashigeandSkoog(MS)solidmediumcontaining100mMand200mMmannitol
and100mMand200mMNaClfordroughtandhighsalinitystressconditions,respectively.
Asacontrol,seedswerecultivatedonplatesonMSmediuminatissuecultureroom.Fortem-
peraturestress,plateswereculturedunderlow(18˚C)andhigh(40˚C)temperatures.After14
daysofgrowthunderdifferentconditions,phenotypicdifferenceswereobservedandanalyzed
statistically.Fortheexperimentofgerminationrate,rootlength,andsurvivalrate,thenumber
ofsampleswas100.
Meanwhile,thetransgenicandWTseedsweresowedinsoilfor4weeks;theseedlingswere
treatedwithdifferentstresses(osmoticstress,highsalinity,andlowandhightemperatures)
for48h.Leaveswerecollectedtoperformenzymeactivitymeasurementsofascorbateperoxi-
dase(APX),peroxidase(POD)andsuperoxidedismutase(SOD),andthecontentrationof
malonaldehyde(MDA)wasalsoquantified.[35].
LactateDehydrogenase(LDH)enzymeactivityassay
WRAB18appendedwitha6×HistagwasexpressedbytheE.colisystemandpurifiedusing
His[Ni2+]resin.TheLDHenzyme(EC1.1.1.27,rabbitmusclelactatedehydrogenase)from
Sigma(USA)wasdilutedtoafinalconcentrationof10μg/mLinsodiumphosphatesolution,
pH7.4.Bovineserumalbumin(BSA,Sigma,USA)andsucrosesolution,asacontrol,werepre-
paredat20μg/mLof20μLwithsodiumphosphate(pH7.4),whichwasthesametoWRAB18.
The20μLLDHsolutionwasmixedwiththesamevolumesofBSA,sucrose,andWRAB18
proteinsolution,respectively,thenprovidedtovariousstresstreatments.Thesereactionsys-
temswerethenexposedtohigh(45˚C)andlow(0˚C)temperaturefor30minand12h,respec-
tively.Droughttreatmentwasperformedusingadryer(Vaccubrand,Germany),andsamples
weredehydratedto70%andrehydratedtotheoriginalvolume.Forhighsalinitytreatment,
samplesweretreatedwithreactionbuffercontaining200mMNaClfor2h.LDHinsolutions
withnotreatmentwasusedascontrol.BeforemeasuringtheLDHactivity,thesampleswere
dilutedtoreachanenzymeconcentrationof0.5μg/mL,thenatotalof750μLofreactionmix
containing10mMpyruvicacid,0.2mMNADH,and10mMsodiumphosphate,pH7.4,was
addedtoeachreactionsystemtoensurethattheconcentrationofNADHineachsystemwas
0.13mM.Reactionmixturewasaddedtothe12wellplates,andtheabsorbanceat340nm
wasmeasuredwithSpectraMaxM2MolecularDevicesover3minatdifferenttimeintervals
accordingtothedifferenttreatments.Eachreactionwasrepeatedthreetimes,andtriplicate
datawereanalyzedusingancillarysoftware(SoftMaxPro,MolecularDevices,USA).
Real-timequantitativePCRanalyses
Real-timequantitativePCRwasperformedusingtheSYBRPrimeScript™RT-PCRKit(TaKaRa)
at25μLvolumeontheCFX96TMReal-timesystem(Bio-Rad,USA).
ThecDNAtemplatesforthisexperimentwerepreparedfromdifferenttissues(roots,stems
andleaves)oftwo-leaf-periodwheatseedlings.PrimerswereshowninS1Table,theexpression
ofβ-actingenewasusedascontrol.PCRthermalcyclesweresetat95˚C,30sfor1cycle;
95˚C,5s;60˚C,30sand72˚C,30sfor40cycles;72˚C,10minfor1cycle.Eachreactionwas
PLOSONE|DOI:10.1371/journal.pone.0171340 February16,2017 5/19
WRAB18enhancesstresstoleranceincells
replicatedthreetimesandrelative-foldexpressionwasanalyzedaccordingtotheLivakmethod
[36].
TransientexpressioninprotoplastsofNicotianabenthamiana
Areformedvector,namedpA7GFP,harboringtheCaMV35SpromoterandGFPgenesequence
wasusedinthisstudy[37,38].ThefulllengthofWRAB18ORFsequence,containingtheXbaI
restrictionenzymesiteatthe50endandtheSmaIrestrictionenzymesiteatthe30end,was
amplified(S1Table).ThePCRproductwasdigestedusingthesetworestrictionendonucleases,
andthetargetfragmentsharboringtheGFPgenesequenceatthe50endwereinsertedinto
pA7GFPvectors.Theidentifiedpositiverecombinantvectorsandplastid-localizationmarker
plasmid(pt-rkCD3-999)[39]weretransferredintotheAgrobacteriumtumefaciensGV3101
strainbyelectricshock.Thesetwopositivebacterialstrainswerecultivatedin100mLLBliquid
mediumunderantibioticselection(50mg/Lrifampicin+50mg/Lkanamycin)at28˚Cfor24h
andthencentrifugedat2616g-forcefor5mintocollectcells.Afterresuspensionin5mlAgro-
mix(10mMMgCl ,10mMMES/KOHpH5.6,and150μMacetosyringone),thebacteriawere
2
culturedindarkat28˚Cfor4h.ThesamplesweredilutedtoanOD of1.0,afterwhichthe
600
twosamplesweremixed.Themixturewasextractedusinganeedlelesssyringeandinfiltrated
intothreefullystretchedbladesofplantsof4weeksold.ThestrainswithemptypA7GFPplas-
midswereusedasacontrol.After2-daycultivation,theinfiltratedleavesweredigestedusing
enzymesolutioncontainingcellulaseandmecerozymeR10toobtainprotoplastsaccordingto
thereferredprotocol[40].Thefluorescenceanalysiswasperformedusinganinvertedconfocal
microscope(NikonA1R).TheGFPfusionswereexcitedwitha488nmargonlaseranddetected
usinga505–530nmband-passemissionfilter,mCherryfusionswereexcitedusinga561nm
laseranddetectedusingacustom-made595–620nmband-passemissionfilter.
Statisticalanalysis
DataanalysisweredoneusingGraphPadPrismversion5.0(USA)withat-testorANOVA.
SignificantdifferenceswereindicatedasaP-valuelessthan0.05.
Results
CloningandsequenceanalysisofWrab18
TodeterminethefunctionofWRAB18undervariousabioticstresses,thenucleicacidse-
quencesofWRAB18(GeneBankaccessionno.AB115914)wereclonedfromTriticumaesti-
vum.cultivarZhengyin1.TheWRAB18cDNAcontainedanopenreadingframeof510bp
encodingaproteinof169aminoacidswithapredictedmolecularweightof17.53kDaanda
pIof5.95.TheWRAB18protein(GeneBankaccessionno.BAC80266.1)isclassifiedasa
group3LEAprotein,similartootherknowngroup3LEAproteins.Theaminoacidsequence
ofWRAB18showedsimilarstructuralcharacteristics,alongwiththeconserveddomain.Mul-
tiplesequencealignmentwasperformedusingtheMEGA5.0softwareandthefinalfigurewas
exportedbyESpript3.0(http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi)(S1AFig).Inaddi-
tion,WRAB18wasfoundtoberichinalanine(20.7%),lysine(11.8%),andthreonine(12.4%)
butlackingcysteine,proline,andtryptophan.The3DstructureofthewheatWRAB18protein
waspredictedtoshowthestructuraldomainsofalpha-helixesinfigureS1BwithPHYRE2at
http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index.(S1BFig).WRAB18proteinwas
expressedintheE.coliandexaminedby12%SDS-PAGEstainedwithCoomassieBrilliant
BlueR-250(S2AFig).Immunoblottinganalysiswasperformedtoconfirmtheexpressionof
WRAB18(S2BFig).
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WRAB18enhancesstresstoleranceincells
OverexpressionofWRAB18enhancedthegrowthofE.coliunderstress
ManystudiesusedtheE.coliproteinexpressionsystemtoobtaininformationontheactivities
oftargetproteinsincells,becausethissystemisconvenient,efficient,andsimpletooperate
[41].ToexaminethefunctionofWRAB18inprokaryoticcellsinvivounderdifferentstresses,
E.colicellseitheroverexpressingWRAB18orharbouringanemptyvectorcontrolwascul-
turedunderfivedifferentconditions(37˚C,with500mMmannitoland500mMNaClat
28˚Cand45˚C),respectively.ThebacterialsolutionofBL21/pET28aandBL21/WRAB18was
culturedat37˚Casacontrol.
TheOD wasmeasuredatevery2htogeneratethegrowthcurveforE.coli,whichcould
600
displaythebacterialviabilityvisually.Thegrowthcurvesofbacteriashoweddifferencesunder
thesefourkindsofstresstreatments.AllstressesledtotheinhibitiontothegrowthofE.coli,how-
evertheextentofinhibitionwasquitedifferent.AsshowninFig1,theOD ofBL21/WRAB18
600
washigherthanthatofBL21/pET28aunderthesefourstressconditions,whilethegrowthwas
consistentat37˚C(theoptimalgrowthtemperatureforE.coli)(Fig1A).BL21/WRAB18showed
bettergrowthkineticsthanthoseofcellscarryingtheemptyvectorunderdrought(Fig1B)andat
45˚C(Fig1E).However,superiorgrowthwasobservedunderhighsalinity(Fig1C)andcold
treatment(Fig1D),andthestatusunderhighsalinitywasmoreexcellent.Thesegrowthcurvesof
E.coliindicatedthatoverexpressionofWRAB18increasedthetoleranceofE.colistrainsto
drought,salinity,heat,andcold,especiallytohighsalinityandlowtemperature.
OverexpressionofWRAB18increasedthestresstoleranceof
transgenicplants
Basedonsemi-quantitativeRT-PCR,wechosethreetransgeniclines,OE1(Lane3),OE2
(Lane5),andOE3(Lane7),withthehighestWRAB18geneexpressionforsubsequentcom-
parisons(Fig2A).
Fig1.GrowthcurvesofEscherichiacoliculturestransformedwithWRAB18orcontrolpET28aunderfourabioticstresses.E.coli
strainsgrownunderstandardcultureconditions(A),inmediumsupplementedwith800mMmannitol(B)or500mMNaCl(C),andunder
exposureto28˚C(D)or45˚C(E).TheOD wasmeasuredasanindicatoroftheincreaseindensityoftheliquidcultures.Eachstress
600
assaywasperformedthreetimes,andstatisticallysignificantdifferenceswereanalyzedusingtheStudent’st-test.
doi:10.1371/journal.pone.0171340.g001
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WRAB18enhancesstresstoleranceincells
Fig2.Droughtandsalinitytoleranceinwild-typeversusWrab18-overexpressingN.benthamiana
plants.(A)Identificationoftransgenictobaccoplantsbysemi-quantitativePCR.(B)Phenotypicdiscrepancy
PLOSONE|DOI:10.1371/journal.pone.0171340 February16,2017 8/19
WRAB18enhancesstresstoleranceincells
ofwild-typeandOElinesonMSmediumcontainingdifferentconcentrationsofmannitolandNaCl.(C)
Germinationrateofwild-typeandOElinesonMSundernormalconditions,200mMmannitol,or200mM
NaCl.(D)Rootlengthand(E)survivalrateofwild-typeandOElinesonMSundernormalconditions,200mM
mannitol,or200mMNaCl.Statisticallysignificantdifferenceswereanalyzedusingone-wayANOVA
(*p<0.05or**p<0.01).
doi:10.1371/journal.pone.0171340.g002
ToexploretheeffectsofoverexpressedWRAB18intobacco,weevaluatedthedevelopmen-
talconditionsandgerminationofthesethreetransgeniclines.TheWTandtransgenictobacco
seedswereplantedindifferentareaofMSsolidmedium,eachmediumcontained0,100,or
200mMmannitolrespectivelyandthesameconcentrationgradientofNaClwassetupin
otherMSsolidmediums.TherewasnovisibledifferencebetweenWTseedlingsandtrans-
geniclinesundernotreatmentconditionsandonlyaslightdifferenceunder100mMNaCl;
however,thereweremoredifferencesunder200mMmannitolandNaCltreatment.In200
mMNaClandmannitolMSmedium,thegrowthoftransgenicseedlingswasbetterthanthat
ofWTseedlings;thiswasalsoobservedinplatesgrownunderlowtemperature(18˚C),butthe
morphologyunderhightemperature(40˚C)(Fig3A)wasmuchbetterthanthoseunderthe
othertreatments(Fig2B).ThegerminationrateonMSplatesweresimilar,upto90%,while
statisticalanalysisofthegerminationraterevealedthat~32%ofWTseedsand~53%oftrans-
genicseedsgerminatedunder200mMmannitolafter14days.GerminationratesofOElines
werenearlytwiceasmuchasWTplantsunder200mMNaCltreatment(Fig2C).However,
thedifferencedecreasedto~4%at40˚Cbutreached~28%at18˚C(Fig3B).
Thetransgenicplantsalsoshowedstresstoleranceintermsoftherootlengthandsurvival
rate.TherootgrowthoftheOEtransgenicplantswashigherthanthatoftheWT,butthephe-
notype,germinationrate,andsurvivalrateweresimilar.Therootlengthat40˚Cwaslonger
thanthatunderothertreatments(Figs2D,3Cand3D).Meanwhile,exceptthatthesurvival
rateofthetransgeniclineswassimilartothatoftheWTat40˚C(Fig3E),survivalrateoftrans-
geniclineswashigherthanthatoftheWTunderotherabioticstresses(Fig2E).
WRAB18overexpressionenhancedoxidativestresstoleranceinplants
Whenplantssufferfromstress,theyoftenshowanacceleratedageingprocess.Oneobvious
observationisadecreaseintheactivitiesofenzymesinvolvedinthedefensesystem,suchas
SOD,APX,andPOD[35,42].ToknowthedifferencesinenzymaticactivitiesbetweenWRAB18
overexpressionplantsandWTlines,theenzymaticactivitiesofAPX,PODandSOD,andthe
contentofMDAweredetectedunderbothnormalandstressconditions(Fig4).Accordingto
thedatasetsinFig4,alittlesignificantdifferenceswereobservedbetweenWTandtransgenic
linesbeforetreatments,whileallenzymaticactivitiesincreasedsignificantlyafterbeingtreated.
However,thechangeswerequitediverse.Thetransgeniclinesshowedasignificantincreasein
APXactivitiesunderstressconditions.TheactivitiesofPODandSODinthetransgenictobacco
lineswereobviouslyhigherthanthoseintheWTlines.MDAcontentincellisanimportant
parameterforevaluatingtheoxidationlevelofliposomesandthedegreeofmembranedamage
[43].WhiletheMDAcontentintheWTplantsweremuchhighercomparedtothetransgenic
plantsafterstresstreatments.Overall,thesedatashowedthatWRAB18overexpressionresulted
inincreasedantioxidantactivityandstabilityunderstress.
Tissue-specifictranscriptaccumulationandplastidlocalizationof
WRAB18
Real-timequantitativePCRwasconductedtoinvestigatethetissue-specificexpressionpat-
ternsofWRAB18intwo-leaf-periodwheatseedlings.TherelativeexpressionofWRAB18was
PLOSONE|DOI:10.1371/journal.pone.0171340 February16,2017 9/19
WRAB18enhancesstresstoleranceincells
PLOSONE|DOI:10.1371/journal.pone.0171340 February16,2017 10/19