Transgenic Alfalfa Plants Expressing the Sweetpotato Orange Gene Exhibit Enhanced Abiotic

17 Pages·2015·3.98 MB·English

RESEARCHARTICLE
Transgenic Alfalfa Plants Expressing the
Sweetpotato Orange Gene Exhibit Enhanced
Abiotic Stress Tolerance
ZhiWang1,2,3,QingboKe1,6,MyoungDuckKim1,SunHaKim1,ChangYoonJi1,6,Jae
CheolJeong1,6,Haeng-SoonLee1,6,WooSungPark5,Mi-JeongAhn5,HongbingLi3,
BingchengXu3,XipingDeng3,Sang-HoonLee4,YongPyoLim2,Sang-SooKwak1,6*
1 PlantSystemsEngineeringResearchCenter,KoreaResearchInstituteofBioscienceandBiotechnology
(KRIBB),Daejeon,305–4432,Korea,2 DepartmentofHorticulture,ChungnamNationalUniversity,Daejeon,
Korea,3 StateKeyLaboratoryofSoilErosionandDrylandFarmingontheLoessPlateau,InstituteofSoil
andWaterConservation,ChineseAcademyofScienceandMinistryofWaterResources,NorthwestA&F
University,Yangling,Shaanxi,P.R.China,4 GrasslandandForagesDivision,NationalInstituteofAnimal
Science,RuralDevelopmentAdministration,Cheonan,Korea,5 CollegeofPharmacyandResearch
InstituteofPharmaceuticalSciences,GyeongsangNationalUniversity,Jinju,Korea,6 DepartmentofGreen
ChemistryandEnvironmentalBiotechnology,KoreaUniversityofScience&Technology,Daejeon,Korea
* [email protected]
OPENACCESS
Abstract
Citation:WangZ,KeQ,KimMD,KimSH,JiCY,
JeongJC,etal.(2015)TransgenicAlfalfaPlants Alfalfa(MedicagosativaL.),aperennialforagecropwithhighnutritionalcontent,iswidely
ExpressingtheSweetpotatoOrangeGeneExhibit
distributedinvariousenvironmentsworldwide.Werecentlydemonstratedthatthesweetpo-
EnhancedAbioticStressTolerance.PLoSONE10
(5):e0126050.doi:10.1371/journal.pone.0126050 tatoOrangegene(IbOr)isinvolvedinincreasingcarotenoidaccumulationandenhancing
resistancetomultipleabioticstresses.Inthisstudy,inanefforttoimprovethenutritional
AcademicEditor:HiroshiEzura,Universityof
Tsukuba,JAPAN qualityandenvironmentalstresstoleranceofalfalfa,wetransferredtheIbOrgeneinto
alfalfa(cv.XinjiangDaye)underthecontrolofanoxidativestress-inducibleperoxidase
Received:January22,2015
(SWPA2)promoterthroughAgrobacteriumtumefaciens-mediatedtransformation.Among
Accepted:March28,2015
the11transgenicalfalfalines(referredtoasSORplants),threelines(SOR2,SOR3,and
Published:May6,2015 SOR8)selectedbasedontheirIbOrtranscriptlevelswereexaminedfortheirtoleranceto
Copyright:©2015Wangetal.Thisisanopen methylviologen(MV)-inducedoxidativestressinaleafdiscassay.TheSORplantsexhib-
accessarticledistributedunderthetermsofthe itedlessdamageinresponsetoMV-mediatedoxidativestressandsaltstressthannon-
CreativeCommonsAttributionLicense,whichpermits
transgenicplants.TheSORplantsalsoexhibitedenhancedtolerancetodroughtstress,
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare alongwithhighertotalcarotenoidlevels.TheresultssuggestthatSORalfalfaplantswould
credited. beusefulasforagecropswithimprovednutritionalvalueandincreasedtolerancetomultiple
DataAvailabilityStatement:Allrelevantdataare abioticstresses,whichwouldenhancethedevelopmentofsustainableagricultureon
withinthepaper. marginallands.
Funding:Thisworkwassupportedbygrantsfrom
theKRIBBInitiativeprogram,theKorea-China
InternationalCollaborationProject,NationalResearch
FoundationofKorea(NRF),theNationalNatural
ScienceFoundationofChina(No.51479189),China
andthe111projectoftheMinistryofEducation, Introduction
China(No.B12007).Thefundershadnoroleinstudy
Soilsalinizationanddesertificationaretwomajorenvironmentalproblemsthatgreatlylimit
design,datacollectionandanalysis,decisionto
publish,orpreparationofthemanuscript. agriculturalproductionworldwide[1].Areasaffectedbysaltanddroughtstressaccountfor
PLOSONE|DOI:10.1371/journal.pone.0126050 May6,2015 1/17
AlfalfaPlantsExpressingIbOrwithEnhancedAbioticStressTolerance
CompetingInterests:Theauthorshavedeclared almosthalfoftheworld’sagriculturallands[2].Formostcrops,highsalinityandwaterdeficit
thatnocompetinginterestsexist. severelydecreasecropgrowth,yield,andqualityduetoincreasedosmoticandoxidativestress.
Duetothedramaticincreaseinthehumanpopulation,togetherwithincreasinglyseriousenvi-
ronmentalproblems,itwillbedifficultforworldagriculturetomeettheworld’sfoodandener-
gyrequirementsinthefuture[3].Therefore,itisnecessarytodevelopnovelcropcultivarswith
excellentenvironmentalstresstoleranceforuseinsustainableagriculture.
Alfalfa(MedicagosativaL.),oneofthemostimportantlegumeforagecrops,iswidelyculti-
vatedthroughouttheworldduetoitssignificanteconomicvalueandexcellentagricultural
traits[4].Asoneofthehighest-yieldingforagecrops,alfalfahasoutstandingnutritionalquali-
ty,withhighlevelsofprotein,minerals,andvitaminsaswellaswell-balancedaminoacids.
Moreover,thenitrogen-fixingabilityofalfalfareducestheamountofenergyrequiredforits
productionandimprovessoilstructure.Inaddition,alfalfahasdeeperrootsthanmostcrops.
Thisdeep,vigorousrootsystemallowsalfalfatoadapttovariousenvironmentalconditions
andincreasesitscapacitytopreventsoilerosion.Thus,alfalfaisconsideredtobeapotential
foragecropforuseinareassubjectedtoenvironmentalstresssuchasdroughtandhighsalinity
[5].However,theperniciouseffectsofabioticstress(suchassaltanddroughtstress)stillrepre-
sentmajorlimitstoalfalfaproduction.Toimprovetheadaptabilityofalfalfatotheseenviron-
mentalstresses,manystudiesfocusedonmodifyingvariousaspectsofalfalfaplants,suchas
theactivationofcascadesofmolecularnetworksinvolvedinstressresponses[6].
Carotenoids,aclassofcolorfulpigmentsandimportantnutrients,arebiosynthesizedinthe
plastids(suchaschloroplastsandchromoplasts)ofplants[7,8].Thesemultifunctionalmetab-
olites,whichhavepowerfulantioxidantactivity,playcriticalrolesinlightcollection,protection
ofphotosyntheticsystems,andabscisicacid(ABA)synthesis[9,10,11].Theaccumulationof
carotenoidsintobaccoandArabidopsisplantshasresultedinenhancedtolerancetoabiotic
stressesincludingUVirradiation,highlight,andsaltstress[9,12,13].
TheOrangegene(Or),whichisinvolvedincarotenoidaccumulation,sharesahighlevelof
homologyinmanycropssuchascauliflower,rice,tomato,andArabidopsisthaliana[14,15].
IntroductionofthecauliflowerOrgeneinducedtheformationofchromoplastsandincreased
thecarotenoidcontentsintransgenicpotatotubers[16].Transgenicricecallusexpressing
AtOraccumulatehigherlevelsofcarotenoidsthanthecontrol[15].Wepreviouslydetermined
thattheOrgene(IbOr)fromorange-fleshedsweetpotatoisnotonlyinvolvedincarotenoidac-
cumulation,butitalsofunctionsinresponsetomultipleabioticstresses[11].Theexpressionof
IbOrrapidlyincreasesafterNaCl,PEG,andH O treatment.Transgenicsweetpotatocalli
2 2
overexpressingIbOrexhibitenhancedtolerancetosaltstress,withincreasedcarotenoidcon-
tentsandantioxidantactivity.Thus,theOrgenemaybeusefulfordevelopingvaluablecrops
withenhancedtolerancetomultipleenvironmentalstressesandincreasednutrientcontents.
Whendesigninganefficientexpressionsystem,itisimportanttoselecttheproperpromoter
[17,18].Wepreviouslyisolatedandcharacterizedthestrong,oxidativestress-induciblesweet-
potatoperoxidaseanionic2(SWPA2)promoter[19].TheSWPA2promoterinduceshigher
levelsofexogenousgeneexpressionthanthe35SpromoterfromtheCauliflowermosaicvirus
(CaMV35Spromoter)inresponsetovariousstresstreatments,anditwassuccessfullyapplied
toseveraltransgenicplantssuchaspoplar,potato,andrice[20–22].TheSWPA2promotercan
beworkedatmostorgansincludingleafandrootinresponsetooxidativestress.Therefore,the
stress-inducibleSWPA2promoterishighlysuitableforgeneratingtransgenicplantswithen-
hancedtolerancetoenvironmentalstresses.
Inthisstudy,toimprovethenutritionalqualityandenvironmentalstresstoleranceofalfal-
fa,wegeneratedtransgenicplantsexpressingIbOrunderthecontroloftheSWPA2promoter
(referredtoasSORplants)throughAgrobacterium-mediatedtransformation.TheSORplants
exhibitedenhancedtolerancetomultipleabioticstresses,alongwithincreasedaccumulationof
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AlfalfaPlantsExpressingIbOrwithEnhancedAbioticStressTolerance
carotenoids.TheresultsindicatethatSORalfalfaplantsmightbeusefulasforagecropsforsus-
tainablecultivationonmarginallands.
MaterialsandMethods
Constructionofplantexpressionvector
IbOr-InscDNAwasutilizedtoconstructtheexpressionvectorusedinthisstudyduetoits
strongabilitytoincreasecarotenoidaccumulationandsalttoleranceinplants[11].Tocon-
structtheplantexpressionvector,achimericgenecassette,containingIbOr-Insandthenos
terminator(NOSt)underthecontroloftheoxidativestress-inducibleSWPA2promoter[19]
withEcoRIandHindIIIsites,wasconstructedandligatedintothecorrespondingsitesofthe
pCAMBIA2300binaryvector,whichharborsthenptⅡgeneasaselectablemarker.Theresul-
tantvector,pSIbOr-Ins,wasmobilizedintoAgrobacteriumtumefaciensstrainEHA105foral-
falfatransformationviaafreeze–thawmethod[23].
Plantmaterialsandtransformation
MedicagosativaL.seeds(cv.XinjiangDaye)wereprovidedbyProf.BoZhangofXinjiangAg-
ricultureUniversity,China[24].Theseedsweresurface-sterilizedwith0.5%mercuricchloride
solutionfor30min,thoroughlyrinsed7–8timeswithdistilledwater,andgerminatedonMur-
ashigeandSkoog(MS)medium(pH5.7)[25]withhalf-strengthvitaminsandsaltundera16/
8hlight/darkcycle,withalightintensityof150μmolm-2s-1andarelativehumidityof65%at
25°C.Fivedaysaftergermination,thehypocotylsofseedlingswereutilizedforplanttransfor-
mationwithA.tumefacienscontainingtheSWPA2promoter-IbOrcassette.
ThetransformedcalliwereinducedfrominfectedhypocotylsandselectedonSchenkand
Hildebrandt(SH)medium[26]containing2.0mgl-12,4-dichlorophenoxyaceticacid(2,4-D),
0.2mgl-1kinetin,250mgl-1cefotaxime,and50mgl-1kanamycin.Shootswereregenerated
fromthecallifollowingtransfertoMSmediumcontaining1.0mgl-1benzylaminopurine
(BAP),0.3mgl-11-naphthylaceticacid(NAA),250mgl-1cefotaxime,and50mgl-1kanamy-
cin.Throughouttheexperiments,theculturesweremaintainedinacultureroomat25±2°C
undera16hphotoperiod.RegeneratedshootsweretransferredtofullMSmediumforrooting.
Therootedplantletsweretransferredtosoilafter1weekofacclimationinpotsinthe
growthchamber.
PCRanalysis
GenomicDNAwasextractedfromtheleavesofalfalfaplantsaspreviouslydescribed[27].
PCRwasconductedwithpurifiedgenomicDNAinaPCRpremix(Cat.no.K-2012,Bioneer,
Korea)usingaspecificprimersetdesignedbasedonthesequenceoftheIbOrgeneandpartof
theSWPA2promotertoconfirmtheintegrationofIbOr(Table1).Theamplificationreactions
consistedof94°Cfor5min(1cycle),followedby30cycles(94°Cfor45s,60°Cfor45sand
72°Cfor1min)andafinalextensioncycleof7minat72°C.ThePCRproductswereseparated
ona1%agarosegel,stainedwithethidiumbromide,andvisualizedunderUVlight.Allsubse-
quentexperimentswereconductedonclonedplantsfromtheT -generationoftransgenic
0
plants.
Geneexpressionanalysis
ToactivatetheSWPA2promoterandinduceIbOrexpression,similarlysizedplantsgrownin
soilfor1monthweretreatedwith5μMMV,250mMNaClsolution,orwithholdingwaterfor
1week.Leaves(fromthesameposition)ofalfalfaplantswereutilizedforRNAextraction.
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AlfalfaPlantsExpressingIbOrwithEnhancedAbioticStressTolerance
Table1. Gene-specificprimersusedforgenomicandRT-PCRanalysis.
Genename Primersequences(5’–3’)
SWPA2-3'_F Forward:GAAACCTTAGAGGCAATTCATGCA
IbOr_R Reverse:CGTGGGTCATGCTCGCTTGCCATAGCCATC
IbOr-RT Forward:ATCTCCATGGAAGGCTCAAATC
Reverse:CGACGGATGAAGAAAAGGAG
MsPSY(KJ955630) Forward:GCACCTGAATCCAAGGCTTC
Reverse:GCATCTTCTCCAACATCTCTGA
MsCHY-β(KJ955631) Forward:ACGGTGTTTGGGATTGCCTACA
Reverse:TTGGTGAGCAGCAGCAACTCTT
MsLCY-β(KJ955632) Forward:GCATTGAAGAAGATGAGCAGTG
Reverse:ACCACCGATTCCAACAACTCT
MsNCED(KJ955633) Forward:AGCCACACTATGTAGCCGTTA
Reverse:TGACCAATCCAATGCCGTTAG
MsActin(JQ028730.1) Forward:TCCTAGGGCTGTGTTTCCAAGT
Reverse:TGGGTGCTCTTCAGGAGCAA
doi:10.1371/journal.pone.0126050.t001
TotalRNAwasextractedusingaGeneAllRibospinPlantkit(GeneAll,Seoul,Korea)andtreat-
edextensivelywithRNase-freeDNaseItoremoveanycontaminatinggenomicDNA.For
semi-quantitativeandquantitativeexpressionanalysisofrelatedgenessuchasIbOrandthe
Actingeneinalfalfaplants,totalRNA(2μg)wasusedtogeneratefirst-strandcDNAusing
Moloneymurineleukemiavirus(MMLV)reversetranscriptasefromanRT-PCRkit(TOP-
scriptRTDryMIX)inaccordancewiththemanufacturer’sinstructions.Forsemi-quantitative
RT-PCRanalysis,thePCRconditionswereasfollows:aninitialdenaturationstepat94°Cfor
5min(1cycle),followedby27cycles(94°Cfor45s,60°Cfor45s,and72°Cfor30s)anda
finalextensioncycleof7minat72°C.ThePCRproductswereseparatedandvisualizedasde-
scribedforgenomicPCRanalysis.Quantitativereal-timePCR(q-RT-PCR)wasperformedina
fluorometricthermalcycler(DNAEngineOpticon2,MJResearch,USA)usingEver-Green20
asafluorescencedyeaccordingtothemanufacturer’sinstructions.ThealfalfaActingenewas
usedasaninternalcontrolforanalysisofgeneexpression.Transcriptlevelswerecalculated
relativetothecontrols.Datarepresentthemeansandstandarderrorsofthreebiological
replicates.TheexpressionlevelsoftheIbOr,Actin,andalfalfacarotenogenicgenes(MsPSY,
MsCHY-β,MsLCY-β,andMsNCED)wereanalyzedbyquantitativereal-timePCRusingthe
gene-specificprimerslistedinTable1.ThePCRproductofeachalfalfacarotenogenicgene
wasconfirmedbysequencinganalysisandfurtheranalyzedthroughalignmentwithother
knownplantcarotenogenicgenes.
Methylviologen(MV)treatmentandionleakageanalysis
Theoxidativestresstoleranceassaywasperformedaspreviouslydescribed[28].Fourleaves
collectedfromthesamepositiononfouralfalfaplantswerefloatedinasolutioncontaining
0.4%(w/v)sorbitoland5μMMV,placedinthedarkfor12htoallowdiffusionofMVintothe
leaves,andsubjectedtocontinuouslight(150μmolm-2s-1)treatmentat25°C.Thelossofcyto-
plasmicsolutesfollowingMVtreatment,basedontheelectricalconductanceofthesolution,
wasmeasuredwithanionconductivitymeter(model455C,IstekCo.,Seoul,Korea)overa
timeperiodrangingfrom0to36handcomparedwiththetotalconductivityofthesolution
followingtissuedestruction.Theextentofcellulardamagewasquantifiedbyionleakage,
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AlfalfaPlantsExpressingIbOrwithEnhancedAbioticStressTolerance
whichisanindicatorofmembranedisruption.Themeasurementswereconductedintriplicate
withthreeindependentplantsofeachline.
Saltanddroughtstresstreatment
One-month-oldalfalfaplantsgrowninagrowthchamberat25°C(60%relativehumidity,
16/8h[light/dark]photoperiodwithlightsuppliedatanintensityof150μmolm-2s-1)were
utilizedfortheabioticstresstoleranceassay.
Forsaltstresstreatment,alfalfaplantswereirrigatedwitha250mMNaClsolutionevery
2daysfor1week.Fordehydrationtreatment,theplantswereirrigatedwithsimilarquantities
ofwaterthroughtraysplacedunderneaththepotsfor1week,followedbywithholdingwater
for7days.Theplantswerethenwateredandallowedtorecoverfromthedroughtconditions.
Analysisofrelativewatercontents
Therelativewatercontents(RWC)weremeasuredasdescribedpreviously[21,29].Thefollow-
ingformulawasutilized:RWC(%)=[(FW-DW)/(TW-DW)]×100,inwhich
FW=immediateweightoffreshlycollectedleaves,TW=turgidweightofleavesafterincuba-
tioninwaterfor6hat20°Cinthelight,andDW=dryweightofthesameleavesafterdrying
at80°Cfor48h.RWCwasmeasuredusingthefourthfullyexpandedleaffromtheshoot
apicalmeristem.
Analysisofchlorophyllcontents
Chlorophyllcontentsweremeasuredwithaportablechlorophyllmeter(SPAD-502,Konica
Minolta,Japan)fromthefifthintact,fullyexpandedleaves(countingfromtheshootapical
meristem)ofindividualplants.Totalchlorophyllcontentsafterstresstreatmentwerecom-
paredwiththoseundernormalconditions.
Analysisoflipidperoxidation
Lipidperoxidationwasestimatedbymeasuringthemalondialdehyde(MDA)contentsusinga
modifiedthiobarbituricacid(TBA)method[30,31].Approximately0.1gofleaftissuewas
groundin10mlof10%trichloroaceticacid(TCA)usingamortarandpestle.Thehomogenate
wascentrifugedat10,000rpmfor20min.Thereactionmixture(containing2mlofextract
and2mlofTBA)washeatedat100°Cfor30min,quicklycooledonice,andcentrifugedagain
at10,000rpmfor20min.Theabsorbanceat450,532,and600nmwasdeterminedusinganul-
travioletspectrophotometer(Spectronic,Genesys2,USA).Themeasurementswereconducted
intriplicatewiththreeindependentplantsofeachline.
AnalysisofH O contents
2 2
FortheH O assay,alfalfaleaves(collectedfromthesameposition)wereincubatedina
2 2
1mgml-1solutionof3,3-diaminobenzidine(DAB)-HCl(pH3.8)for6hat25°Cundercontin-
uouslightaccordingtothemethodsdescribedpreviously[10,32,33].Thechlorophyllwasre-
movedviaincubationat80°Cfor2hin80%ethanol.
Analysisofprolinecontents
Thefreeprolinecontentsofalfalfaplantsweremeasuredspectrophotometricallyaspreviously
described[34,35].Leaftissueswerehomogenizedin1.5mlofaqueoussulfosalicylicacid(3%),
andtheresiduewasremovedbycentrifugationat12,000gfor10min.Onemilliliterofthesu-
pernatantwith1mlofacid-ninhydrinand1mlofglacialaceticacidwasboiledinawaterbath
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AlfalfaPlantsExpressingIbOrwithEnhancedAbioticStressTolerance
at100°Cfor1h.Aftercoolingthereactionmixture,2mloftoluenewasadded,andthemixture
wasvortexedvigorouslyandincubatedatroomtemperaturefor30minuntilitseparatedinto
twophases.Theupperphase(containingproline)wasmeasuredwithanultravioletspectro-
photometer(Spectronic,Genesys2,USA)at520nmusingtolueneastheblank.Theproline
concentrationwasquantifiedbasedonastandardcurveusingD-proline.
Analysisofcarotenoidcontents
Carotenoidswereextractedfromleavesof1-month-oldalfalfaplantsandanalyzedusingan
Agilent1100HPLC(high-performanceliquidchromatography)system(Hewlett-Packard,
PaloAlto,CA,USA)aspreviouslydescribed[11].Allextractionprocedureswereperformed
undersubduedlighttoavoidpigmentdegradationandloss.Twentymicrolitersofstandardor
samplewasinjecteddirectlyontoaYMCC carotenoidcolumn(3μm,4.6×250mm)with
18
solventA(MeOH-tert-butylmethylether[MTBE]-H O[81:15:4])andsolventB(MeOH-MT-
2
BE-H O[6:90:4])usingastepgradientelutionof100%solventAforthefirst15min,followed
2
by100%solventAto100%solventBoverthenext35min.Aconditioningphase(50–60min)
servedtoreturnthecolumntoitsinitialstate.Theflowratewas0.7ml/min,andthecolumn
temperaturewas30°C.Theeluentwasdetectedat450nmusingaUV-visibledetector.The
HPLC-DADsystemwasoperatedviachemstationsoftware(Hewlett-Packard).Carotenoids
werequantifiedusinganexternalcalibrationmethod.Carotenoidstandardswerepurchased
fromCaroteNature(Lupsingen,Switzerland).Themeasurementsforeachlinewereconducted
induplicatewithfourbiologicalrepeats.
Statisticalanalysis
Datawerestatisticallyanalyzedbyone-wayANOVA.Subsequentmultiplecomparisonswere
examinedbasedontheleastsignificantdifference(LSD)test.Allstatisticalanalyseswerecar-
riedoutusingtheStatisticalPackageforSocialSciences(SPSS12);statisticalsignificancewas
setat(cid:1)P<0.05and(cid:1)(cid:1)P<0.01.
Results
GenerationoftransgenicIbOralfalfaplants
TransgenicalfalfaplantsexpressingIbOrunderthecontroloftheoxidativestress-inducible
SWPA2promoter(referredtoasSORplants)weresuccessfullygeneratedviaAgrobacterium-
mediatedtransformation(Fig1A).Elevenkan-resistantalfalfalinescontainingtheIbOrgene
wereobtainedandinitiallyverifiedviagenomicDNAPCRanalysisusingaspecificprimerset
(Table1).AsshowninFig1B,therecombinantIbOrgenehadbeenintegratedintothege-
nomesofall11independenttransgenicalfalfalines,whilenointegrationwasdetectedinnon-
transgenic(NT)plants.Regeneratedalfalfaplantsgrowninagrowthchamberfor1month
werethenanalyzedbysemi-quantitativereversetranscriptionpolymerasechainreaction
(RT-PCR)usingMV-treatedleafdiscs.TheinducedexpressionofIbOrwasdetectedinall11
transgeniclines,butnotinNTplants.Amongthe11SORlines,IbOrtranscriptlevelswere
highestinlinesSOR2,SOR3,andSOR8(Fig1C).Theselineswere,therefore,selectedfor
furtheranalysis.
EnhancedtolerancetoMV-mediatedoxidativestressinSORalfalfa
ToinvestigatetheMV-mediated-oxidativestresstoleranceoftheSORplants,leaves(fromthe
sameposition)of1-month-oldSORandNTplantsweretreatedwith5μMMVsolutionfor
variousperiodsoftime(0,12,24,and36h).MVisanon-selectiveherbicidethatinducesthe
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AlfalfaPlantsExpressingIbOrwithEnhancedAbioticStressTolerance
Fig1.DevelopmentoftransgenicalfalfaexpressingIbOrunderthecontroloftheSWPA2promoter
(SORplants).(A)Diagramoftheoxidativestress-inducibleSWPA2promoter:IbOrconstructusedforalfalfa
transformation.Verticalbarshowstheprimerset(A2pro::IbOr)usedforgenomicPCRanalysis.(B)Genomic
DNAPCRanalysisusingtheA2pro::IbOrprimerset.Numbers(1–11)representindependenttransgenic
lines.M,sizemarkers;NT,non-transgenicplant;PC,positivecontrol.(C)RT-PCRanalysisof11lines
expressingstableIbOrgeneintegrationintransgenicplantsfollowing2hof5μMMVtreatment.
doi:10.1371/journal.pone.0126050.g001
productionofmassiveburstsofreactiveoxygenspecies(ROS)inplants,whichdisruptsmem-
braneintegrity,leadingtocelldeath.At24hfollowingMVtreatment,seriousvisibledamage
wasdetectedinNTleaves,whereasSORleafdiscsexhibitedonlypartialnecrosis(Fig2A).We
quantifiedthedegreeofcellulardamagebasedonsoluteionleakage,arecognizedindicatorof
membranestabilityagainstoxidativestress[36].ThetransgeniclinesSOR2,SOR3,andSOR8
exhibitedsignificantlylowerlevelsofionleakage(32%,22.8%,and31.7%,respectively)than
NTplants(68.2%)at24hunderMVtreatment(Fig2B).SORplantsdisplayedbettermem-
branestabilitythanNTplantsunderMV-mediatedoxidativestress.Wethenperformedq-
RT-PCRtoanalyzeIbOrexpressioninleavestreatedwithMVfor12h.TheIbOrtranscriptlev-
elsweresignificantlyhigherintransgeniclinesSOR2,SOR3,andSOR8thaninNTplants
underbothnormalandMV-treatmentconditions(Fig2C).TheSORplantsexhibitedhigher
IbOrexpressionafterstresstreatmentthanundernormalconditions.Theseresultsindicate
thattheexpressionofIbOrincreasedthetoleranceofSORalfalfaplantstoMV-mediated
oxidativestress.
IncreasedtolerancetosaltstressinSORalfalfa
ToanalyzethesaltstresstoleranceoftheSORalfalfaplants,4-week-oldNTandSORplants
wereirrigatedwith250mMNaClsolutionsfor7days.Noobviousdifferenceswereobserved
betweentheNTandSORlinesundernormalconditions.After4daysofhighsalttreatment,
thegrowthofNTplantswasseverelyinhibitedcomparedwiththeSORlines.AllthreeSOR
linesgrewwell,withonlyafewslightlyyellowleavesobserved,whereasmostleavesofNT
plantsshowedseverewiltingandchlorosis.Atday7ofNaCltreatment,theSORlinesexhibited
lessdamagethantheNTplants,whichwerealmostdead(Fig3A).
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AlfalfaPlantsExpressingIbOrwithEnhancedAbioticStressTolerance
Fig2.EffectsofMV-mediatedoxidativestressonleavesofSORandNTplants.(A)Visibledamagein
leavesafter5μMMVtreatmentfor24h.(B)Ionleakagewasmeasuredafter0,12,24,and36hofMV
treatment.Percentageofrelativemembranepermeabilitywascalculatedusing100%torepresentthevalues
obtainedafterautoclaving.(C)IbOrtranscriptlevelsafter12hof5μMMVtreatment.Theexpressionlevelsof
IbOrwerenormalizedtothatofthealfalfaActingeneastheinternalcontrol.Dataareexpressedasthe
mean±SDofthreeindependentbiologicalreplicates.Barslabeledwithasterisksshowsignificantdifferences
fromthatofNTat*P<0.05or**P<0.01byt-test.
doi:10.1371/journal.pone.0126050.g002
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AlfalfaPlantsExpressingIbOrwithEnhancedAbioticStressTolerance
Fig3.EnhancedtolerancetohighsalinityinSORplants.(A)Plantgrowthundernormalconditions(upper
panel)andsaltstress(250mMNaCl)for4days(middlepanel)and7days(lowerpanel).(B)Relative
transcriptlevelsofIbOrinleaves.(C)Relativechlorophyllcontentsofalfalfaplantsafter3daysofsalt
treatment.(D)MDAcontentsinleavesafter3daysofsalttreatment.(E)Prolinecontentsofalfalfaplantsafter
3daysofsalttreatment.(F)DABstainingforH O accumulationinthethirddetachedleavesafter3daysof
2 2
250mMNaCltreatment.Thevaluesrepresentthemean±SDofthreeindependentreplicates.Asterisks
indicateasignificantdifferencefromthatofNTat*P<0.05or**P<0.01byt-test.
doi:10.1371/journal.pone.0126050.g003
ToconfirmthelevelsofIbOrtranscriptsaftersaltstresstreatment,leavesofNTandSOR
plantssubjectedtoNaCltreatmentfor2dayswereexaminedbyq-RT-PCR.Underbothnon-
stressedandstressedconditions,noexpressionofIbOrwasdetectedintheNTplants,whereas
theexpressionofIbOrinSORplantswasstronglyinducedundersaltstressversusnormalcon-
ditions(Fig3B).
Wemeasuredthechlorophyllcontentsinthecentralregionsofthefifthleaves(fromthe
shootapicalmeristem)ofbothNTandSORplantsintheabsenceorpresenceofsalttreatment.
TherewasnosignificantdifferenceinchlorophyllcontentsbetweenNTandSORplantsbefore
saltstresstreatment.After3daysofhighsalttreatment,thechlorophyllcontentsofallSOR
andNTplantswerereduced;however,thethreeSORlinesmaintainedrelativelyhighlevelsof
chlorophyll(21–25%reduction),whileadramaticdecreaseinchlorophylllevels(34–46%)was
detectedintheNTplants(Fig3C).MDA,anaturallyoccurringproductoflipidperoxidation
causedbyacceleratedROSproduction[37],isanimportantindicatorofthedegreeofcell
membranedamageunderstressconditions[38].Undernormalconditions,theMDAcontents
ofSOR3weresimilartothoseofNTplants,whileSOR2andSOR3hadslightlylowerlevelsof
MDA.However,whentreatedwith250mMNaClfor3days,higherlevelsofMDAwerede-
tectedinNTplantsthanintheSORlines(Fig3D).Thedegreeofcellmembranedamagewas
greaterinNTplantsthaninSORplantsundersaltstressconditions.Theseresultsindicatethat
thepresenceoftheIbOrgenecaninhibitoreliminatetheaccumulationofROSinthecell.
Moreover,proline,animportantcompatibleosmolyte[39],playsacriticalroleinplantadapta-
tiontoenvironmentalstress.WethereforemeasuredtheprolinecontentsofNTandSOR
PLOSONE|DOI:10.1371/journal.pone.0126050 May6,2015 9/17
AlfalfaPlantsExpressingIbOrwithEnhancedAbioticStressTolerance
Fig4.EnhancedtolerancetodroughtstressinSORplants.(A)Phenotypesof1-month-oldNTandSOR
plantsbeforetreatment(upperpanel),afterwithholdingwaterfor4days(middlepanel),andrecovered
phenotypeafterre-wateringfor7days(lowerpanel).(B)TranscriptlevelsofIbOrafterwithholdingwaterfor2
days.(C)RWC(%)inleavesafter3daysofwaterwithholding.(D)MDAcontentsinleavesafter3daysof
droughttreatment.(E)Prolinecontentsinleavesafter3daysofdroughttreatment.(F)DABstainingforH O
2 2
accumulationinthethirddetachedleavesofalfalfaplantsafter3daysofdroughttreatment.Valuesrepresent
themean±SDofthreeindependentreplicates.AsterisksindicateasignificantdifferencefromthatofNTat*
P<0.05or**P<0.01byt-test.
doi:10.1371/journal.pone.0126050.g004
plants(Fig3E).Undernon-stressconditions,NTandallSORlineshadsimilarprolinecon-
tents.However,aftersaltstresstreatment,theSORlineshad2.9–3.7timeshigherlevelsoffree
prolinethanNTplants.Inaddition,theleavesofSORalfalfaplantshadlowerlevelsofH O
2 2
thanNTplants.SORleavesofsaltstress-treatedplantsexhibitedlittlecolorchangeafterDAB
staining,whilethoseofstressedNTplantswerebrownduetohighH O levels(Fig3F).These
2 2
resultsindicatethatIbOrexpressionincreasessaltstresstoleranceinalfalfaplants.
SORalfalfaexhibitsincreaseddroughtstresstolerance
ToevaluatethedroughtstresstoleranceofSORplants,1-month-oldNTandSORplantswere
subjectedtowaterdeficitfor7days.Beforewithholdingthewatersupply,theplantswereirri-
gatedwithsimilarquantitiesofwaterfor1week;noobviousdifferenceswereobservedbetween
SORandNTplants.After4daysofwithholdingwater,weobservedseverewiltingoftheNT
plants,whiletheSORplantsexhibitedlesswilting.Whentheplantswerere-wateredafter
droughtstresstreatment,allSORlinesrecoveredsuccessfully,withonlyafewwitheredleaves,
whereasNTplantswerealmostdeadandfailedtorecoverfromdehydrationconditions(Fig
4A).IbOrtranscriptlevelsweresignificantlyhigherinSORplantsthaninNTplantsafter
droughttreatment(Fig4B).Toassessthedegreeofdroughtstress,theRWCvalues,agoodin-
dicatoroftheturgiditymaintainedbyplants,wereestimatedusingleavesfromalfalfaplants
beforeandafterwithholdingwaterfor3days.After3daysofdroughtstresstreatment,severe
waterlosswasobservedintheleavesofNTplants,withonly61.2%watercontent,whileSOR2,
PLOSONE|DOI:10.1371/journal.pone.0126050 May6,2015 10/17