Overexpression of Arabidopsis AnnAt8 Alleviates Abiotic Stress in Transgenic Arabidopsis and ...

25 Pages·2016·7.15 MB·English

plants
Article
Overexpression of Arabidopsis AnnAt8 Alleviates
Abiotic Stress in Transgenic Arabidopsis and Tobacco
DeepankerYadav*,IsrarAhmed,PawanShukla†,PrasannaBoyidiand
PulugurthaBharadwajaKirti*
DepartmentofPlantSciences,SchoolofLifeSciences,UniversityofHyderabad,Hyderabad500046,India;
[email protected](I.A.);[email protected](P.S.);[email protected](P.B.)
* Correspondence:[email protected]@gmail.com(D.Y.);
[email protected](P.B.K.);Tel.:[phone](P.B.K.);Fax:[phone](P.B.K.)
† PresentAddress:CentralSericulturalResearchandTrainingInstitute,CentralSilkBoard,NH-1A,Gallandar,
Srinagar192121,India
AcademicEditor:SylvieRenault
Received:5November2015;Accepted:1April2016;Published:14April2016
Abstract: Abioticstressresultsinmassivelossofcropproductivitythroughouttheworld. Because
ofourlimitedknowledgeoftheplantdefensemechanisms,itisverydifficulttoexploittheplant
geneticresourcesformanipulationoftraitsthatcouldbenefitmultiplestresstoleranceinplants. To
achievethis,weneedadeeperunderstandingoftheplantgeneregulatorymechanismsinvolved
instressresponses. Understandingtherolesofdifferentmembersofplantgenefamiliesinvolved
indifferentstressresponses,wouldbeastepinthisdirection. Arabidopsis,whichservedasamodel
systemfortheplantresearch,isalsothemostsuitablesystemforthefunctionalcharacterizationof
plant gene families. Annexin family in Arabidopsis also is one gene family which has not been
fully explored. Eight annexin genes have been reported in the genome of Arabidopsis thaliana.
Expression studies of different Arabidopsis annexins revealed their differential regulation under
various abiotic stress conditions. AnnAt8 (At5g12380), a member of this family has been shown
to exhibit ~433and~175fold increase in transcript levels under NaCl and dehydration stress
respectively. TocharacterizeAnnexin8(AnnAt8)further,wehavegeneratedtransgenicArabidopsis
andtobaccoplantsconstitutivelyexpressingAnnAt8,whichwereevaluatedunderdifferentabiotic
stressconditions. AnnAt8overexpressingtransgenicplantsexhibitedhigherseedgerminationrates,
betterplantgrowth, andhigherchlorophyllretentionwhencomparedtowildtypeplantsunder
abioticstresstreatments. Understressconditionstransgenicplantsshowedcomparativelyhigher
levelsofprolineandlowerlevelsofmalondialdehydecomparedtothewild-typeplants. Real-Time
PCRanalysesrevealedthattheexpressionofseveralstress-regulatedgeneswasalteredinAnnAt8
over-expressingtransgenictobaccoplants,andtheenhancedtoleranceexhibitedbythetransgenic
plants can be correlated with altered expressions of these stress-regulated genes. Our findings
suggestaroleforAnnAt8inenhancingabioticstresstoleranceatdifferentstagesofplantgrowth
anddevelopment.
Keywords: annexin;seedlinggrowth;osmoticstress;methylviologenstress;saltstress;seedgermination
1. Introduction
Abioticstressincludesphysicalandchemicalchangesintheenvironment,whichaffectnormal
growth and productivity of the plant. Due to shifts in the climatic conditions and improper
management in farming, abiotic stress has become a challenge for the global agriculture industry.
Abioticstressultimatelycouldleadtothelossofcellhomeostasisandcelldeath. Inordertomaintain
stabilityincellstructureandfunctionalmachineryinsidethecell[1],plantshaveevolvedagroup
Plants2016,5,18;doi:10.3390/plants5020018 www.mdpi.com/journal/plants
Plants2016,5, 18 2of25
ofgenes,whoseexpressionhelpsthemsurvivetheadverseconditions[2]. Salinityanddroughtare
commonstresses,whichplantsfaceduringtheirlifecycle. Fewplantspecieshaveevolvedinsuch
awaythattheyhavedevelopedadaptations,andcancompletetheirlifecycleunderthesestressful
conditions. However,inotherspecies,theirdefensesystembecomesoperationalwhenexposedtothe
stress[3–5].Thedefenseresponsesalsovarywiththeseverityanddurationofstressgiventoaplant.In
mildstressconditions,expressionsofstress-inducedgenesdonotoverlapeachother. However,under
severestressconditions,acommonsetofgenesmayupregulate,whichcouldbeduetothegeneration
ofreactiveoxygenspecies(ROS)[6]. Manygenesinvolvedindefensemechanismsagainstabiotic
stresseshave beenidentifiedsofar. Annexinsarealsoamongthem. Theyarepresentthroughout
plants,animalsandfungi. Recentlytheirpresencehasalsobeenreportedinprokaryotes. Annexin
isasuperfamilyofgenes,andplantannexinswhichbelongtofamilyD,havebeenfurthergrouped
into different subfamilies [7,8]. In the course of evolution, plant annexins have become diverse in
their structure and function [8]. They have been shown to play a significant role in plant growth
anddevelopmentundernormalandstressconditions[8–11]. Inplants,thefirstmultigeneannexin
family was identified in Arabidopsis with eight members representing this family, and expression
and characterization studies have established them as a multifunctional protein family [8,11–18].
TheypossessdifferentpropertieslikeCa2+binding,ionpermeability,andperoxidaseactivity,which
havebeencorrelatedwiththeirresponsesduringplantdevelopmentandstresscondition[19]. An
increaseinplantannexinabundanceandtheirrecruitmenttomembranehasbeenreportedunder
differentstressconditions[20–23]. Thisrecruitmentofannexinstothemembranehasbeenlinkedto
manyfunctionslikechannelproperties,protectionofmembraneandROS-inducedsignaling[24,25].
AnotherimportantpropertyofannexinisCa2+dependentlipidbinding. Recentreportssuggestthat
annexins can mediate the ROS-induced changes in Ca2+ permeability of membrane [26–29]. This
annexin-mediatedCa2+ modulationissupposedtoplayasignificantroleinabioticstresssignaling
(Cyt)
andgeneregulation. Annexinregulatoryresponsescanalsobemediatedbychangesinphytohormone
levelduringplantgrowthanddevelopmentinstressconditions[14]. Thereislimitedinformation
onhowthesepropertiesworkinacumulativemannerduringstressresponses. Previousexpression
studiesofannexinfamilymembersindifferentplantsshoweddifferentialexpressionpatternunder
normalandstressconditions[12,16,30–37]. Proteomicstudiesinmanyplantspeciesalsodisplayed
their upregulation under salinity [18,38,39] and heat stress [15,40,41]. In a transcriptome study in
Arabidopsis, annexin members showed a differential expression pattern during seed and seedling
stages, and most of them were shown to be up-regulated in various abiotic stress treatments [31].
Previous studies on AnnAt1, AnnAt2, and AnnAt4 have indicated their role in salt and drought
tolerance[14,20,21],whereasAnnAt5playsaroleinpollendevelopment[16,17]. Amongtheother
members of Arabidopsis annexin family, AnnAt8 (AT5G12380) has been reported for its high fold
transcriptaccumulationduringseedlingstageundersaltanddehydrationstressandshowedsimilar
responsecomparabletoRd29andP5Cs(twomarkergenesforsaltstressinArabidopsis)[31]. Some
tissue-specificgeneexpressionstudiesinArabidopsisalsoshowedthepresenceofAnnAt8transcriptin
femalegametophyte[42,43]. Inarecentstudy,ithasbeendemonstratedthatAnnAt8expressionin
ArabidopsisrootsdoesnotchangeontreatmentwithH O [29]. Abovementionedexpressionstudies
2 2
suggestapossibleroleofAnnAt8inplantgrowthanddevelopmentinnormalaswellasinstress
conditions. Sincegeneexpressionstudiesdonotalwaysleadtoconclusionsaboutthegenefunction,
we followed the overexpression strategy of gene characterization. Further to this, we generated
AnnAt8overexpressingtransgenictobaccoandArabidopsisplantsandanalyzedtheirperformance
underdifferentstressconditions. ThecurrentstudyprovidesevidencefortheinvolvementofAnnAt8
inalleviatingabioticstressintransgenicArabidopsisandtobacco.
Plants2016,5, 18 3of25
2. MaterialsandMethods
2.1. AnnAt8ConstructPreparation
Thefull-lengthcDNAoftheAnnAt8gene(Locus: At5g12380andNCBIGenBankAccessionNo:
NM_121276) was amplified by using following primers (ORF1 For and ORF1 Rev harboring NcoI
and XbaI sites respectively) and cloned in plant expression vector pRT100. Primers employed for
theamplificationofORFIarelistedinSupplementaryTableS1. ExpressioncassettewithCaMV35S
promoterandpoly-adenylationsignalwasreleasedfromthepRT100usingPstIdigestion,andthe
samesitewasusedtoclonetheexpressioncassetteintothebinaryvectorpCAMBIA2300.
2.2. PlantMaterialandExperimentalConditions
ArabidopsisthalianaecotypeCol0wasusedforgeneratingthetransgenicplantsconstitutively
expressingAnnAt8. TheArabidopsisplantsweregrowninagrowthroomunderstandardcontrolled
light conditions. For vegetative growth of plants controlled temperature (21 ˘ 2 ˝C) and light
conditions (8 h of light/16 h of dark period) were used in the growth chamber. For reproductive
growth,16hoflight/8hofdarkperiodwasused. Lightintensityingrowthchamberwasmaintained
at100–150µmol¨m´2¨s´1.
NicotianatabacumLinncultivarSamsunwasusedfortransgenicgeneration. Thetobaccoplants
weregrowninthegreenhouseundernaturallightconditions. Seedgerminationwasperformed,and
seedlingsweremaintainedinagrowthroomat26˘2˝Cwith16hoflight/8hofdark.
2.3. PlantTransformation
Arabidopsis plant transformation was performed by the floral dip method with minor
modifications[44]. AgrobacteriumstrainEHA105harboringtheconstructpCAMBIA2300::AnnAt8
wasusedtotransformtheArabidopsisfollowingstandardinplantaprotocol[44]. Forthefloraldip
transformation, anappropriatefloweringstagewasselected, andabunchoffloweringstalkswas
invertedtodipinAgrobacteriumcell(harboringpCAMBIA2300::AnnAt8)suspensioncontaining5%
sucroseand0.05%SilwetL-77fortwomin. Further,theplantswerewrappedwithplasticfilmsto
maintainhighhumidityfor16–24hinthedark. Finally,theplasticcoverswereremoved,andplants
wereallowedtogrowtosiliqueformationandmaturity,whichwasfollowedbydryingandharvesting
ofseeds. PrimarytransformantswereselectedinT generationusingkanamycin(50mg/L).Theseeds
1
fromtheputativetransgenicswereagainselfedtoobtainT progeniesthroughT .
3 2
Tobaccoplanttransformationwasperformedbyleafdisctransformationmethodwithminor
modifications [45]. Small pieces of fully grown leaves were surface sterilized by 0.01% HgCl for
2
3–5min,whichwasfollowedby4–5washeswithsterilizedwater. Theexplantswerethenincubated
in Agrobacterium (strain EHA105) cell (harboring pCAMBIA2300::AnnAt8 construct) suspension.
Subsequently,thetreatedexplantsweredriedwithsterilizedtissuepaperandplacedonfullMS-Agar
medium(with2.0mg/LBAPand0.1mg/LNAA)forcocultivation. Aftertwodaysofcocultivation,
theexplantsweretransferredtotheshootinitiationmedium(MS-Agarmediumwith2.0mg/LBAP
and0.1mg/LNAA)withkanamycin(125mg/L)fortransformedshootselectionandCefotaxime
(250mg/L)tocheckAgrobacteriumovergrowth. Afterfourweeksontheshootinductionmedium,
newlyformedshootsfromthecallusweretransferredtoshootelongation(MS-Agar)medium. Further,
theelongatedshootsweretransferredtoarootinitiationmedium(MS-Agarmediumwith0.1mg/L
NAA); both media contained Kanamycin for selection. Finally, the selected putative transgenic
plantlets were kept for hardening in the growth room and then transferred to the greenhouse for
furthergrowthandanalysis. Theseedsfromtheputativeprimarytransgenicplantswerecollected
through self-pollination and were germinated on Kanamycin containing medium to screen the T
1
plants,theconfirmedplantswereselfedtoobtainT progeny.
2
Plants2016,5, 18 4of25
2.4. SeedGerminationAnalysisinSaltandOsmoticStress
Forseedgerminationassay,AnnAt8overexpressingtransgenic(T generation)andWTArabidopsis
3
seedsweresurfacesterilizedby1%sodiumhypochlorite(NaOCl)foroneminandthenwashedwith
sterile water4–5 times. The seeds were transferred tothe ½ MS-Agar mediumand maintainedin
thegrowthchamberwithaphotoperiodof8/16hlight/darkregimeat21˘2˝Croomtemp. For
salttreatment,weused100mMofNaClinhalfstrengthMSmedium;forosmoticstresstreatment,
250mMsorbitolandforABAtreatment,5µMofABAwasused.
Totesttheseedgerminationratesintobacco,T transgenic(T-14.3andT-15.2)andWT(Control)
2
seeds were surface sterilized in 75% ethanol for one min, which was followed by treatment with
sodium hypochlorite (NaOCl) for ten min. After sterilization, seeds were washed 4–5 times with
sterilewaterandtransferredtothehalfstrengthMS+0.8%Agar(HiMediaLab. Pvt. Ltd. Mumbai,
India)platesandmaintainedinacultureroomwithaphotoperiodof16hlightand8hdarkatroom
temp27˘1˝C.Forsalttreatment,weused0,100,150and200mMofNaClinhalfstrengthMS-Agar
medium. Forinducingosmoticstress,0,300and400mMsorbitolconcentrationswereused.
2.5. SeedlingStressAssay
ForArabidopsisseedlingassays,AnnAt8overexpressingArabidopsistransgeniclinesandWTseeds
weresurfacesterilizedbyNaOClandwereallowedtogerminateinagrowthchambermaintained
at21˘2˝Cwithaphotoperiodof8/16hlight/dark. Aftersevendaysofpost-germinationgrowth
onhalfstrengthMS-Agarmedium, WTandAnnAt8overexpressingseedlingsweretransferredto
stressmediaandweregrownonverticallyplacedagarplates. SeedlingsofWTandtransgeniclines
wereallowedtogrowfurtherononlyhalfstrengthMSmediumforcontrol, andhalfstrengthMS
mediawith100mMNaClforsaltstressand250mMsorbitolforosmoticstressrespectively. Forall
experiments,excepttheNaCltreatment,seedlingsweregrownonsemi-verticalagarplates.
Toevaluatethestresstoleranceoftransgenictobaccoplantsattheseedlingstage,weexposed
the transgenic and WT seedlings to different stress conditions for different time periods. For all
thebioassaysperformedinthecurrentstudy;weusedT generationseeds. Forseedlingassay,T
2 2
generationseedsoftransgeniclines,andWTweresurfacesterilizedbyNaOClandplacedonhalf
strengthMS(withoutorganics)+0.8%Agar(HiMediaLab. Pvt. Ltd. India). Seedlingswereallowed
to grow for ten days in a growth chamber maintained at 27 ˘ 1 ˝C with a photoperiod of 16/8 h
light/dark. Ten days old seedlings of WT and transgenic lines were transferred on half strength
MS-Agarmediumwith100and200mMNaClforsaltstress. Dehydrationstresswasgivenby200and
300mMsorbitolinhalfstrengthMS-Agarmedium. Forphotooxidativestress,10µMMethylViologen
(MV)wasusedinhalfstrengthMS-Agarmedium. Foreachexperiment,seedlingsweregrownon
horizontalagarplates.
2.6. LeafDiscSenescenceAssay
Fullygrownleavesfromtwo-montholdtobaccoplantsweretakenforleafdiscsenescenceassays.
Theleafdiscsof1cmdiameterwereexcisedwiththehelpofacorkborerandusedfurtherinstress
treatments.Sorbitol(300,400and500mMconc)wasusedtoinducedehydrationstress.Similarly,NaCl
solutionsof100,200and300mMconcentrationswereusedforsaltstress. Waterwasusedasacontrol.
Thetreatmentswerecarriedoutincontinuouslightuntilidentifiabledifferenceswereobserved.
2.7. BiochemicalAnalyses: DeterminationofChlorophyllContentandLipidPeroxidationAssay
Tomeasurethetotalchlorophyllcontentofseedlingsandleafdiscs,equalquantitiesoftreated
andnon-treatedsamplesweretaken.Thetotalchlorophyllcontentofseedlingsandsenescingleafdiscs
wasestimatedaccordingtoHiscoxandIsraelstam[46]. Lipidperoxidationintheformofthiobarbituric
acidreactivesubstanceswasestimatedfollowingtheMDAestimationprotocol[47].
Plants2016,5, 18 5of25
2.8. EstimationofProlineContentinTransgenicandWTTobaccoSeedlings
Fifteenday-oldseedlingsofWTandtransgeniclinesweretransferredontohalfstrengthMS-Agar
mediumwith200mMNaCl,andseedlingsgrownonlyonhalfstrengthMS-Agarmediumwereused
asacontrol. AfterfourdaysofNaCltreatment,seedlingswereusedforestimationofprolineaccording
toBatesetal.[48].
2.9. RealTimeExpressionAnalysisofStress-InducibleGenesinWTandTransgenicTobaccoPlants
Fortheevaluationoftranscriptlevelsofdifferentstressmarkergenes,15day-oldtransgenic,and
WTtobaccoseedlingswereincubatedwithhalfstrengthMS(control)and200mMNaCl(treatment)for
6handsampleswerecollectedforRNAisolation. TotalRNAwasextractedusingTRIZOL(Invitrogen,
Waltham,MA,USA).ThefirststrandofcDNAwaspreparedfrom2µgoftotalRNAusingMMLV
ReverseTranscriptase(Clontech,MountainView,CA,USA)inareactionvolumeof20µL.Forthe
real-timeexpressionanalysis,FASTSTART-SYBRMIX(Roche,Penzberg,Germany)wasused. All
thereactionswereperformedinMastercycler® 204eprealplex4(Eppendorf,Hamburg,Germany).
PCR program used for all the amplifications was 95 ˝C for 10 min and 40 cycles of 95 ˝C for 15 s
(denaturation),57˝Cfor30s(annealing),72˝Cfor30s(elongation).PrimersusedintheqPCRanalysis
are listed in Supplementary Table S3. Each sample was amplified in three independent biological
replicateswiththreetechnicalreplicates. Relativegeneexpressionwascalculatedaccordingtothe
∆∆C method[49]. NtActinwasusedasareferencegenetonormalizegeneexpressionintobacco.
T
∆C forWT(untreated)wasusedasacontrolforthecalculationofrelativechangesinexpressionof
T
transgeniclines(untreated)andNaCl-treatedWTandtransgeniclines.
2.10. SubcellularLocalizationofAnnAt8Protein
TocheckthesubcellularlocalizationofAnnAt8protein,theORFwasreamplifiedwithORF2-F
andORF2-RprimersharboringEcoRIandSmaIsitesrespectively. Theamplifiedproductwasfurther
digestedandclonedinthepEGADvectoratthesamesites,asaC-terminalfusionprotein. Primers
usedintheamplificationofORF2arelistedinSupplementaryTableS1. Further,theconfirmedvectors
were mobilized into Agrobacterium tumefaciens strain EHA105. The Agrobacterium cell (harboring
thepEGAD-AnnAt8construct)suspensionwasinfiltratedintobaccoleaves(NicotianatabacumLinn
cultivarSamsun)andGFPexpressionwasvisualizedundertheconfocalmicroscope(CarlZeissLSM
710NLOConfoCor3,Germany)at480to515nmwavelengthaftera48hincubationperiod,according
toKumarandKirti[50].
2.11. StatisticalAnalysis
Allthedatawasanalyzedbythestatisticalanalysisofvariance(one-wayANOVA)bySIGMA
PLOT,St. Louis,MO,USAver. 11.0.
3. Results
3.1. MolecularAnalysisofTransgenicArabidopsisandTobaccoPlants
ForthemolecularandfunctionalcharacterizationofAnnAt8,AnnAt8overexpressingtransgenic
ArabidopsisandtobaccoplantsweregeneratedthroughAgrobacterium-mediatedgenetictransformation
ofArabidopsisthalianaecotypeCol0andNicotianatabacumL.cultivarSamsun. TransgenicArabidopsis
plantsweregeneratedbythestandardfloraldipmethod[44]. InT generation,putativetransgenic
1
plantswereselectedonhalfstrengthMSmediumcontainingKanamycin(50mg/L),andtheputative
transformedplantswereanalyzedforthepresenceoftransgenebyCaMV35SandAnnAt8gene-specific
PCR(SupplementaryFigureS1). Alongwiththis,theT-DNAintegrationwascrossconfirmedbyNptII
genespecificPCR(datanotshown). PrimersequencesusedforthePCRanalysisoftransgenesare
mentionedintheSupplementaryTableS2. Further,onthebasisofAnnAt8geneexpressionanalysis
Plants2016,5, 18 6of25
Plants 2016, 5, 18 6 of 25
iPnlatnhtse 20T16, g5,e 1n8e ration (L-3, L-4, L-7, L-8, L-10, L-14, L-15 and L-19) (Figure 1), two transgenic6li onfe 2s5
1
(Ltw-3o, Ltr-a7n)swgeenreicfi lninaellsy (Lse-3le, cLte-7d) fworerdee ftianialelldy asnealelyctseids ifnort hdeeTta3ilgeedn aenraatlyiosnis (ionb tthaien Te3d gtehnroeruagthiosne l(fionbgtaainnedd
cttrwhorosos turcaghnhes csgkeeilnnfiginc) g.li nanesd ( cLr-o3s, sL c-7h)e wckeirneg f)i.n ally selected for detailed analysis in the T3 generation (obtained
through selfing and cross checking).

Figure 1. Expression analysis of AnnAt8 by semi-quantitative RT-PCR in AnnAt8 overexpressing
FFiigguurree 11.. EExxpprreessssiioonn aannaallyyssiiss ooff AAnnnnAAtt88 bbyy sseemmii--qquuaannttiittaattiivvee RRTT--PPCCRR iinn AAnnnnAAtt88 oovveerreexxpprreessssiinngg
ttrtraraannnsssgggeeennniicicc A AArraraabbbiididdoopoppssisisiss p ppllalaannnttstssc cocoonnnfiffirirmrmmeededda atattT TT111 g ggeeennneeerrraaattitioioonnn... W WWTTT i isiss t tataakkkeeennn a aasss a aa nn nooonnn--t-trtrraaannnsssgggeeennniicicc c ccooonnnttrtrorooll,l,, BB B i isiss
non-template control. 18S used as reference gene.
non-templatecontrol.18Susedasreferencegene.
non-template control. 18S used as reference gene.
Since the functional analysis of AnnAt8 in Arabidopsis thaliana transgenic showed the AnnAt8
SSiinnccee tthhee ffuunnccttiioonnaall aannaallyyssiiss ooff AAnnnnAAtt88 iinn AArraabbiiddooppssiiss tthhaalliiaannaa ttrraannssggeenniicc sshhoowweedd tthhee AAnnnnAAtt88
involvement in abiotic stress tolerance, we proceeded to see whether AnnAt8 can be used for the crop
iinnvvoollvveemmeenntt iinn aabbiioottiicc ssttrreessss ttoolleerraannccee,, wwee pprroocceeeeddeedd ttoo sseeee wwhheetthheerr AAnnnnAAtt88 ccaann bbee uusseedd ffoorr tthhee ccrroopp
transformation purposes for the improving stress tolerance. Hence, tobacco was used as a model
ttrraannssffoorrmmaattiioonn ppuurrppoosseess ffoorr tthhee iimmpprroovviinngg ssttrreessss ttoolleerraannccee.. HHeennccee,, ttoobbaaccccoo wwaass uusseedd aass aa mmooddeell
system, and we generated transgenic tobacco plants expressing AnnAt8 in a constitutive manner and
ssyysstteemm,, aanndd wwee ggeenneerraatteedd ttrraannssggeenniicc ttoobbaaccccoo ppllaannttss eexxpprreessssiinngg AAnnnnAAtt88 iinn aa ccoonnssttiittuuttiivvee mmaannnneerr aanndd
evaluated the plants for stress tolerance at different stages of growth. Tobacco transformation was
eevvaalluuaatteedd tthhee ppllaannttss ffoorr ssttrreessss ttoolleerraannccee aatt ddiiffffeerreenntt ssttaaggeess ooff ggrroowwtthh.. TToobbaaccccoo ttrraannssffoorrmmaattiioonn wwaass
done by standard leaf disc protocol [45]. In T0 generation, 16 independent putative transformants
ddwoonenreee b bayyn sastltayanzndeddaar rdfdo lrle eatahffe dd ipisscrce psperronotctoeoc cooolfl [t4[r45a5]n.].s IgInne nTTe00s g gbeenyne egrraeatntiioeo-nns,p, 1e16c6i ifinincdd ePepCpeeRnn ddueesnninttg pp uuNttapatttiIivIv ee(S ttruraapnnpsslffeoomrrmmenaatnnattrssy
wweerreea annaalylyzzededf ofrotrh ethper epsreensceenocef toraf ntsrgaennsegsenbyesg benye -gsepneec-isfipcePcCifiRc uPsCinRg uNspitnIgI (SNupptIpIl e(mSuepnptalerymFeingtuarrey
Figure S2) and AnnAt8 gene specific primers. Primer sequences used for the PCR analysis of
SF2ig)uanred SA2n)n aAntd8 gAennneAspt8e cgifiecnep rsimpeecrisfi.cP rpirmimerersse.q uPerinmceesr usseeqduefonrceths euPsCedR afonra ltyhseis PoCftRr aannsgaleynseisa roef
transgene are mentioned in the Supplementary Table S2. Further, on the basis of AnnAt8 gene
mtraennstigoenneed ainret hmeeSnutpiopnleedm einn tathrye TSaubpleplSe2m.eFnutratrhye rT,oanblteh eS2b.a FsiusrothfeArn, noAnt 8thgee nbeaseixsp oref sAsinonnAatn8a lgyesnise
expression analysis using semi-quantitative RT-PCR in putative transgenic plants of T0 generation
ue(sxFipingrguessrseeim o2n)i,- qaeunigaahnlytt isptiaslat iunvstesin R(gTT --sP1e1Cm, R1i-2iqn,u 1pa4nu, tt1iat5tai, tv1iev6et, r 1Ra7nT,s -1gP9eC naRinc dipn lT ap-n2ut0st)a otwifveTer0 etg rsaeennlesegcrateetnidoic nf op(rFl afinguutrstr heoef2r ) T,a0en igagelhyntseiprsla aitnnio tTns1
((TgF-ei1gn1ue,rr1ae2t 2i,o)1,n4 e., iB1g5ah,ste1 pd6l, ao1nn7t ,st1 h(9eT a-e1nx1dp, rT1e2-s2,s 01io)4nw, 1ae5rn,e a1lsy6e,sl ei1sc7 ta,e n1dd9f oasrtnrdfeu sTrst -ha2se0sr)a awyn eparleeyr sfsioesrlemincetTedd1 i gnfoe tnrh efeur arTtt1ih ogenre. naBenaraasletyidosinos noinnth TTe1-
eg1xe1pn.1ree,r saTst-ii1oo2nn.1.a ,Bn Taa-sl1ye4ds.i 3so, anTn -tdh15es. t2er,xe Tpss-r1ea7sss.s1iao, ynT p-a1en9ra.f1loy rasmnisde a dTn-idn2 0st.ht1re,e tsThs1 raegsees natryear npastegiroefnonriomcn leiTdn- e1isn1 .w1th,eeTr e-T1 f12i ng.1ae,nlTley-r1 as4et.il3oe,ncTt e-o1dn5 f.T2o-,r
T1-11.71.,1 T,T-1-129.1.1, Tan-1d4.T3-,2 T0.-11,5t.h2,r eTe-1tr7a.n1,s gTe-n19ic.1l inanesdw Te-2re0.fi1n, athllryesee tlreacntesdgefonricd leitnaeilse dwaenrea lfyinsiasll(yT -s1e4l.e3c,tTe-d1 5f.o2r,
detailed analysis (T-14.3, T-15.2, T-19.1) in the T2 generation. Transgenic lines T-14.3 and T-15.2 were
Tdu-e1st9ea.d1il )einidn aatlnhl aealsTyss2aiygs se( nTwe-1hra4ilt.e3io ,l niTn.-e1T 5rTa.-2n1,s 9Tg.1-e1 nw9i.ca1s)li nianles stohT ei-n 1Tc4l2.u 3gdeaennded riaTnt- i1soo5n.m2. Tew reeaxrnepseugrseiemndieci nnlitnsa.el ls aTs-s1a4y.s3 wanhdil eTl-i1n5e.2T w-1e9r.1e
wusaesda lisno ailnl calsusdaeyds winhsiolem lieneex Tp-e1r9im.1 ewnatss. also included in some experiments.

Figure 2. Expression analysis of AnnAt8 in AnnAt8 overexpressing transgenic tobacco plants by semi-
FFiigguurree 22.. EExxppreresssisoionn anaanlaylsyissi osfo AfnAnnAnt8A itn8 AinnnAAntn8A otv8eroevxeprreexspsrinesgs tinragnstrgaennsigc etonbicactcoob apclacontps lbayn stsembyi-
quantitative RT-PCR. WT is taken as non-transgenic control. 18S was used as a reference.
sqeumain-qtiutaatnivtiet aRtiTv-ePRCTR-.P WCRT. iWs tTakiesnta akse nnoans-tnroann-stgreanniscg ceonnictrcooln. t1r8oSl .w18aSs uwsaesdu asse ad raesfearreenfceer.e nce.
3.2. Enhanced Seed Germination of Arabidopsis Transgenic Plants under Stress Conditions
33..22.. EEnnhhaanncceedd SSeeeedd GGeerrmmiinnaattiioonn ooff AArraabbiiddooppssiiss TTrraannssggeennicicP Plalannttssu unnddeerrS SttrreessssC Coonndditiitoionnss
Seed germination rates of WT and AnnAt8 overexpressing Arabidopsis seeds were checked under
SSeeeedd ggeerrmmiinnaattiioonn rraatteess ooff WWTT aanndd AAnnnnAAtt88 oovveerreexxpprreessssiinngg AArraabbiiddooppssiiss sseeeeddss wweerree cchheecckkeedd uunnddeerr
different stress treatments. On regular half strength MS medium, more than 90% of WT and
ddiiffffeerreenntts tsrtersessstr etaretmatemnetsn.tOs. nOreng urelagruhlaarlf hstarlefn gsttrheMngSthm eMdSiu mm,emdiourmet, hmano9re0 %thoafnW 9T0%an doft raWnsTg eannidc
transgenic seeds completed germination at day 3. In the case of NaCl (100 mM) treatment, WT
steraednssgceonmicp lseeteeddsg ceormmipnlaettieodn gaterdmayin3a.tIionnt haet cdaasey o3f. NInaC tlh(e1 0c0asme Mof) tNreaaCtml e(1n0t,0W mTMsh) otrweeadtmoennlyt, 2W0%T
showed only 20% seed germination, while transgenic lines showed more than 90% of seed
sseheodwgeedrm oinnalyti o2n0,%w hsieleedtr agnesrgmeninicaltiinoens, swhohwileed tmraonrsegtehnainc 9l0in%eso fssheoedwgeder mminoareti otnh.aAn t950µ%M oAf BsAe,ead
germination. At 5 µM ABA, a strong inhibition was observed in the germination of WT seeds, and
sgterormnginianthioibni.t Aiotn 5w µaMs oAbBseAr,v ae dstrinontgh eingheirbmitiionna twioans oofbWserTvesede idns ,thaen dgeormnliyna2t0i%on soefe dWgTe sremeidnsa, taionnd
only 20% seed germination was observed. However, the transgenic lines showed more than 90% seed
wonalsy o2b0s%e rsveeedd. geHrmowineavtieorn, twhaes torabnsesrgveendic. Hlionwesevsehro, wtheed trmanosrgeenthica nlin9e0s% shoseweeddg meromrei nthaatino n90a%t steheids
germination at this concentration of ABA. The osmotic stress condition generated by adding sorbitol
cgoenrmceinntartaitoionn ato tfhAisB cAon.cTehnetroatsimono toifc AstBrAes.s Tchoen odsimtioonticg estnreersast ceodnbdyitiaodnd gienngesraotrebdit obly iandtdhiengm seodrbiuitmol
in the medium showed comparatively less inhibition in seed germination; 50% of seed germination
sihno twhee dmceodmiupma rsahtoivweelyd lceossmipnahriabtiitvioenlyi nlessse eindhgibeirtmioinn ainti osnee;d5 0g%eromfisneaetdiogne; r5m0%in aotfi osenedw agserombsinearvtieodn
was observed in the case of WT at 250 mM sorbitol concentration while AnnAt8 overexpressing (OE)
iwnaths eobcaseservoefdW inT tahte 2c5a0sem oMf WsoTr baitt 2o5l0c omnMce nstorrabtiitoonl cwohnicleenAtrnantAiotn8 wovheilree xApnrneAssti8n gov(OerEex)plirneesssisnhgo w(OeEd)
lines showed more than 95% seed germination at the same concentration. In each case, observations
mlinoerse sthhaonw9e5d% msoereed tghearnm 9i5n%at isoeneda tgtehremsianmateiocno nacte tnhter astaimone. cInonecaecnhtrcaatsieo,no. bIsne ervacahti ocnassew, oebresemrvaadteioonns
were made on day 3 after initiation of the treatment (Figure 3).
dwaeyre3 mafatedrei nointi dataiyon 3 oaffttehre intrietaiattmioenn to(fF tihgeu trreea3t)m. ent (Figure 3).
Plants 2016, 5, 18 7 of 25
Plants2016,5, 18 7of25
Plants 2016, 5, 18 7 of 25
100 WL-T3 ** ** ** ** ** **
%) 100 WLL--T37 ** ** ** ** ** **
80 L-7
)(
%
n 80
(o
on ati 60
in
atmi 60
n
mier 40
g
erd 40
ge
d e 20
S
e
e 20
S
0
0 1/2 MS NaCl (100 mM) ABA (5 µM)Sorbitol (250 mM)
1/2 MS NaCl (100 mM) ABA (5 µM)Sorbitol (250 mM)
Figure 3. Seed germination of WT and AnnAt8 overexpressing (OE) Arabidopsis plants under different
FFstiirggeuusrrsee c 33o..n SSdeeieetiddo nggsee.rr mmSeiienndaa ttgiiooenrnm ooiffn WWatTTio aannn pdde AArcnnennnAAtattg88e oo ovvfee rrWeexxTpp arreenssdss iiAnnggn n((OOAtEE8)) OAAErraa bAbiirddaoobppissdiiossp ppslliaas nntrttssa nuusnngddeeenrri cdd liiiffnffeeerrsee nLntt-
ss3tt rareenssdss cLcoo-n7n dudinittidiooennrs sn.. oSSeremeedda lgg (eehrrammlfii nsnatartteiionongnt phpe eMrrcceSen)n tatanaggdee so aoflftW W(1T0T0a a nmnddMA A)n,n nAnABAtA8t8 O( O5E µEA MAra)r baabindiddopo opsisssmist rotartnaicns gsstgerneeniscsic l(i slnionersebsLi tL-o3-l
a32n 5ad0n dLm -L7M-u7) n.u dnInedr eenr aoncrohmr maclaa(slh e(a,h lafthlsfe tsr teronebgnstgehrthvM aMtSi)So)an nasn dwds aesarltelt( 1(m1000a0dm meM Mo)n,), A AtBhBeAA t( (h55iµ rµdMM )d)a aanynd da oofstsemmr ooittniicccu sstbtrraeetssisso (n(ss oourrbnbidittoeolrl
22g55e00rm mmiMnMa)t.)iI.o nnIen ac ocehnadcchaits ieoc,nats.h eFe, ootrbh eseea rcovhba sbtieioronvlosagtwiiocenarels rmwepaedlriecea otmne at(hdne)e, t h1o0inr0d tsdheaeey dtsah foitrefd rW indTca uyab naadtfi toternra nuisnngdceuenrbiagct eilroimnne isnu awntdieoerner
cgtaoeknremdniit.n iDoanatit.oanF ro ecrpoerneadsciehtniobt nitoh. leFo omgrie ceaaanlcsrh eo pbf linioc =lao t3ge ±ic( SnaEl) ,.r 1Te0pw0liocs aeatesetde s(rnios),fk 1sW 0in0Td saiecneaddtest rtohafna tWs gthTeen amincedlai ntnre avsnawslugeeersne oitcfa ltkirneanens.s gDweaentriaec
rtleianpkeresen sw.e Dnetarettah s eriegmpnreiefaisnceasnnott ftlhyn eh= mi3geh˘aenrSs E toh.faT nnw =to h3a a±st t SeoErfi. sWTkwsToi n( acdsoitcneatrtrieosklt)hs awinthdtheincea matene taahlnyaztv etahdlue bemsye ooafnnter va-awnlsuagyees nA oicNf tlOrinaVnessAgw e(enpr ie<c
sl0ii.ng0en1si)fi . wcaenretl ysihgingihfiecranthtlayn hthigahteorf tWhaTn( cthonatt roofl )WwTh e(ncoanntarolyl)z ewdhbeyn oannea-lwyzaeydA bNyO oVnAe-w(pa<y 0A.0N1)O.VA (p <
0.01).
33..33.. SSaalltt aanndd DDeehhyyddrraattiioonn SSttrreessss TToolleerraannccee ooff AAnnnnAAtt88T TrraannssggeennicicA ArraabbididooppsissisP Plalanntsts
3.3. Salt and Dehydration Stress Tolerance of AnnAt8 Transgenic Arabidopsis Plants
DDiiffffeerreenntt ggrroowwththp paraarmameteetresrlsi kliekreo orotogtr ogwrothwathn danfrde sfhrewshei gwhetiwghetr ewaesrsea yaesdsatyoeodb steor voebsthereveef ftehcet
oefffsetcrtDe soisfff cseotrrneendstis t igcoornonswdoittnhio tpnhaesr oganrmo tewhtetehr gso rlfoiksweete hrd oloionft g sgeseraodnwldinthyg osa uannndgd f pryleoasunhtn swgo epfilWgahnTtt saw onefdr WeA aTnsn saAanytd8e dAO nEtonl Aiontbe8ss OeLrE-v3 elai ntnhedes
LeLf--f73e. caAtn fodtfe Lrs-t47r4e. sAdsa fcyteosrno 4df4igt dirooanwysst ohonfo gnthrNoew agCtrhol w(o1nt0h 0N omafC Msle )e(1md0lei0nd mgiusMm a)n, mwd eeydooiubusnmegr, v pweldaen tohtbsa sotefNr vWaeCdTl tachanaudts NeAdnaClneAls sct8ain uOhsEeibd il tilnieoesnss
oLinn-h3t ihabneitdgio rLno- w7o.nt Ah thfotefe rAg 4rno4n wAdtath8y soe xfo pfA rgnernsosAwintt8gh e poxlnpa nrNetssasCiinnl gc(1o p0ml0ap nmatrMsi si)no m ncoetomdiWpuamTri,ps wolanen totosb (WsFeirTgv uperdlea tn4htAas t,( BNF)i.agCurl ec a4uAs,eBd). less
inhibition on the growth of AnnAt8 expressing plants in comparison to WT plants (Figure 4A,B).
B
B 20
WT
20 L-3
m) 15 WLL--37T
m)h (c 15 * L-7
(cgt 10 *
ot length Root len 105 * *
Ro 5
0
1/2 MS NaCl (100 mM)
0
1/2 MS NaCl (100 mM)

Figure 4. The growth of WT and AnnAt8 overexpressing Arabidopsis plants under normal and salt
Figure4. ThegrowthofWTandAnnAt8overexpressingArabidopsisplantsundernormalandsalt
F(1i0g0u rme M4.) Tshtree sgsr ocwonthd iotifo nWs T( Aa)n dP lAanntn gArto8w otvhe roefx WprTes asinndg AAnrnabAidt8o pesxisp rpelsasnitnsg u Anrdaebri dnooprsmis atrl aannsdg esnailct
(100mM)stressconditions(A)PlantgrowthofWTandAnnAt8expressingArabidopsistransgeniclines
(li1n0e0s mL-M3 )a sntdre Lss- 7c ounnddietiro Nnsa C(Al )( 1P0l0a nmt Mgr)o wsttrhe sos.f TWhTre ea nrdep AlincantAets8 w eexrper eussseindg p Aerr atbriedaotpmsiesn ttr,a annsgde tnhiec
L-3andL-7underNaCl(100mM)stress.Threereplicateswereusedpertreatment,andtheexperiment
leixnpees rLim-3e natn wd aLs- 7re upneadteerd NtharCicle (. 1(B00) RmoMot) l esntrgetshs .o Tf hWreTe a rnedp lAicnanteAs t8w eexrpe ruessesidn gp eArr atrbeidaotpmsiesn ttr,a annsdge tnhiec
wasrepeatedthrice.(B)RootlengthofWTandAnnAt8expressingArabidopsistransgenicplantsunder
epxlapnetrsi muenndte wr anso rrempeaal teadn dth rsiaclet . s(Btr)e Rsso. oItn l eenagcthh ocfa sWe,T oabnsde rAvnatniAont8s ewxperree smsinagd eA raafbtiedro p4s4i s dtraaynss gfreonmic
normalandsaltstress.Ineachcase,observationsweremadeafter44daysfromgermination.Forroot
pgelarnmtsin autniodne.r Fnoor rrmooatl leanngdt hs amlte asstruersesm. Ienn t,e aacvhe racgasee v, aolubsee frovra 1ti5o pnsla nwtleertes omf aWdeT aanftde rt r4a4n sdgaeynsic flrinoems
lengthmeasurement,averagevaluefor15plantletsofWTandtransgeniclineswastakenforeach
bgwieoarlsmo tgianikcaaetlnior efnop.r lF iecoaarct erho (bnoit)o .lleDongagtiacthar lem rpeerpealssieucnartetemm (eenna).nt ,Ds aoavfteanr ar=egpe3r v˘easeSlunEet. Omfoner ea1na5ss p toelfar innstk =le i3nts d± oi cSfa EWt.e OTs tnahnea dta tsthtreearnmisskge aeinnndivcia clliaunteeesss
owthfaatsrt attnahksege mnen efioacrn le ivnaaechlsu wbesieo roleof gstiircgaannli srfigecepanlniicctal yltienh e(insg )hw. eDerartetha as ringepnthrifeaiscteaonnftt Wlmy Tehai(ngcsho enorft rntoh l=a) ,n3w ±thh SaeEtn .o aOfn nWael yTaz s(etcedornibstykro iolnn)d,e iw-cwahtaeeyns
AtahnNaatOl ytVhzAeed m( pbey<a on0n .0ve5a-)wl.uaeys AofN tOraVnsAg e(pn i<c 0l.i0n5e)s. were significantly higher than that of WT (control), when
analyzed by one-way ANOVA (p < 0.05).
As it is known that NaCl imposes osmotic stress as well as ion toxicity on the plants, we used
AsitisknownthatNaClimposesosmoticstressaswellasiontoxicityontheplants,weused
sorbiAtosl iitn i sh aklnf oswtrenn tghtaht MNaSC-Al gimarp moseedsi uomsm toot icch setcrkes fso ar st hwee olls amso itoinc sttorxeiscsi teyf foenc tt hane dp lmanotnsi, tworee du stehde
sorbitolinhalfstrengthMS-Agarmediumtocheckfortheosmoticstresseffectandmonitoredthe
spolarbnitt oglr oinw htha laf ts dtriefnfegrtehn tM tiSm-Ae gpaori nmtse.d Aiutm th teo e cahrleyc ks tfaogre t hofe oobssmerovtiact isotnre assft eerff 1e5ct daanyds mofo gnriotowrethd, twhee
plant growth at different time points. At the early stage of observation after 15 days of growth, we
Plants2016,5, 18 8of25
Plants 2016, 5, 18 8 of 25
plantgrowthatdifferenttimepoints. Attheearlystageofobservationafter15daysofgrowth,we
found that the growth of WT seedlings was more hampered than AnnAt8 expressing lines L-3 and L-
foundthatthegrowthofWTseedlingswasmorehamperedthanAnnAt8expressinglinesL-3andL-7
7 (data not shown). We also noted that the growth inhibition caused by 250 mM Sorbitol was less
(datanotshown). Wealsonotedthatthegrowthinhibitioncausedby250mMSorbitolwaslessthan
than that of 100 mM NaCl treatment (data not shown). Another observation made at day 28 showed
thatof100mMNaCltreatment(datanotshown). Anotherobservationmadeatday28showedthat
that the transgenic plants not only survived under high osmotic stress but also showed better root
thetransgenicplantsnotonlysurvivedunderhighosmoticstressbutalsoshowedbetterrootgrowth,
growth, with about threefold increase as compared to WT (Figure 5), while in the normal growth
withaboutthreefoldincreaseascomparedtoWT(Figure5),whileinthenormalgrowthcondition,a
condition, a negligible difference was seen between the growth of WT and transgenic plant. With the
negligibledifferencewasseenbetweenthegrowthofWTandtransgenicplant. Withtheadvancement
advancement of time, we observed that the transgenic plants showed better adaptation to the
oftime,weobservedthatthetransgenicplantsshowedbetteradaptationtothedehydrationstress
dehydration stress while strong inhibition was observed in the growth of WT plants.
whilestronginhibitionwasobservedinthegrowthofWTplants.

C D
10 30
WT WT
8 LL--37 mg) 25 LL--37
gth (cm) 6 ** ** weight ( 1250 **
Root len 24 nt fresh 10 **
a 5
Pl
0 0
1/2 MS Sorb 250 1/2 MS Sorb 250

FFigiguurree5 5.. EEffffeecctt ooff oosmsmootitci cstsrtersess osno tnheth gerogwrotwh tohf WofTW anTda AnndnAAnt8n eAxtp8reexsspirnegs sAinragbiAdorapbsiisd opplasinstsp.l (aAn,tBs.)
(ARo,Bo)t Raonodt sahnodost hgorootwgtrho woft hploafnptsl aunntsdeurn ndoerrmnoalr m(haallf( hsatrlfenstgrtehn gMthS)M aSn)da onsdmoostmico sttircesstsr e(sssor(bsoitrobli t2o5l0
2m50Mm),M re),srpeescptievcetilvye. lIyn. Ienaechac hcacsaes, et,hteh eobosbesrevravtaitoinosn swwereere mmaaddee oonn ddaayy 2288 aafftteerr ggeerrmmininaattioionn.. TThhrreeee
rreepplilcicaatetessw wereereu suesdedp epretrr etaretmatemnet,natn, dantdh ethexe peexrpimereimntewnta swraesp eraetpeedattehdri cteh;r(iCce); R(Coo) tRleonogt tlhenogftWh Tofa WndT
traannds gtreanniscgleinneics lLin-3esa nLd-3L a-n7du Lnd-7e runndoremr naolramndalo asnmdo otiscmstorteiscs sctroensds ictoionndsi;ti(oDn)sF; r(Des)h Fwreesihg hwteoifgWht Tofa WndT
traannds gtreanniscgleinneics ulinnedse runnodremr anloarmndalo asmndo toicsmstorteiscs sctorensdsi tcioonnds.itFioonrsr.o Footrl ernogotth leanngdthfr aenshd wfreeisghh wtdeaigtah,t
odbastear,v oabtisoenrvsawtieornes mwaedree monaddea yon2 8daany d28fo arneda cfohrr eeapclhic raetepl(inca)tteh e(na)v tehrea agveevraalguee vsafloure1s5 foprl a1n5t psloafnWts Tof
aWndTt arannds gtreanniscgleinneics lwineerse wtaekreen t.aDkeanta. Dreaptare rseepnrtemseenatn mseoafnns =of3 n˘ =S 3E .±T SwEo. Tawstoer aissktesriinskdsi ciantdeitchaatet tthheat
mtheea nmveaalnu vesaloufetsr aonf strgaennsigcelinniecs liwneesr ewseigren sifiigcnaniftilcyanhtilgyh heirgthhearn ththaant tohfaWt oTf W(coTn (tcroonl)trwohl)e wnhaenna laynzaeldyzbeyd
obnye -ownaey-wAaNy OAVNAO(VpA< (0p.0 <1 )0..01).
AAnno obbsseerrvvaattiioonnm maaddeea attd daayy4 444s shhoowweeddt thhaattt htheeg grroowwththo offW WTTw waassc coommppleletetelylyi ninhhibibitieteddw whhiliele
tthheet rtraannssggeennicicp plalannttssm maaininttaainineeddt thheeirirn noorrmmaallg grroowwththu unnddeerro ossmmootitcics strtreessss( 2(25500m mMMS Soorbrbitiotol)l.).R Roooott
lelennggtthha annddf frreesshhw weeigighhttm meeaassuurreemmeennttss ooff WWTT aanndd lliinneess LL--33,, LL--77 wweerree aallssoo ppeerrffoorrmmeedd ((FFiigguurree 66CC,,DD))..
WWeem meaesausurerdedth tehela ltaetrearlarlo rootodte dnesnitsyitoyf opfl apnltasnatsn danfodu fnodunthda tthtahte tlhaete lraatlerroaol trodoetn sdietynswitays whiagsh heirgihnetrh ien
trthane stgraenniscgpenlaicn tpslcaonmtsp caormedptaoretdh etoW tTheu nWdTer uonsdmeor toicsmstroetsics (sdtraetsasn (odtastha onwont )s.hForwesnh).w Feriegshht wofepiglahnt tosf
wpalasnatlss owmase aalssuor emdeaatsudrifefder aetn dtitfifmereepnot itnimtseo pfostirnetsss otfr esatrtemsse ntrte(aFtimguernets (5FDiguarneds 65DD) .and 6D).
Plants2016,5, 18 9of25
Plants 2016, 5, 18 9 of 25

C D
20 100
WT WT
L-3 g) L-3
cm) 15 ****** ** L-7 ht (m 80 ** L-7
ht ( eig 60 **
g 10 w
ot len resh 40
Ro 5 nt f 20
a
Pl
0 0
1/2 MS Sorb 250 1/2 MS Sorb 250

FFiigguurree 66.. EEffffeecctt ooff oossmmoottiicc ssttrreessss oonn tthhee ggrroowwtthh ooff WWTT aanndd ttrraannssggeenniicc AArraabbiiddooppssiiss ppllaannttss ((AA,,BB)) ggrroowwtthh
ooff pplalannttu nudnedrecro nctornoltr(ohla lf(hsatrlef nsgttrhenMgSth) anMdSo) smanodti cosstmreossti(cs osrtbrietsosl 2(5s0ormbiMto)l co2n50d itmioMns), rceospnedcittiivoenlsy,.
Trehsrpeeecrtievpelliyca. tTehsrweee rreepulsiceadtepse wrterreea utmseedn tpaenr dtrethaetmeexnpte rainmde tnhtew eaxspereripmeaetnetd wthasr irceep.e(aCt)edR othortilceen. g(Cth)
oRfoWot Tleanngdtht roafn WsgTe nanicdL t-r3anasngdenLi-c7 Lu-n3 daenrdn Lo-r7m uanldaenrd noosrmmoalt iacnsdtr oessmscootnicd sittrioesnss ;co(Dnd)iFtiroenshs; w(De)ig Fhrtesohf
pwleaingths.t oInf pelaacnhtsc. aInse eaocbhs ecravsea toiobnsewrvaastimona wdeaso mnaddaey o4n4 daafyte 4r4g aefrtmeri gneartmionin.aFtioorn.r oFootr rleonogt tlehndgathta d,athtae,
athvee raavgeeravgaelu veasloufe1s 5opf 1la5n ptslaonftWs oTf aWnTd atrnadn stgraennsicgelinniecs lwineerse wtaekreen tafkoerne afcohr ebaioclho gbiicoalolgreicpalilc raetpel(inca).teD (ant)a.
representmeansofn=3˘SE.Twoasterisksindicatethatthemeanvaluesoftransgeniclineswere
Data represent means of n = 3 ± SE. Two asterisks indicate that the mean values of transgenic lines
significantlyhigherthanthatofWT(control)whenanalyzedbyone-wayANOVA(p<0.01).
were significantly higher than that of WT (control) when analyzed by one-way ANOVA (p < 0.01).
33..44.. OOssmmoottiicc aanndd SSaalltt SSttrreessss TToolleerraannccee ooff AAnnnnAAtt88 TTrraannssggeennicicT ToobbaacccocoP Plalanntsts
WWee eevvaalluuaatteedd tthhee ttoolleerraannccee ooff AAnnnnAAtt88 ttrraannssggeenniicc ttoobbaaccccoo ppllaannttss uunnddeerr oossmmoottiicc ssttrreessss aatt ddiiffffeerreenntt
ssttaaggeess ooff ppllaanntt ggrroowwtthh.. SSoorrbbiittooll wwaass uusseedd iinn tthhiiss ssttuuddyy ttoo iinndduuccee oossmmoottiicc ssttrreessss.. SSeeeedd ggeerrmmiinnaattiioonn,,
sseeeeddlliinngg,, aanndd mmaattuurreep plalanntts stataggeessw weerereo obbsesrevrvededin inth tehep rperseesnetnstt ustduyd.yT. hTehgee grmerimnaintiaotniopne prceerncteangteasgeosf
Wof TWaTn dantrda ntrsagnesngicenliinc elsinwese rweearlem aolsmtsoismt isliamriulanrd uenrdcoenr tcroonltcroonl dciotniodnit(iwonit h(wouitthsoourtb sitoorlb),it(oFli)g, u(Freig7uCre).
M7Co)r. eMthoarne 9th5apne 9rc5e npterocfetnhte osfe ethdes soefetdhse oWf Tthaen WdtTr aannsdg etnraicnsligneensicg elrinmeisn gateerdmainftaetred3 daaftyesr o3f dpalaytsi nogf
opnlattihnegc oonn ttrhoel cmonetdrioul mm,ewdihuimle, iwnh3i0le0 iann 3d004 0a0ndm 4M00s morMbi tsoolrcboitnotla cionnintaginhianlgf shtarelfn sgtrthenMgtSh mMeSd miae,dthiae,
othvee roavllegraerllm gineramtioinnawtioans dwelaasy eddelianyWedT iann WdtTr aannsgde ntriacnlisngeesn,iicn lcinomesp, ainri scoonmtopathriesocno nttor otlhceo ncdointtioronl.
Acotn3d0i0timonM. Asto 3r0b0it omlMco snocrebnittoral tcioonnc,etnhterasteioedn, gtheerm seienda tgioenrmsitnaratteiodno sntadrtaeyd 4o,na dnady i4t, caonndt iint ucoendtiunpuetdo
tuhpe tdo atyhe8 doafy o8b osfe rovbasteirovna.tiTonh.e Tdhaey dwayi sweioseb soebrsveartvioatniosnssh sohwoewdedth tahtatth tehet rtarnansgsgenenicicl ilnineess sshhoowweedd aa
ssiiggnniifificcaannttllyy hhiigghheerr ppeerrcceennttaaggee ooff ggeerrmmiinnaattiioonn ccoommppaarreedd ttoo WWTT ((FFiigguurree 77AA)).. SSiimmiillaarrllyy,, oonn 440000 mmMM
ssoorrbbiittooll mmeeddiiuumm,, tthhee ddaayy wwiissee ppeerrcceenntt ggeerrmmiinnaattiioonn ooff ttrraannssggeenniicc sseeeeddss wwaass hhiigghheerr tthhaann tthhee WWTT sseeeeddss
((FFiigguurree 77BB))..
Plants2016,5, 18 10of25
Plants 2016, 5, 18 10 of 25
A B
100 * 100 *
* *
80 80
%) * %)
Germination ( 246000 ** WT Germination ( 246000 ** * * WT
T-14.3 T-14.3
T-15.2 ** T-15.2
0 0
4 5 6 7 8 6 7 8 9
Time (Day) Time (Day)
C
100
80
%) *
n ( 60 **
o
ati
n
mi 40
er
G 20 WT
T-14.3
T-15.2
0
0 100 200 300 400 500
Sorbitol conc (mM)
FiFgiugurere7 .7S. eSeededg egremrmininataitoionnr artaetseso fofW WTTa nadndA AnnnAnA88o voevreerxepxprersessisninggt rtarnasngsegnenicict otboabcaccocop plalnantstsu unndderer
osomsmootitcics sttrreessss ccoonnddiittiioonn.. (A(A, B,)B a)rea rdeayd wayiswe piseercpeenrtcaegnetsa ogfe sseeodf gseeermdigneartmioinn raetsiopnecrtievsepleyc otinv e3l0y0 oannd
304000a nmdM40 0somrbMitoslo; rb(Cit)o lS;o(Crb)iStoolr bditoosled roesseproenspseo nosne otnhet hseeseede dgegremrmininataitoionn, ,ppeerrcceennttaaggee vvaalluuee ooffs eseeded
gegremrminiantaiotinono non0 ,03, 0300a0n adnd40 400m0 mMMso srobritboiltowl ewreertea ktaenkeonn odna yda4y, 74,a 7n dan8d,r 8e,s preescptievcetliyv,eflryo,m frothme tdhaet edoafte
polaft ipnlgatoinngt hoenm theed ima,efdoirae, afcohr ebaioclho gbiicoallorgeipcalilc raetepl(inc)at1e5 0(ns)e e1d50s osefeWdsT oafn WdtTra annsdg etnraicnlsigneensiwc elirneetsa kweenr.e
Dtaatkaerne.p Dreastean rtempereasnesnot fmne=an3s˘ oSf En. =A n3 a±s SteEr.i sAknin adsitceartiessk tihnadtitchaetems ethanatv tahleu emseoafntr avnaslugeesn iocfl itnraensswgeerneic
silginneifis cwaenrtely sihgingihfiecranthtlayn htihgahteor fthWanT t(hcoatn otrfo Wl)Tw (hceonntarnoal)l ywzheednb aynoanlyez-ewda byyA oNneO-wVAay( pA<N0O.0V5A). (p < 0.05).
For seedling stress assay, 10 day-old seedlings were transferred to the half strength MS medium
Forseedlingstressassay,10day-oldseedlingsweretransferredtothehalfstrengthMSmedium
with different concentrations (100–200–300 mM) of sorbitol. No differences were observed between
withdifferentconcentrations(100–200–300mM)ofsorbitol. Nodifferenceswereobservedbetween
the WT and the transgenic lines at 100 mM sorbitol concentration. At 200 mM, there were significant
theWTandthetransgeniclinesat100mMsorbitolconcentration. At200mM,thereweresignificant
differences in the root and shoot growth of transgenic seedlings compared to WT (data not shown).
differencesintherootandshootgrowthoftransgenicseedlingscomparedtoWT(datanotshown).
Increased root and shoot growth was observed in the transgenic lines with enhanced root branching.
Increasedrootandshootgrowthwasobservedinthetransgeniclineswithenhancedrootbranching.
At 300 mM sorbitol concentration, the seedling growth of WT and transgenic plants was slow in the
At300mMsorbitolconcentration,theseedlinggrowthofWTandtransgenicplantswasslowinthe
beginning. However, the transgenic lines resumed active growth with the passage of time showing
beginning. However,thetransgeniclinesresumedactivegrowthwiththepassageoftimeshowing
overall enhanced growth compared to the WT (Figure 8). A significant difference in the seedlings
overallenhancedgrowthcomparedtotheWT(Figure8). Asignificantdifferenceintheseedlings
biomass was also observed under osmotic stress with a higher fresh weight of transgenic lines in
biomass was also observed under osmotic stress with a higher fresh weight of transgenic lines in
comparison to WT (Figure 9A).
comparisontoWT(Figure9A).
Leaf disc senescence assay was performed to evaluate the total chlorophyll content of WT and
LeafdiscsenescenceassaywasperformedtoevaluatethetotalchlorophyllcontentofWTand
transgenic lines under normal and stress conditions; leaf discs from the mature leaves of two-month-
transgeniclinesundernormalandstressconditions;leafdiscsfromthematureleavesoftwo-month-old
old transgenic and WT plants were taken for analysis. At 300 mM concentration, we did not find any
transgenicandWTplantsweretakenforanalysis. At300mMconcentration, wedidnotfindany
significant difference in chlorophyll content of WT and transgenic lines. At 400 mM concentration,
significantdifferenceinchlorophyllcontentofWTandtransgeniclines. At400mMconcentration,one
one transgenic line showed significantly higher chlorophyll content compared to WT. The total
transgeniclineshowedsignificantlyhigherchlorophyllcontentcomparedtoWT.Thetotalchlorophyll
chlorophyll content of both transgenic lines was significantly higher than WT at 500 mM sorbitol
contentofbothtransgeniclineswassignificantlyhigherthanWTat500mMsorbitolconcentration
concentration (Figure 9B).
(Figure9B).
As salt tolerance in crop plants is of commercial significance, we studied the response of AnnAt8
As salt tolerance in crop plants is of commercial significance, we studied the response of
expressing transgenic tobacco plants towards sodium chloride treatment. In the case of seed
AnnAt8 expressing transgenic tobacco plants towards sodium chloride treatment. In the case of
germination, three different concentrations of sodium chloride (100, 150 and 200 mM) were used to
seedgermination,threedifferentconcentrationsofsodiumchloride(100,150and200mM)wereused
check the germination rates. We observed a delay in the germination of WT and transgenic seeds on
sodium chloride containing germination media. On the germination medium without NaCl, seeds of