Ocimum metabolomics in response to abiotic stresses

26 Pages·2017·3.14 MB·English

RESEARCHARTICLE
Ocimum metabolomics in response to abiotic
stresses: Cold, flood, drought and salinity
ShubhraRastogi1,SaumyaShah2,RiteshKumar2,DivyaVashisth2,MdQussenAkhtarID2,
AjayKumar2,UpendraNathDwivedi1,AjitKumarShasanyID2*
1 DepartmentofBiochemistry,UniversityofLucknow,Lucknow,UttarPradesh,India,2 Biotechnology
Division,CSIR-CentralInstituteofMedicinalandAromaticPlants,Lucknow,UttarPradesh,India
*[email protected]
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Abstract
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a1111111111 Ocimumtenuiflorumisawidelyusedmedicinalplantsinceancienttimesandstillcontinues
tobeirreplaceableduetoitsproperties.Theplanthasbeenexploredchemicallyand
pharmacologically,however,themolecularstudieshavebeenstartedlately.Inanattempt
togetacomprehensiveoverviewoftheabioticstressresponseinO.tenuiflorum,denovo
transcriptomesequencingofplantleavesunderthecold,drought,floodandsalinitystresses
OPENACCESS
wascarriedout.Acomparativedifferentialgeneexpression(DGE)studywascarriedout
Citation:RastogiS,ShahS,KumarR,VashisthD,
betweenthecommontranscriptsineachstresswithrespecttothecontrol.KEGGpathway
AkhtarMQ,KumarA,etal.(2019)Ocimum
metabolomicsinresponsetoabioticstresses: analysisandgeneontology(GO)enrichmentstudiesexhibitedseveralmodificationsinmet-
Cold,flood,droughtandsalinity.PLoSONE14(2): abolicpathwaysastheresultoffourabioticstresses.Besidesthis,acomparativemetabolite
e0210903.https://doi.org/10.1371/journal.
profilingofstressandcontrolsampleswasperformed.Amongthecold,drought,floodand
pone.0210903
salinitystresses,theplantwasmostsusceptibletothecoldstress.Severetreatmentsofall
Editor:NiranjanBaisakh,LouisianaState
theseabioticstressesalsodecreasedeugenolwhichisthemainsecondarymetabolitepres-
University,UNITEDSTATES
entintheO.tenuiflorumplant.Thisinvestigationpresentsacomprehensiveanalysisofthe
Received:October12,2018
abioticstresseffectsinO.tenuiflorum.Currentstudyprovidesaninsighttothestatusof
Accepted:January3,2019 pathwaygenes’expressionthathelpsynthesizingeconomicallyvaluablephenylpropanoids
Published:February6,2019 andterpenoidsrelatedtotheadaptationoftheplant.Thisstudyidentifiedseveralputative
abioticstresstolerantgeneswhichcanbeutilizedtoeitherbreedstresstolerantO.tenui-
Copyright:©2019Rastogietal.Thisisanopen
accessarticledistributedunderthetermsofthe florumthroughpyramidingorgeneratingtransgenicplants.
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited.
DataAvailabilityStatement:NGSsequencingdata
Introduction
generatedinthecurrentinvestigationissubmitted
toNCBISRA(ShortReadArchive).Thebioproject Abioticstresseslike-drought,flood,salinityandcoldaretheenvironmentalfactorswhich
IDassignedis:(PRJNA390220)(https://www.ncbi.
holdanadverseimpactovertheplants’growthandproductivity.Inresponse,plantshave
nlm.nih.gov/bioproject/PRJNA390220).
developednumerousphysiologicalandbiochemicalhomeostaticbalancetoattaintolerance
Funding:SRreceivedStartUpResearchGrant andadaptationagainstsuchstresses[1].Theseremediesincludemolecularrearrangementsat
(Grantnumber-YSS/2014/001011)byScienceand
somestageingeneexpressioninitiatingfromregulationattranscriptionallevelleadingtopro-
EngineeringResearchBoardtoconductthisstudy
cessingofmRNA,post-transcriptionallevelormodificationofprotein.Undertheseabiotic
(http://www.serbonline.in/SERB/HomePage.do).
stresses,plantsexhibitprecisetranscriptionalregulationaffectingitstranscriptome[2–5].Abi-
SRdesigned,conceived,conductedtheresearch
plansandwrotethemanuscript.SR,SS,RK,DV, oticstressesaffectdifferentlyplanttoplant,likedroughtstressinpotatoreducestheeconomic
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Ocimummetabolomicsinresponsetoabioticstresses
MQA,AKperformedtheabioticstressexperiments yieldofplantby13%while,incaseofrice,maizeandcommonbeansyieldreductionranges
andbioinformaticsanalysis.AKSandUNDgave from24to92%[6].
directionsandsuggestionsforimplementationof
Coldstressisoneofthemostdamagingabioticstresses.Transcriptomeandthemetabolism
theexperiments.Alltheauthorshavereadand
oftheplantareseverelyaffectedbythecoldstresswhichmaybeduetothedirectinhibition
approvedthefinalmanuscript.
ofmetabolicenzymesatcoldtemperaturesaswellasreprogrammedgeneexpression[7].As
Competinginterests:Theauthorshavedeclared
reportedearlier,coldstressraisestheproductionofphenolicsandfacilitatestheirassimilation
thatnocompetinginterestsexist.
intothecellwallaseitherligninorsuberin[8].Whereasdroughtstressduetowaterdeficit
conditionsisgenerallyassociatedwithelevatedtemperaturesandsolarheat.Thoughthetoler-
ancetodroughtstressisobservedinallplants,whiletheextentoftolerancediffersfromspecies
tospecies[9].Onthecontrary,theconditionoffloodalsoimposesacriticalprobleminterms
ofstresswhichiscausedbyoxygeninsufficiencyleadingtowiltingthoughtherootissur-
roundedbysurpluswater.Thisconditionsubsequentlyinfluencesnutrientuptakebyreducing
thepotassium/sodiumionuptakeinhibitingthetranslocationofpotassiumiontotheplant
shoot[10,11].Thestateoflowoxygenconcentrationsorhypoxiainsoilsisduetothelowoxy-
gensolubilityinwater[12].Ontheotherhand,salinitycausescelldehydrationbyosmotic
imbalanceandcytoplasmicwatereliminationandthisleadstodropinvacuolarandcytosolic
volumes.Becauseofsalinitystress,theremaybeaccretionorreductionofaparticularsecond-
arymetaboliteinplanta[13]likeanthocyaninsshowanincreaseincontentinresponsetosalt
stress[14].Onthecontrary,salt-sensitivespeciesexhibitadecreasedlevelofanthocyanin
undersalinitystress[15].
Previously,immenseresearchhasbeencarriedoutinanefforttounderstandthestresstol-
erancemechanisminplants.Significantcandidateproteins,genesandpathwayshavebeen
recognizedutilisingOmicsapproachesreferringtohugesetsofbiologicalmolecules[16].
GenOmicsimplyforidentificationofgenes,transcriptOmicsformRNA,proteomicsfor
protein,andmetabolOmicsformetabolitesidentification[16].Despitethefactthatseveral
molecularmechanismsrelatedtostresshavebeenrevealedinotherplants[7–13],however,
completeunderstandinginO.tenuiflorumisstilllacking.Inthisregard,transcriptomicstrate-
gieshaveopenedtheavenuesincomprehendingplants’responseagainstabioticstresses[17–
18].Aschematicoverviewoftheprocessofabioticstressandactivationofthegenesin
responsetothesestresseshasbeenprovidedinFig1.
Ocimumtenuiflorum(2n=16)isoneofanimportantmedicinalandaromaticplantbelong-
ingtothefamilylamiaceaewhichisgrownwidelyworldwide[19].Lately,O.tenuiflorumhas
beeninvestigatedforitsgenomeinformationandthesecondarymetabolicpathwayrelated
genes[20–22].However,inthecurrentscenarioofclimatechange,themolecularstudiesof
theplantagainstthemajorabioticstressesstillremainelusive.ItiswellestablishedthatO.
tenuiflorumismostsusceptibletocoldstressbuttheeffectofdrought,floodingandsalinity
stressesonthisplanthasyetnotbeeninvestigated.Consideringalltheseabovepoints,the
presentstudywasexecutedtogetaninsightofthetranscriptomicchangesoccurringintheO.
tenuiflorumplantundercold,drought,floodandsalinitystresses,separately.Transcriptscom-
monaswellasexclusiveineachoftheabioticstresseswerealsoanalysed.
Materialsandmethods
Abioticstresstreatmenttotheplantsandtheirsampling
Seedsof‘CIM-AYU’varietyofO.tenuiflorumwerecollectedfromNationalGeneBankof
CSIR-CentralInstituteofMedicinalandAromaticPlants,India.Thepotexperimentwascon-
ductedatGlass-housefacilityofCIMAP,Lucknow,U.P.India(26.8946˚N,80.9815˚E).The
seedsweresowninearthenpots(3000cm3)intheglasshouseprovidingaphotoperiodof14h
naturallight/10hdark,85%relativehumidityandtemperatureof250C/200C(day/night).
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Ocimummetabolomicsinresponsetoabioticstresses
Fig1.ASchematicoverviewoftheactivationofthegenesinresponsetofourabioticstresses(Cold,Flood,
DroughtandSalinity).Abbreviations:ROS(ReactiveOxygenspecies),cAMP(cyclicadenosinemonophosphate),
CDPK(Calciumdependentproteinkinases),cdk(cyclindependentkinase),cdkR(cyclindependentkinases
regulatory),CaCmpk(Cacalmodulindependentproteinkinases),MAPK(mitogenactivatedproteinkinases,PLC
(phospholipaseC),SOS(saltoverlaystress),PLD(phospholipaseD),NAC(NAM(noapicalmeristem),
ArabidopsisATAF1/2andCUC2),ICE(inducerofCBFexpression1),bZIP(basicLeucineZipperDomain),ABA
(abscisicacid),CaM(calmodulin),AP2EREBP(AP2(APETALA2)andEREBPs(ethylene-responsiveelementbinding
proteins),LEA(lateembryogenesisabundant),andCOR(coldresponsive).
https://doi.org/10.1371/journal.pone.0210903.g001
Twomonthsoldplantsaftergerminationwereusedforthestresstreatment.Eachtreatment
offlood,drought,saltandcoldwasprovidedto10plantsalongwithcontrolofsamedevelop-
mentalstageandpooledsampleswerecollectedforlibrarypreparation.Stresstreatmentswere
giveninthesoil(sandyloamtype)mixedwithvermi-compostintheratioof3:1.Conditions
ofallthestresstreatmentswereoptimizedwithdifferentparametersasshowninS1Fig.and
onlysinglesampleofeachabioticstressexhibitingseverestresswerechosenfortranscriptome
sequencing.Floodtreatmentwasgiventotheplantsbyconstantlymaintainingthewaterlevel
0.5inchesabovethesoilintheearthenpots[23].0.5inchwaterlevelwasmaintainedinflood
treatmentplantssoastoavoidthewaterlossduetoevaporation.Thetreatmentwascontinued
upto30daysandthereafterthesampleswerecollected.Incaseofdroughtstress,plantswere
maintainedat20%fieldcapacityfor30daysasdescribedbyChauhanandAbugho[24].O.
tenuiflorumplantsweresubjectedtodroughtstresswith20%,40%and60%fieldcapacities
andoutofthis20%fieldcapacitysamplewasselectedforthetranscriptomesequencing.
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Ocimummetabolomicsinresponsetoabioticstresses
Salinitystresswasgivenin5concentrationsviz.25mM,50mM,75mM,100mMand125mM
NaClbyirrigatingthetestplantpotswith100mlofNaClsolution[25]constantlyfor10days
andateveryalternatedayduringthecompletetreatmentcycleof30days.Topreventthe
plantsfromosmoticshock,treatmentwasstartedwithlowerconcentrations,gradually
increasingtheconcentrationdaily,untileachgroup.However,sampleof125mMsaltwith
severestresswasselectedfortranscriptomesequencing.Coldtreatmentwasgiventotheplants
byincubatingtheminacoldroomat40Cbymaintainingtheoptimallightrequiredbythe
plant[26].Thecoldtreatmentsamplewascollectedafter72hoursasstandardizedbytheprior
experimentsbecauseexceedingtheincubationtimeleadstoplantdeath.
Commoncontrolplantswerekeptinnormalconditionswithregularwateringtomaintain
thesoilmoistureuptothewaterholdingcapacityofthesoil.Leafsamplesofdrought,flood
andsalinitystresstreatmentswereharvestedafter30dayswhilethecoldtreatmentwaspro-
videdatthethirdlastdaysuchthatallthecontrolshadthesametimepointforsamplecollec-
tion.Leafsamplesfromthewholeplantwerecollectedandpooledforsequencingintriplicates
aswellasothermolecularandmetaboliteanalysis.SampleswerestoredinRNAlater(Sigma
Aldrich)at-800Cuntiluse[27].
TotalRNAisolationanditsquantitativeandqualitativeanalysis
TotalRNAwasisolatedfromtheleafsamplesbyZRPlantRNAMiniprep(ZymoResearch,
Irvine,CA,USA)aspersupplier’sprotocol[28].QualitativeanalysisoftheisolatedRNAwas
carriedoutusing1%DenaturingRNAagarosegelandwasquantifiedusingNanoDrop.
Librarypreparation,transcriptomesequencingandassembly
IlluminaNextSeqpairedend(PE)librarieswerepreparedfromthequalitycheckpassedRNA
samplesviailluminaTruSeqstrandedmRNAsamplepreparationkit(Part#15008136;Rev.A;
Nov2010)followingtheprotocolasstatedin“TruSeqRNASamplePreparationGuide”[29].
ThePCRenrichedlibrarieswereanalysedin4200TapeStationsystem(AgilentTechnologies)
withHighsensitivityD1000Screentapeasperthemakers’instructions.PEilluminalibraries
wereloadedontoNextSeq500forclustergenerationandsequencinginbothdirections,apply-
ing2x75bpchemistry.Therawsequencingdatawasthenprocessedtogetthehighquality
cleanreadsusingTrimmomaticv0.35[30]togetridofadaptersequences,confusingreads
(unidentifiednucleotidesbiggerthan5%),andthebadqualityreads(havingqualitythreshold
(QV)morethan10%andphredscorelessthan20).Aminimumlengthof50nucleotides
aftertrimmingwasapplied.Denovoassemblywasthencarriedoutwiththesehighquality
(QV<20),paired-endreadsusingTrinityRNA-Seqassemblersetatdefaultparameters.
Greatquantityofmisassembledtranscriptsanderroneoustranscriptsmayappearduringthe
sequenceassembly;hence,allhighqualitysequencereadsweremappedbacktothecorre-
spondingassembledtranscriptsusingBWAv0.7.12[31]forvalidationoftranscript.Thenon-
redundanttranscriptswerefurtherclusteredtogetherusingCD-HIT-EST-4.5.5[32]at95%
identityandquerycoverage.Thesearecalledtheunigenes(thesequenceswhichcouldnolon-
gerbeextended).NGSsequencingdatageneratedinthecurrentinvestigationissubmittedto
NCBISRA(ShortReadArchive).ThebioprojectIDassignedis:(PRJNA390220)https://www.
ncbi.nlm.nih.gov/bioproject/PRJNA390220).
Codingsequencepredictionandfunctionalannotation
TheTransDecoder(http://transdecoder.github.io)wasusedtopredictcodingsequences
(CDS)fromtheunigenes[33].NCBInonredundant(NR)proteindatabasewasusedto
searchthepredictedCDSswiththehelpofBasiclocalalignmentsearchtool(BlastX)
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Ocimummetabolomicsinresponsetoabioticstresses
(Evalue1e-05).Blast2GOprogramwasusedtorevealtheGeneontology(GO)annotations
oftheCDSs.Further,thepredictedCDSswereclassifiedfortheirrolesthroughGOassign-
ments.ThreecriterionswerefollowedtoretrieveGOtermsfromtheannotatedCDSsinthe
courseofGOmapping.First,accessionIDsfromtheBlastXresultwereutilizedtoreclaim
genesymbolsornames,identifiedgenesymbolsornameswerethenexploredinthespecies
specificrecordsofthegeneproducttablesfromGOdatabase.Secondly,UniProtIDswere
retrievedusingtheBlastXresultaccessionIDswiththehelpofProteinInformationResource
(PIR)whichconsistsofdatabaseslike-ProteinSequenceDatabase(PSD),UniProt,Swis-
sProt,TrEMBL,RefSeq,GenPeptandProteinDataBank(PDB).Thirdly,dbxrefandgene
producttablesofGOdatabaseweredirectlyexploredtosearchfortheaccessionIDs.
ToidentifythepotentialinvolvementofCDSoffourabioticstresssamples(cold,flood,
drought,salt)andcontrolinbiologicalpathways,CDSweremappedtostandardpathways
inKEGGdatabase.TheresultofKEGGstudyconsistsofKEGGOrthology(KO)assign-
mentsandCorrespondingECnumbersandmetabolicpathwaysofthepredictedCDSusing
KEGGautomatedannotationserverKAAS[34].Asupplementarydatasetforalltheannota-
tionsoftranscriptsincontrol,cold,flood,droughtandsaltstresseshavebeenprovidedin
S1Dataset.
Differentialgeneexpression
ThegoodqualityreadsofeachstresssampleweremappedtotheircorrespondingsetofCDS
usingBWAalignerforreadcountcalculation.Thecommonhitaccessionswererecognized
fordifferentialgeneexpressionstudiesonthebasisofBLASTresultsagainstNRdatabase.A
negativebinomialdistributionmodel(DESeq1.8.1package)(http://www-huber.embl.de/
users/anders/DESeq/)[35]wasemployedtoperformdifferentialexpressionanalysis.Method-
blind,sharingmode-fitonlyandfittype-localparameterswereusedtoestimatethedisper-
sionvalues.FurtheronthebasisofformulaFC=Log2(Treated/Control)thelogfoldchange
(FC)wasdeterminedinordertoclassifythegenesasup-ordown-regulated.Acomprehensive
analysisoflinkagehierarchicalclusterwasexecutedusingmultipleexperimentviewer(MEV
v4.8.1)[36]onbesthundreddifferentiallyexpressedgenes.Heatmapclustershowstheexpres-
sionofgenes.Levelofexpressionisrepresentedlog2ratioofgeneabundancebetweencontrol
vs.cold,controlvs.flood,controlvs.drought,andcontrolvs.salt.Log-transformedandnor-
malizedvalueofgeneswasusedtoconstructtheheatmaponthebasisofPearsonuncentered
correlationdistanceaswellasbasedoncompletelinkagemethod.Venndiagramswerepre-
paredusingVenny’son-line[37].AsupplementarydatasetfortheDGEofControlvs.Cold,
Controlvs.Flood,Controlvs.Drought,andControlvs.Saltwithannotationshavebeenpro-
videdintheS2,S3,S4andS5Datasets,respectively.
Metaboliteanalysisandcomparativeprofiling
Comparativemetaboliteprofilingofdifferentstresssamples(Cold,flood,droughtandsalt)
wasperformedasdescribedbyAkhtaretal.[38]usingGasChromatography(MSD7890A,
AgilentTechnologies)equippedwithautosampler,HP-5MScolumnand7977Amassdetector.
Foridentificationandquantificationofmetabolites,100mgoffreshleavesweregroundinice
coldMTBE(tert-butylmethylether)(SiscoResearchLaboratory,India)alongwithtoluene
(SiscoResearchLaboratory,India)asinternalstandard.Theextractsweredehydratedbyadd-
inganhydroussodiumsulfateand1μloftheeachextractswereinjectedtoGC-MSsystem.All
thesampleswererunintriplicatesandtheaveragewithstandarddeviationwereconsidered
fordatainterpretation.
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Ocimummetabolomicsinresponsetoabioticstresses
Quantitativeandsemi-quantitativeRT–PCRanalysis
SYBRGreenchemistry(AppliedBiosystems)wasappliedforperformingthequantitativeRT–
PCRasdescribedbyRastogietal.[21].PrimerdesigningforthevalidationofDGEandto
studytheeffectoffourabioticstressesonthephenylpropanoidpathwaygenesthrough
qRT-PCRwascarriedoutwiththehelpofsoftwarePrimerExpressversion2.0(AppliedBio-
systems)andwereprocuredfromEurofinsGenomicsIndiaPvt.Ltd.(S1Table).Triplicatesof
eachreactionwererunonFastRealTimePCRSystemmodel‘7900HT’(AppliedBiosystems)
[21,22].
Resultsanddiscussion
Denovotranscriptomesequencingandassembly
TheRNAseqPEsequencinglibrariesweremadeusingtheRNAsamplesofallthefourstresses
(cold,drought,flood,andsalt)andacontrolleafsample.PEilluminalibrariesweresequenced
onNextSeq500using2x75bpchemistry.Themeanofthelibrariesfragmentsizedistribu-
tionswere434bp,399bp,419bp,467bp,and381bpforcontrol,cold,drought,flood,andsalt
samples,respectively.ThesequencedrawdatawasprocessedusingTrimmomaticv0.35in
ordertoobtaincleanhighqualityreads.StatisticalsummaryofRNAsequencingandassembly
hasbeenprovidedinS2Table.Denovoassemblywasthencarriedoutusingthesepairedend
reads.ThefilteredhighqualityreadswereassembledintotranscriptsusingTrinityRNAseq
assembler.Atotalof40,560(control),32,731(cold),36,502(drought),34,650(flood)and
27,811(salt)codingsequences(CDS)wereidentifiedfromwithin64,603(control),55,891
(cold),58,018(drought),55,956(flood)and46,235(salt)unigenetranscripts(S3Table)using
TransDecoder.SummaryofunigeneandCDSstatisticsispresentedinS3Table.Deviationin
thenumberofassembledunigenetranscriptofcontrolandstress(cold,drought,floodand
salt)samplesmaypossiblybebecauseoftheinconsistentresponseoftheplantsagainstthe
stresses,ormighthaveresultedduetosometechnicalnoisearisenatsomestepofsequencing
procedure[39].Theassembledunigenetranscriptsrangedbetween201to>13,338basesin
length.Distributionnumberofcodingsequences(CDS)andunigeneaccordingtotheirlength
hasbeenprovidedintheS2Fig,wherethehighesttranscriptsamongtheassembledtranscript
dataofallthesampleswereinthesizerangeofbelow500bp,subsequentlytranscriptsranging
between1,001–1,500bp.Thecomparablerangeoftranscriptlengthandabundancehasalso
beenpriorreportedfromO.tenuifloruminacomparisonofitstranscriptomedatawiththatof
O.basilicum[22].
Differentialgeneexpression
Differentialgeneexpressionanalysiswascarriedoutbasedontheidentificationofthecom-
monhitaccessionsbasedonBLASTagainstNRdatabaseperformedusingDESeq.Atotalof
14,639(cold),16,664(flood),15,181(drought)and12,899(salt)transcriptswerecommonin
eachstresswithrespecttothecontrol.Further,thesegeneswerecategorizedasup-regulated
ordown-regulatedbasedontheirfoldchangevalues.Geneswithfoldchangevaluemore
thanzerowereconsideredtobeup-regulatedwhereaslessthanzeroasdown-regulated.Sta-
tisticallysignificantresultswerefilteredusingthethresholdp-valueandp-adjustedvalue
�0.05.Inflood,droughtandsalttreatmentssignificantBenjamini-HochbergadjustedPval-
ues�0.05[40]wereobtained,whileitwasnotsoincaseofcoldstresstreatment,hence,the
transcriptswithp-value�0.05wereconsideredforanalyzingthedifferentiallyexpressed
transcriptsundercoldstress.VenndiagramintheFig2Asummarizesthenumberofgenes
whichareup-regulated,down-regulated,havingnochangeandalsothegenesexclusiveto
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Ocimummetabolomicsinresponsetoabioticstresses
Fig2.Differentiallyexpressedgenesdistribution.(A)Venndiagramsrepresentingcommonlyup-regulated,down-
regulated,neutralandexclusivetranscriptsineachstressconditionwithrespecttocontrol.(B)Volcanoplotrepresents
significantlytheup-anddown-regulatedtranscriptshavingfoldchanges>1and<-1,respectivelywhereeachpoint
signifiesaDEG.Pointslyingabovethehorizontallineandlefttotheverticallineweresignificantlydown-regulated
(p<0.05)whiletheonesbelowthehorizontallineonthesamesidewerenon-significantlydown-regulated.Onthe
otherhand,pointsabovethehorizontallineandrighttotheverticallinewerefoundtobesignificantlyup-regulated
whiletheonesbelowthehorizontallineontherespectivesidewerenon-significantlyup-regulated.
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Fig3.Differentiallyup-regulatedanddown-regulatedtranscriptsinallthe4stresses.(A)Venndiagram
illustratingthecommonandexclusivetranscriptsthataredifferentiallyup-regulated.(B)Venndiagramillustrating
thecommonandexclusivetranscriptsthataredifferentiallydown-regulated.(C)Bargraphrepresentingthe
differentiallyup-regulatedtranscriptsclassifiedontheirfoldchangeexpressionbasis.(D)Bargraphrepresenting
differentiallydown-regulatedtranscriptsclassifiedontheirfoldchangeexpressionbasis.
https://doi.org/10.1371/journal.pone.0210903.g003
eachofthefourabioticstresses(cold,flood,drought,salt).Fromthevenndiagram,itcould
beinferredthatmaximumnumberofcommonlyexpressedtranscriptsshowednochangein
allthefourabioticstresseswithrespecttocontrol.However,transcriptsshowingdown-regu-
lationwerehigherthantheup-regulatedonesinallthestressesexceptthefloodingstress
andthetranscriptsexclusivetostresswerelowerthancontrolexceptindroughtstresswhere
theexclusivetranscriptsweremoreinnumberascomparedtocontrol.Whereasthevolcano
plotsintheFig2Brepresentsignificantlytheup-anddown-regulatedtranscriptshavingfold
changes>1and<1,respectively.Transcriptsabovethehorizontallineandlefttothevertical
lineweresignificantlydown-regulatedwhiletheonesbelowthehorizontallineonthesame
sidewerenon-significantlydown-regulated.Ontheotherhand,transcriptsabovethehori-
zontallineandrighttotheverticallinewerefoundtobesignificantlyup-regulatedwhilethe
onesbelowthehorizontallineontherespectivesidewerenon-significantlyup-regulated.
However,thetranscriptsthatliebetweenthetwoverticallineswereneutralinexpression.A
totalof255(cold),693(flood),601(drought),and369(salt)geneswerefoundtobeup-regu-
latedinthedifferentialgeneexpressionwhile332(cold),561(flood),665(drought),and539
(salt)genesweredown-regulated.Fig3Aand3BrepresentsseparateVenndiagramofthe
up-anddown-regulatedgenesshowingthecommonaswellastheexclusivegenesexpressed
inthecourseofstresscondition.Itwasobservedthat33transcriptswerecommonlyup-
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Ocimummetabolomicsinresponsetoabioticstresses
regulatedwhileonly7transcriptswerecommonlydown-regulatedinallthefourstresses,
theannotationsofthesetranscriptsareprovidedinS4TableandsequencesprovidedinS5
Table.Inallthefourabioticstressesofthepresentinvestigation,outofthe33commonly
up-regulatedand7commonlydown-regulatedtranscripts,20and3transcripts,respectively
(S4Table),weretheoneswhichwerepreviouslyreportedtobeinvolvedinstressresponse
[41–48].PrimaryroleofPP2Cgenesisinstresstolerance,particularlyABAresponse,hence
up-regulationofthistranscript(CDS_21223_Unigene_34825)inallthelongtermstresses
mayeasilybeattributed[49].F-boxproteins(CDS_721_Unigene_1749)inplantsare
reportedtobeinducedbyabioticandbioticstresses[41].Na+/H+(AtNHX1)andK+/H+
(AtNHX3)antiportersfromArabidopsishavebeendemonstratedforimprovedtolerance
(CDS_39398_Unigene_61545)againstsaltanddroughtstressesintransgenicpoplar[42].
Cytosolicsulfotransferase(OsSOT9)genefromricehasbeenreportedasanovelcandidate
(CDS_38924_Unigene_60033)concernedtostresstolerance[43].Wangetal.[44]have
demonstratedthatRPN1a,theArabidopsis26Sproteasomesubunit(CDS_12189_Unigene_
22188)isnecessaryforoptimalgrowthofplantaswellasstressresponses.Though,cyclic
nucleotide-gatedionchannel(CNGC)familymembers(CDS_14008_Unigene_24746)are
recognizedfortheirroleinthecationsuptakeofNa+,K+andCa2+ionsthereby,regulating
growthanddevelopmentofplant,Jhaetal.[45],havealsodemonstrateditsroleinimparting
abioticandbioticstresstolerance.Commonlydown-regulatedtranscriptsamongallthe
fourabioticstressesinthepresentinvestigationare-coiledcoildomain-containing150
(CDS_26797_Unigene_42479),flavanone3-dioxygenase-like(CDS_20547_Unigene_33835),
methylthioribosekinase(CDS_1507_Unigene_3270),andGDSLesteraselipaseAt4g18970-
like(CDS_24654_Unigene_39576).Plantlongcoiled-coilprotein,F3H(2-oxoglutartate-
dependentdioxygenase)andGDSLlipasehavebeenreportedtobeinvolvedinaplant
defenseresponse[46–49]andsincethesetranscriptswerefoundtobedown-regulatedinall
thestressesofthepresentinvestigation,makingthedefensesystemofplantweak,toresist
theabioticstressesimposedontheplant.Ahighestrangeof10–25foldchangeexpression
wasobservedinbothup-anddown-regulatedgenesfollowedby25–50fold(Fig3Cand3D).
Acompletelinkagehierarchicalclusteranalysiswasalsocarriedoutontop100differentially
expressedgenesusingMEVv4.8.1,representingtop50up-regulatedandtop50down-regu-
latedtranscriptsineachstress.Levelofexpressionintheheatmapgeneratedrepresentslog2
ratioofgeneabundancebetweencontrolvs.eachofstresssamples(cold,flood,drought,
salt)discretely(Fig4,S6Table).
GeneontologyandKEGGpathwayanalysis
CandidatecodingregionsortheCDSpredicted(S7Table)fromtheunigenesweresearched
againstthenon-redundantproteindatabaseofNCBIusingBlastx.Morethan94%oftheCDS
ineachsamplegotannotatedwhilelessthan4%showednosignificantBlasthit(S7Table).
Blast2GOprogramwasusedforgeneontology(GO)andassigningrolestothepredictedCDS
(S8Table).PredictedCDSofcontrol,cold,drought,flood,andsaltweremappedontothe
standardpathwaysavailableintheKEGGdatabase[50].CDSofallthesampleswerecatego-
rizedunderfivecategories:Metabolism,Cellularprocesses,Geneticinformationandprocess-
ing,EnvironmentalinformationprocessingandOrganismalSystems.Atotalof11,107
(control),9,678(cold),10,201(flood),8,073(drought)and6,890(salt)CDSgotfunctionally
assignedinKEGGoutofwhichonly441(control),379(cold),413(flood),298(drought)and
260(salt)CDSwereattributedtothemetabolismandbiosynthesisofsecondarymetabolites
(S3Fig,S6Dataset).CDSexhibitingsignificantupanddown-regulationwithpvalue�0.05in
allfourstresseswerealsomappedinKEGGdatabaseagainsttheterms.
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Ocimummetabolomicsinresponsetoabioticstresses
Fig4.Heatmapofdifferentiallyexpressedgenes.(A)Controlvs.Cold(B)Controlvs.Flood(C)Controlvs.Drought
(D)Controlvs.Salt.
https://doi.org/10.1371/journal.pone.0210903.g004
Differentiallyexpressedgenes(DEGs)—Enrichmentanalysis
EachofthefourstresssampleswereexploredandanalyzedtorecognizethatwhichoftheGO
termsandKEGGpathwaysgotenriched.TheFDRwasalsocalculatedinordertoidentifythe
percentageoffalsepredictionsinthepredictiondataset.Incold,droughtandsaltstresses46
GOtermswhileinfloodstress33GOtermsfortheDEGswerefoundsignificantlyenriched
(FDR�0.05).Underthebiologicalprocesscategory,“cellularprocess”,“metabolicprocess”
andthe“localization”GOtermswerethemostaffectedandthereforeenrichedsignificantlyin
allthefourabioticstresses(Fig5).Theseprocessesareimperativetothegrowthofplantand
itssurvival[39];however,theymayalsobeindicativeoftheplantresponseagainstfourdiffer-
entstressesgiventotheplantinthisinvestigation.Inthemolecularfunctioncategory,a
noticeableeffectwasobservedintheGOtermsexhibiting“catalyticactivity”,“binding”,
“transporteractivity”,and“structuralmoleculeactivity”(S4Fig)representingthedominionof
signaltransductionandthegeneticregulationoftheactiveenzymaticprocesses[39].Inaddi-
tiontotheGOtermsenrichment,theKEGGpathwayenrichmentintheup-regulatedandthe
down-regulatedDEGswasalsocarriedoutusingKOBAS3.0webserver(S9Table,S7Data-
set).InKOBASanalysis,only10and4transcriptsup-regulatedinfloodingandsalttreatments,
respectively,werefoundtobehighlysignificanti.e.withcorrectedp-value(padjusted)
of�0.05.Theimportantfloodup-regulatedDEGsweretheGOterms-“Biosynthesisofsec-
ondarymetabolites”(correctedp-value=0.0002),“Flavonoidbiosynthesis”(correctedp-
value=0.0295),“Limoneneandpinenedegradation”(correctedp-value=0.0544),and“Stilbe-
noid,diarylheptanoidandgingerolbiosynthesis”(correctedp-value=0.0205).Since,second-
arymetabolitesinplantsplayanimportantroleinstressresponses[51],thereforetheseterms
relatedtosecondarymetabolitesmighthavegotup-regulated.Highlysignificantup-regulated
DEGsGOtermsinsalinitystressi.e.withcorrectedp-value�0.05,were“Carbonmetabo-
lism”,“Spliceosome”,“Carbonfixationinphotosyntheticorganisms”,“Pentosephosphate
pathway”etc.Inadditiontothechangesincarbonandstarch/sugarmetabolism,ribosomes
alsoplayanimportantrole.Inresponsetotheenvironmentalstressestheplantsorganizethe
interactionbetweeneukaryoticandprokaryoticribosomestomaintainthebalanceofprotein
PLOSONE|https://doi.org/10.1371/journal.pone.0210903 February6,2019 10/26