Comparative Genomics of NAC Transcriptional Factors in Angiosperms: Implications for the

21 Pages·2015·2.72 MB·English

RESEARCHARTICLE
Comparative Genomics of NAC
Transcriptional Factors in Angiosperms:
Implications for the Adaptation and
Diversification of Flowering Plants
AlejandroPereira-Santana1,LuisDavidAlcaraz3,EnriqueCastaño2,LeninSanchez-
Calderon4,FelipeSanchez-Teyer1,LuisRodriguez-Zapata1*
1 UnidaddeBiotecnología,CentrodeInvestigaciónCientíficadeYucatán,Mérida,Yucatán,México,
2 UnidaddeBioquímicayBiologíaMoleculardePlantas,CentrodeInvestigaciónCientíficadeYucatán,
Mérida,Yucatán,México,3 DepartamentodeEcologíadelaBiodiversidad,InstitutodeEcología,
UniversidadNacionalAutónomadeMéxico,MéxicoCity,D.F.,México,4 LaboratoriodeBiologíaMolecular
dePlantas,UnidadAcadémicadeCienciasBiológicas,UniversidadAutónomadeZacatecas,Zacatecas,
Zacatecas,México
*[email protected]
OPENACCESS
Abstract
Citation:Pereira-SantanaA,AlcarazLD,CastañoE,
Sanchez-CalderonL,Sanchez-TeyerF,Rodriguez- NACproteinsconstituteoneofthelargestgroupsofplant-specifictranscriptionfactorsand
ZapataL(2015)ComparativeGenomicsofNAC
areknowntoplayessentialrolesinvariousdevelopmentalprocesses.Theyarealsoimpor-
TranscriptionalFactorsinAngiosperms:Implications
fortheAdaptationandDiversificationofFlowering tantinplantresponsestostressessuchasdrought,soilsalinity,cold,andheat,which
Plants.PLoSONE10(11):e0141866.doi:10.1371/ adverselyaffectgrowth.ThecurrentknowledgeregardingthedistributionofNACproteins
journal.pone.0141866
inplantlineagescomesfromrelativelysmallsamplingsfromtheavailabledata.Inthepres-
Editor:Jin-SongZhang,InstituteofGeneticsand entstudy,webroadenedthenumberofplantspeciescontainingtheNACfamilyoriginand
DevelopmentalBiology,ChineseAcademyof
evolutiontoshednewlightontheevolutionaryhistoryofthisfamilyinangiosperms.Acom-
Sciences,CHINA
parativegenomeanalysiswasperformedon24landplantspecies,andNACortholog
Received:June18,2015
groupswereidentifiedbymeansofbidirectionalBLASThits.LargeNACgenefamiliesare
Accepted:October14,2015 foundinthosespeciesthathaveexperiencedmorewhole-genomeduplicationevents,
Published:November16,2015 pointingtoanexpansionoftheNACfamilywithdivergentfunctionsinfloweringplants.A
totalof3,187NACtranscriptionfactorsthatclusteredintosixmajorgroupswereusedinthe
Copyright:©2015Pereira-Santanaetal.Thisisan
openaccessarticledistributedunderthetermsofthe phylogeneticanalysis.Manyorthologousgroupswerefoundinthemonocotandeudicotlin-
CreativeCommonsAttributionLicense,whichpermits eages,butonlyfiveorthologousgroupswerefoundbetweenP.patensandeachrepresen-
unrestricteduse,distribution,andreproductioninany
tativetaxaoffloweringplants.Thesegroupswerecalledbasalorthologousgroupsand
medium,providedtheoriginalauthorandsourceare
likelyexpandedintomorerecenttaxatocopewiththeirenvironmentalneeds.Thisanalysis
credited.
ontheangiospermNACfamilyrepresentsanefforttograsptheevolutionaryandfunctional
DataAvailabilityStatement:Allrelevantdataand
diversitywithinthisgenefamilywhileprovidingabasisforfurtherfunctionalresearchon
annotationsusedinthisworkareinthepaperand
supportinginformationfiles. vascularplantgenefamilies.
Funding:ThisworkwassupportedbyDGAPA-
PAPIIT-UNAMIA200514,LDA;andSEP-CONACYT
59097and153556,LRZ.
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist.
PLOSONE|DOI:10.1371/journal.pone.0141866 November16,2015 1/21
ComparativeGenomicsofNACTranscriptionalFactorsinAngiosperms
Introduction
Environmentalabioticstresses,suchaswaterdeficit,soilsalinity,andextremetemperatures,
haveadverseeffectsonthegrowth,development,andgrainyieldsofcropsworldwide[1,2].To
copewiththesestresses,landplantshavedevelopedmolecularmechanismstoprotectcellular
activitiesandmaintainplantintegrity[3].Transcriptionalcontrolunderenvironmental
stressesplaysamajorroleinplantadaptation.Duringabioticstress,manytranscriptionfactors
(TF)areinvolvedintheinductionofstress-responsivegenesthatplayimportantrolesunder
stressconditionssuchasdrought,cold,heat,soilsalinity,andflooding.Amongtheproteins
involvedinabioticstressresponsesaretheAP2/ERF[4,5,6],ABF[7,8],HSF[9],bZIP[7,10],
MYB[11],NAC[12,13,14],andtheWRKYfamilies[10,15].
NACproteinsconstituteoneofthelargestgroupsofplantTFs.Theyareubiquitously
expressedacrossplantorganismsandareknowntoparticipateinvariousdevelopmentalpro-
cessesandstressresponses.TheNACacronymisderivedfromthreegenesthatwereinitially
discoveredtocontainaparticulardomain(theNACdomain):NAM(noapicalmeristem),
ATAF1/2(Arabidopsistranscriptionactivatorfactor1/2)andCUC2(cup-shapedcotyledon,
[16,17]).Typically,theNACfamilycontainsahighlyconservedNACDNA-bindingdomain
locatedintheN-terminalregionandconsistsofapproximately150–160aminoacidsdivided
intofivesub-domains(A-E)encodingatwistedβ-sheetsurroundedbyafewhelicalelements
[18].TheC-terminaldomaincontainsthetranscriptionalactivationregion(TAR),whichis
non-conservedamongplantsandcanactaseitheratranscriptionalactivatororrepressor[19,
20].Inmanycases,NACTFsareinactivelystoredinthecytoplasmandtranslocatedintothe
nucleusafterstimulation.TheNAC'sTARcontainstransmembranemotifs(TMM)thathelp
anchortheproteintotheplasmamembraneandprovideanefficientmethodforgeneregula-
tion,whichisanadaptivestrategythatallowsforpromptresponsestoenvironmentalchanges
[21,22,23].NACTFsplayseveralessentialrolesinplantdevelopment[24],senescence[25],
auxinsignaling[26],floraltimecontrol[27],floralmorphogenesis[28,29],lateralrootdevel-
opment[26],nutrientmobilization[30],leafmargindevelopment[31],fruitripening[32],lon-
gevitycontrol[33],embryodevelopment[34],postembryonicshootmeristemformation[35],
organboundaryformation[35],leafmovement[36],vascularelementformation[37],andtol-
erancetomultiplebiotic[38,39]andabioticstresses[25,26,27,40,41],andsoon.Although
NACTFshavebeencharacterizedanddescribedinseveralplantsspecies,suchasArabidopsis
[19],populus[42],citrus[43],rice[19],andbarley[44](rangingfrom16–289NACsequences
[45]),asystematicclassificationisstilllacking,andgenome-wideanalysisofthisgenefamily
keepsbeingappliedwithintheplantlineage.
Polyploidyorwhole-genomeduplication(WGD)isnowrecognizedasamajorevolutionary
forceinangiospermgenomedevelopment[46].Infact,researchershavelongrecognizedthat
polyploidyisaninseparablepartofangiospermbiology.Overtime,polyploidsmaybecome
diploidized,suchthattheybehavelikediploidsatthecytogeneticandgeneticlevels[47].Given
thisgenomeduplicationtrend,thegenomeconservationintermsofgenenumberandchromo-
somalorganizationisastonishing.AftertheduplicationofallgenesbytheWDGprocess,
redundantcopiesgetlost(fractionation),retained,orfixedwithmodifiedfunctionalproperties
incontrasttotheoriginalgenes.Therefore,thegenefamilysizecanvarygreatlybetweenspe-
ciesduetothenumberofWGDeventsthattheirgenomeshaveexperiencedthroughevolution
[48,49].DespitetherelativelyrecentoriginofangiospermsduringtheEarlyCretaceousperiod
~130to>385millionyears(MYr)ago,[50,51,52]landplantevolutionhasbecomeextremely
diversebothinmorphologicalandecologicalterms.Overthepast130and150MYr,angio-
spermshavediversifiedtooccupyallhabitableterrestrialandmanyaquaticenvironments[53].
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ComparativeGenomicsofNACTranscriptionalFactorsinAngiosperms
ThecurrentknowledgeregardingtheNACdistributionintheplantlineagecomesfroma
relativelysmallsampling[1,49,54,55].Toshednewlightintotheevolutionaryhistoryofthe
NACfamilyinangiosperms,webroadenedthenumberofplantspeciestoassesstheoriginand
evolutionofthisfamilyinthepresentstudy.Weconductedacomprehensivephylogenetic
analysisthroughthislineage,anddefinedtheorthologousgroups(OGs)between24green
plantgenomesinordertoexaminethegroupsthatwereidentifiedfromtheearly-diverging
plantPhyscomitrellapatensasanout-groupgenometoinspectthespecies-specificexpansion
alongtheangiospermslineage.Ourresultsrevealedthatanincreasednumberofpredicted
NACfamilymemberswithprobabledivergentfunctionsarepresentinthosespecieswith
moreWGDevents,suggestingthatasignificantexpansionoftheNACfamilyoccurredshortly
beforetherapidradiationoffloweringplants.
ThelackofknowledgeregardingtheoriginoftheNACfamilyhasimpededourunderstand-
ingoftheevolutionofthisfamilyacrossthehistoryofplantdevelopment.Therefore,givenour
conceptionoftheevolutionaryhistoryoftheNACfamilyaspartofamuchmorediversesetof
evolutionarilyplants,wecanupdateourknowledgeofthisfamilyanddeterminetheexpansion
ofthislineageovertime.Themainfocusofthisworkwastoconductacomparativegenomics
analysisoftheNACfamilyacrosstheangiosperms.
MaterialsandMethods
NACgenefamilysearchesandretrieval
AHiddenMarkovModel(HMM)profilewasconstructedfortheidentificationofnewmem-
bersoftheNACTFfamilyinfloweringplants.TheHMMprofilewasbuiltusingthePlant
TranscriptionFactorDatabasev3.0[45],inwhich436NACproteinswerepredictedfromfive
plants(O.sativa,V.vinifera,A.thaliana,S.moellendorffii,andP.patens).Sequenceswere
retrieved,andamultiplealignmentwascompletedwithClustalOmega[56].Subsequently,the
alignmentwasmanuallycurated,andthepositionsthatcontainedgapsormissingdatawere
notconsideredtobuildandcalibratetheHMMmodelbyHMMERpackage(version3.1;[57]).
Themodelwascalibratedwiththerequiredcut-offvaluesandwasusedtodetectNAC
sequencesin24plantspecies.Genemodelsoftheplantspeciesanalyzedinthisstudyare
showninS1Table.TheEMBOSS3.0suite[58]wasusedtomanipulatethesequences.The
genomesizeandtheploidylevelofeachplantwereobtainedfrompublishedgenomedataand
thePlantDNAC-valuesdatabase[59].
Sequenceanalysis,multiplesequencealignmentsandevolutionary
modeltesting
AminoacidsequencesweresubjectedtoamotifscaninthePfamdatabasev27.0[60]tocon-
firmthepresenceoftheNACdomain.Allretrievedsequenceswereclusteredusingthe
CD-HITclusteringprogram[61]withanidentitycut-offof0.7inordertoexcludeisoforms
andreducesequenceredundancy.MultiplesequencealignmentswerecompletedusingMUS-
CLE[62]andweremanuallycurated.TheywerethentestedwiththeProtTest2.4statistical
package[63]tofindthebestevolutionarymodelforthemaximumlikelihood(ML)analysis.
TheMEMEsoftware[64]wasusedtoidentifyconservedmotifsintheaminoacidsequencesof
interest,withanoccurrenceparameterforasinglemotifof“onepersequence”andthemaxi-
mumnumberofmotifs.TheTMHMMServerv.2.0(http://www.cbs.dtu.dk/services/
TMHMM/)wasusedtopredicttheTMMforallretrievedNACsequences.HMMlogosofthe
NACdomainwereplottedwiththeSkyligntool[65]foreachofthe24plantspeciesaswellas
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ComparativeGenomicsofNACTranscriptionalFactorsinAngiosperms
fortheglobalsetofalltheNACsequences.Theaminoacidalignmentsoftheselected
sequenceswerevisualizedusingBOXSHADEv3.31C(http://boxshade.sourceforge.net/).
Definingorthologousgroups(OGs)
WeusedareciprocalbestBLASThits(BBH)approach[66,67]toidentifypossibleorthologous
groupsamongthe24plantgenomes.Significanthitswerefilteredaccordingtothefollowing
criteria:weretainedallpotentialOGswheretheBLASTPbit-scoreofthecomparedproteins
wasmorethan200andhadsequencecoverageofatleast50%oftheirlengths[66].Protein
pairswerelabeledasorthologswhenbothsequenceswereeachother'sbi-directionalbesthit.
MossP.patens,abasallineageoflandplants,wasusedasareferenceoutgrouptoperformall
thecomparisonanalysisandtoestablishtheBBHapproach.Additionally,O.sativaandV.
viniferaNACproteinswereusedasreferencesequencesforanalysisintomonocotandeudicot
species,respectively[48].EachNACproteinofP.patenswascomparedwiththeNACprotein
setofthebasalMagnoliopsida(Vitisvinifera)andLiliopsida(Oryzasativa)species.Onthe
otherhand,aBLASTPsearchwasperformedagainstCoccomyxasubellipsoidea,Chlamydomo-
nasreinhardtii,Ostreococcuslucimarinus,Cyanidioschyzonmerolae(http://www.phytozome.
net,[68]),Cyanobacteria(NCBItaxid:1117),andAlphaproteobacteria(NCBItaxid:28211)to
determinetheoriginoftheNACproteins.AVenndiagramwascreatedonR(v3.0.1;[69])
usingtheVennerablepackage(https://r-forge.r-project.org/projects/vennerable/).
EvolutionaryanalysisandAngiospermtreeoflife
ThephylogeneticreconstructionswereRAxMLv.8.0.26suite[70]withaJTTsubstitution
modeland100parametricbootstrap(BS)replicates.Thegamma-distributedrateswereesti-
matedfromthedataset.Thetopologyofthebest-scoringtreewasvisualizedinFigTreev1.4
(http://www.molecularevolution.org)andPhyloWidget(http://www.phylowidget.org,[71]).
Anevolutionarytreeofthe24plantsusedintheanalysisoftheNACproteinswasconstructed
withthePhyloTtreegenerator(http://phylot.biobyte.de/index.html)andmanuallycorrected
accordingtotheAPGIII[72]treetopology.
ResultsandDiscussion
Genome-wideidentificationofNACgenes
Weanalyzedwhole-genomesequencesofalgaeandbacteria,andnosequencescontainingthe
NACdomainwerefound.Inearlydivergingplants,weidentified35NACproteinsinthemoss
P.patensusedasanout-group,andsevenofthosesequencescontainedaTMM,providingevi-
denceofthisbasalmechanismingreenplants.However,noTMMswerefoundinthe20pre-
dictedNACsofS.moellendorffii,theearliestevolutionarybranchofvascularplantsforwhich
genomeinformationisavailable.Incontrast,studiedfloweringplantspossessanaverageof
140NACgenes,withaminimumof71andamaximumof288genes,suggestingalineage-spe-
cificexpansionintotheNACfamilyshortlybeforetherapidradiationinAngiospermshistory
[53,73],likelyduetothenumerousWGDsthathaveoccurredthroughtheangiospermlineage
[46,74].Accordingtoourresults,theextentandnatureofthisexpansionvariesbetweenthe
differentplantlineages.Atotalof3,187sequenceswereidentifiedandretrievedfrom24plant
species(Fig1).Thesespeciesincluded;16eudicotsplants(ClassMagnoliopsida),6monocots
(ClassLiliopsida),onefern(Lycopodiopsida),andamoss(Bryopsida).AllidentifiedNAC
homologshosttheexpectedNAMdomain(PfamPF02365),whilesomecontainedadditional
domains.ThecompletelistofNACproteinsanalyzedinthisstudy,theadditionalannotation
ofeachsequence,andthesequencesusedinthephylogeneticanalysisareavailableinS2Table.
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ComparativeGenomicsofNACTranscriptionalFactorsinAngiosperms
Fig1.Phylogeneticrelationshipsamong24landplantspeciesandthedistributionofNACproteinsidentifiedinthisstudy,basedontheHMM-
generatedprofile.ThetotalnumberofNACproteinsidentifiedineachgenome;theplantgenomesize,ploidyandchromosomenumberofeachspecies;
andthenumberofNACsequenceswithTransmembranemotifs(TMM)foreachgenomeareindicatedontherightincoloredsquares.AncientWGDsare
representedbycoloredstars(detailsweretakenfromCoGepedia,https://genomevolution.org).Speciesnamesarecolor-codedasfollows:blue–Moss,
purple–pseudofern,red—monocots,andgreen—eudicots.*GenomesizeinGb.
doi:10.1371/journal.pone.0141866.g001
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ComparativeGenomicsofNACTranscriptionalFactorsinAngiosperms
AHMMLOGOoftheNACdomainwascompletedforeachspeciestoanalyzetheprevalence
ofspecificresiduesperlineage(S1Fig).
ThemonocotlineagerevealedasimilarnumberofNACsequencesinthesixanalyzedspe-
cies,regardlessoftheirgenomesize.MaizewasfoundtocontainthegreatestnumberofNAC
sequencesfromtheanalyzedmonocotspecies,with186NACgenes.However,italsowas
foundtocontainanadditionalWGDeventcomparedtotheothergrassspecies.Regardingthe
TMMsequences,nocorrelationwasfoundbetweenthenumberofthesesequencesandthe
genomesize,asseeninthecaseofA.tauschii,whichonlypossessesthreeTMMsequencesina
4.3Gbgenome,andB.distachtion,whichhassevenTMMsequencesina0.27Gbgenome.The
regulatedactivationofpreexistingdormantTFsbyTMMisaversatilesystemforaccurate
stresssignalperceptionandtransduction.Itprovidesanefficientmethodforgeneregulation
andisconsideredtobeanadaptivemechanisminresponsetoenvironmentalchanges[23,75].
Ontheotherhand,43ofthe3,187NACsequencescontainedadditionaldomains,mainlyin
theC-terminalregion(seeS2Table).TheAtaus42NACsequenceofA.tauschiicontaineda
zinc-bindingmotifinitsreversetranscriptasedomain(zf-RVT,PfamPF13966)andaReverse
transcriptase-likedomain(RVT-3,PfamPF13456)onitsC-terminus.TheZmays146NAC
sequencehasfourTMMhelixesandanadditionalmotifcalledMatE,whichisannotatedasa
Multiantimicrobialextrusionprotein(PfamPF01554)andisinvolvedinthedetoxificationof
secondarymetabolitesincludingalkaloids.Thislastmechanismispoorlystudiedinplantsys-
temsandcanhelpusunderstandhowplantshaveevolvedtocopewithtoxinsintheirenviron-
ment[76,77].
Itiswellknownthateudicotplantshaveasmallergenomesizethanmonocotplants;never-
theless,wefoundthatthenumberofNACsequenceswasgreaterintheeudicotlineage.Inthe
caseoftheTMMsequences,wefounda1:2ratiointhemonocotplantscomparedtotheeudi-
cotplants.Insomecases,theTMMsequencesfurtherincreasedforthosespeciesthatexperi-
encedmoreWGDevents,suchassomerosidspecies(e.g.,appletree,populus,andB.rapa).
Adetailedstudyofgenomesizerevealsadynamicpatternofgenomeevolution.Anincrease
inthegenomesizeoccurredoverthecourseofvascularplantdevelopment,withanindepen-
dentgenomereductioninSelaginellaandangiospermsandsubsequentincreaseswithinsome
groupsofmonocots[78].Hence,thereappearstobeagreaterrelativeexpansionoftheNAC
genefamily(140:35:20)inthespermatophyta(seedplants)comparedtomossandfernspecies
[79],demonstratingthatduplicatedgenescontributedtotheexpansionoflargefamiliesin
grapevinespecies.TheseduplicationswereproducedbyWGDandtandem,proximal,retro-
transposed,andDNA-basedtransposedduplications.InArabidopsis,ithasbeenshownthat
severalgenefamilieshaveexpandedbytandemduplication[80],retainingmanyoftheirgenes
afterWGDandtransposition[81].Eachofthesegenefamilyexpansionmodescreatesparalogs
thatpotentiallyduplicatesthefunctionoftheoriginalgene.Ifretained,thisfunctionalduplica-
tionsetsthestageforbiasedgeneexpansionandsubsequentsubfunctionalization[82].Some
genefamiliesineudicotplantsaremuchlarger,suggestingdifferentialexpansion(e.g.,the
NACfamilyinPoplarisrepresentedby288members,soybeanby246sequences,andapple
treeby245NACsequences).Ontheotherhand,somerosidspecies,suchasgrapevine,papaya
tree,andsoybean,containthelowestNACsequencenumbersoftheirfamilies,with71,77,and
75sequences,respectively.TheEurosidgroupcontainsmanyeconomicallyimportantflower-
ingplants,suchaslegumesandbrassicales;allofthesespeciesshareapalaeo-hexaploidcom-
monancestorthatemergedafterdivergencefromthemonocotsandbeforetheradiationofthe
eurosids[83].Grapevineandpapayatreeserveasevolutionaryimportantmodelplantsfor
fruittreegenomics[83,84].Withonly24,746predictedgenesforthepapayatreegenome(the
lowestpredictedgenenumberfortheeudicotplants[85])and26,346predictedgenesforthe
grapevine[83],thesetwoplantsappeartohaveexperiencedonlyoneWGDsharedbyall
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ComparativeGenomicsofNACTranscriptionalFactorsinAngiosperms
monocotandeudicotlineages.Thisobservationallowsustobetterunderstandtheancestral
traitsofthislineage.However,thegrapevinegenomeisalsotheclosesttothepalaeo-hexaploid
ancestralarrangementbyfar[83],suggestingthattheduplicationpatternsofancestralgenes
andtheadditionofnewactivitiesintheirfunctionswerecommonoccurrencesintheevolution
ofthisgenome[79].Forthesereasons,thepapayatreewasproposedasanexcellentoutgroup
forArabidopsis,whilegrapevinewasproposedasausefuloutgroupforBrassicales[86].A
resentreport[85]demonstratedareductioninmostgenefamiliesandbiosyntheticpathways
inthepapayaplantandhighlightedthevalueofthisplantasmodeltostudycomplexpathways
andnetworks.PapayacontainsnorecentWGDs,explainingitsloweropportunitiesforsub-
functionalizationandimplyingthatpapayagenesmaybemorerepresentativeofbasalangio-
spermsthanArabidopsisgenesandthoseofmanyothersequencedplants.Here,wefoundthat
thepapayatree'sgenomecontains77NACproteins.Thisstudyisthefirsttoreportandchar-
acterizetheNACfamilyinthepapayatreegenome.AlltheidentifiedNACgeneswerenamed
usingtheprefixCpNAC,andthecompletesetofthepapaya'sNACproteinsisavailablein
S3Table.
ThemajordifferencebetweentheNACproteinfamilynumberswithintheangiosperms
andwithintheoutgroupP.patenscouldbeduetocommonselectivepressuressuchasenviron-
mentalstresses,whichmayhaveguidedtheregulationofplantgrowthanddevelopment[87].
AnotherexplanationfortheNACgenefamilydifferencescouldbethereleaseofselectivepres-
suresduetogeneredundancy.Theeaseofsub-functionalityamongtheduplicatedTFscould
alsobeacontributingfactor.Afterduplication,TFsarecapableofacquiringdifferentfunctions
fromtheirancestorsandcouldbenaturallyselectedfortheirnewfunctions,thusresultingin
theindispensabilityofbothcopies[82].Genefamilynumberdifferencessuggestthegenesin
questionmaybeinvolvedinplant-specificregulatoryfunctions.Thedifferencesalsohighlight
theimportanceofTFduplication,dueitscontributiontotheregulatorynoveltiesthatcouldbe
involvedindevelopment,andresponsestoexternalstimuli[88,89].Otherauthors[79]have
foundthatlargegenefamiliesingrapevineandothersplantspeciesareessentialandare
involvedinthebasicprocessesofplantgrowth.Theseauthorshavealsosuggestedthatthese
familieshaveundergonelargeexpansionsduringtheirevolutionaryhistory,assupportedby
ourreportfortheNACfamily.
OrthologousgroupsandduplicationsintheNACfamily
OrthologousGroups(OGs)werecalculatedbymeansofaBBHusingall24analyzedprote-
omes.Then,amatrixcontainingpresenceinformationfromallpredictedOGswasbuiltand
usedtodefinethesharedcoreofOGsfortheNACproteins.Accordingtopreviouswork[90],
evolutionarygenomicscomparesasetofgenomeswithanoutgroupgenome.Totestthemini-
mumcontentoforthologousproteinsinmonocots,weusedrice(O.sativa)asareference
becauseitsphylogeneticpositionisclosesttothetestedplantsintermsofthehypothetical
basalmonocotsgenome.O.sativaisthoughttohaveanoriginalmonocotchromosomenum-
ber(12)andhasnotevolvedthroughindependentnestedchromosomefusion(NCF)events
[48].Ontheotherhand,grapevine(V.vinifera)wasusedasapivotalgenomeforeudicots
becausethegrapevinegenomecontainstheancientgenome-wideduplicationssharedbyall
eudicotsandhasthelowestnumberofNCFevents[48,83,91,92].P.patenswasusedasan
outgroupforcomparingthesetwopivotalgenomesformonocotsandeudicotsandtoidentify
OGsintheS.moellendorffiiplant.Thecomparisonacrossmultipleoutgroupsisausefultool
thatpermitsustounambiguouslydiscriminationbetweengeneadditionsandlossesinany
group[86].Weruledoutthe39homologousgenesthatdidnothavesignificanthitsaccording
tothecriteriapreviouslyestablished(seeMaterialsandMethods).
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ComparativeGenomicsofNACTranscriptionalFactorsinAngiosperms
FourOGswereidentifiedbetweenP.patensandS.moellendorffii,whichbranchedoffearlier
inplantevolution.TwoofthesequencesinS.moellendorffiiwereorthologoustothePpVNS1
andPpVNS5proteinsofP.patens.Thesegeneswerefoundtobeinvolvedinhydroidcelldif-
ferentiationbyinducingcelldeath,waterconductingandtissuesupportinvascularplants
[54].PpVNS1geneexpressionhasbeenfoundintherhizoidtissuesofP.patens,althoughthis
geneisnotsharedwithvascularplants.Infloweringplants,weidentifiedsevenOGsshared
betweenP.patensandeachrepresentativetaxaofmonocots(rice)andeudicots(grapevines)
(Fig2).AmongthesevenOGssharedbyP.patensandrice,twoofthembelongingtothe
PpVNSrelatedproteins(PpVNS6andPpVNS8)werefoundtoactascell-specializedregula-
torsforwater-conductionandplantsupport.Allsixanalyzedmonocotsshare24OGs,among
whicharethericecharacterizedsequencesOsNAC7(SND-relatedprotein),OsNAC4,
OsNAC6,andOsNAC10(stress-relatedproteins).However,onlysixOGsweresharedpairwise
(Osat-otherspecies).Atotalof43NACsequenceswerericespecific(notsharedwithotherspe-
cies).ThecompletelistofOGsandtheirparalogousgenesinmonocotsisavailableinS4Table.
Inthecaseofeudicotplants,wefoundsevenOGsamongtheP.patensandgrapevine
sequences,twoofwhichbelongedtothePpVNSrelatedproteins(PpVNS6andPpVNS7,
[54]),whichplayakeyroleinwaterconducting.Theseproteinsarepreferentiallyexpressedin
themidribofthecentralregionofnewlyemergedleavesandinthedevelopingleafmidripofP.
patens.WefoundthatthePpVNS6proteinwassharedforbothmonocotandeudicotplants,
unlikePpVNS7andPpVNS8proteinsthatwerespecificforeudicotsandmonocots,respec-
tively.Weidentified13OGsamongthe16analyzedeudicotspecies(S5Table),includingthe
Arabidopsis-characterizedsequencesANAC029(celldeathrelatedprotein),ANAC033(SOM-
BRERO),ANAC037(VND1),andANAC040(NTL8).OnlytwoOGsweresharedpairwise
(Vvin-otherspecies),and10sequenceswerespecifictograpevine.Ontheotherhand,we
found31OGssharedbetweenriceandgrapevine,thetwoselectedrepresentativetaxafor
monocotsandeudicots,respectively(S6Table).Allstudiedtaxainfloweringplantshostpara-
logousgenes,showingevidenceoflargeexpansionsinthisfamily.Finally,onlyfiveOGswere
sharedamongtheP.patens-rice-grapevine(seeFig2).WedecidedtocallthesetheBasal
OrthologousGroups(BOGs).
TheBOGsweretakenasthecoreNACsequences,fromwhichevolutionprogressed
throughdifferenteventsofgenome-duplication.Functionaldivergenceeventsamongthe
duplicatedgeneswerelikelythesourceofevolutionaryinnovationsandspecificadaptation
processesforeachlineage[46].
ComparativeanalysisofeachBOGwasdonebysequencealignment.P.patenssequences
wereusedasbasalgrouptounderlinecharacterchangesalongplantevolution.Weincluded
theorthologoussequencesfromtheanalyzedangiospermplantswiththeorthologousproteins
sharedinriceandgrapevinetobroadenthescopeoftheanalysis.Plesiomorphicaminoacids
weredetectedalongtheNACdomainandintheC-terminalregionthroughdiverselineagesof
plants,andlineage-specificsynapomorphiesformonocot/eudicotspeciesweredetectedinthe
NACdomain(S2Fig).However,theevolutionarydistanceoftheplantlineagesandamino
acidsintheNACdomainwereverysimilar,confirmingtheimportanceofthisdomainalong
itsevolutionaryhistory.Overall,theNACdomaininallfiveBOGshasstructuralconservation,
givingevidenceoftheimportanceofthisDNA-bindingdomainthroughtheplantlineage.
Furthermore,avisualinspectionoftheC-terminalregionofeachBOGwasperformed
usingtheMEMEsoftware[64],identifyingseveralconservedmotifsthatcouldbeconsidered
asthesignatureofthesebasalgroups(S3Fig).InthecaseofBOG1,allitsmembershavethe
signatureLP-box(LP[QX]L[ED]SP)andtheWQ-box(W[RA]ALD[KR][FL][VL]ASQL)that
werepreviouslydefinedastheresponsiblemotifsforthetranscriptionalactivityofgene
ANAC012(SND1;[93]).Someconservedmotifswerepresentbothinearlydivergingplants
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ComparativeGenomicsofNACTranscriptionalFactorsinAngiosperms
Fig2.SchematicrepresentationofNACOGssharedamongAngiosperms.A)Venndiagramshowingtheorthologousgenenumbersharedbetween
theP.patensoutgroupandriceandgrapevinegenomesastheselectedrepresentativesformonocotandeudicotplants,respectively.Thetotaloverlap
consistedoffiveproteinsthatdefinethebasalcoreoftheNACgenes.B)ThenumberoforthologousproteinsdetectedbymeansofBBHin24greenplants.
GreenlinesindicatetheOGssharedbetweenthemossP.paten,S.moellendorffii,andthebasaltaxaofthemonocotandeudicotlineages.Thenumber
belowtheredlineindicatesthenumberofOGssharedbetweenthericeandgrapevinespecies.BlacklinesindicatethenumberofOGssharedamongthe
monocotsandeudicotswiththeirrespectiveselectedpivotalspecies.
doi:10.1371/journal.pone.0141866.g002
andinmorerecentfloweringplantlineages,suggestingtheyoriginatedbeforethesplitofthe
monocotandeudicotlineages.MembersofeachOGaresupposedtobederivedfromthesame
ancestorgene,sharingwell-conservedmotifs,structuresandlikelyfunctions[49].Ourfindings
revealthepresenceoffiveancestralproteinspreservedbothinmonocotandeudicotlineages.
Theseproteinsprobablyplayedkeyrolesintheadaptationandsurvivaloffloweringplantline-
ageandexpandedthroughdifferentduplicationmechanismstocopewiththeenvironment
challenges.
NACphylogeneticanalyses
Weconductedaphylogenetictreereconstructionfrom24greenplantsinordertounderstand
theNACfamilyevolutionanddistributionthroughtheangiospermslineageandidentifyspe-
cies-specificNACgeneexpansionsorreductions.TheglobalsetofNACsequenceswas
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ComparativeGenomicsofNACTranscriptionalFactorsinAngiosperms
clusteredbyCD-HITsoftware([61];seeMaterialsandMethods)toreducesequenceredun-
dancy,resultingin2016non-redundantsequencesusedforthephylogeneticanalyses.The
phylogenetictreewasarrangedintosixmajorclusters,asshownintheFig3.
ThereweresomeconservedresiduesintheNACdomain,suggestingavitalroleintheir
structureduetotheirprevalencethroughdistinctlineages,asinthecaseoftheGxxFxPresi-
duesintoA-motif,theWresidueintotheC-motif,andtheWxMHEYsignatureintheD-
motif.Theimportanceoftheseresiduesshouldberegardedinfuturestudiestounraveltheori-
ginandevolutionoftheNACdomaininplantlineage.
Inthephylogeneticanalysis,sequencesfrommonocotsandeudicotstendtoclusterinthe
sameclades,suggestingalineage-specificexpansionafterdivergencefromtheircommon
ancestor(seeS4Fig).Arabidopsissequenceswereusedasreferencesforthephylogenomic
analysisoftheNACfamilybecauseoftheexperimentalsupportofthismodelspecies.The
NACgroupI(seeFig3)containsproteinsinvolvedincontrollingcellwallcomposition,bio-
synthesis,andxylemdevelopment[94].Accordingtoourresults,GroupIisproposedasthe
basalNACgroup,giventheessentialangiosperm-specificinnovationsinwaterconduction
controlledbyaVascularNACDomain(VND)subgroupofNACfamily.Previousauthors[54]
determinedthatNACtranscriptionfactorsmayhavecontributedtotheevolutionofboth
waterconductingandcellsupportingduringtheadaptationofplantstoland.InGroupI,P.
patens'12sequenceswereclustered,withonlyonesequencewithaTMM,andthisgroupcon-
tains8grapevineand12ricesequences.WedeterminedthatOGVvin21-Athal22(ANAC070,
BEARSKIN2,BRN2;S7Table)isinvolvedinrootcapmaturationandcellwallmodifications.
Athal22containsonlyoneparalogoussequence:Athal35(ANAC070,BEARSKIN1).Alleudi-
cotspeciessharetheorthologoussequenceBRN2,exceptforthepapayatree[95].TheOGVvi-
n27-Athal40(ANAC043,NST1)hastwoparalogoussequences,Athal44(ANAC012,NST3)
andAthal46(ANAC066,NST2),whichareinvolvedinsecondarywallsynthesis,andhave
redundancywiththeVNDproteinsinArabidopsis[96].WecouldonlydetectoneNSTin
papayatree,cucumber,andcacao;twosequencesforrice,soybeanandtomato;threesequences
inbarley,Arabidopsis,cassavaandPoplartree;foursequencesformaize;andfivesequences
forbananatree.
WedeterminedtheOGVvin25-Athal34(ANAC037,VND1)hastwoparalogoussequences,
Athal30(ANAC076,VND2)andAthal45(ANAC105,VND3).TheotherVNDsequences
ofArabidopsisareintheOGsVvin16-Athal28(VND4,ANAC007),whichhasonlyone
paralogoussequenceAthal33(VND5/ANAC026),andVvin28-Athal42(VND7,ANAC030).
Otherresearchers[54]foundeightNACsequencesinP.patenswithsimilaritytotheVND/
NST/SNDproteins,andtheynamedthesequencesPpVNS[VND-,NST/SND-,SMB
(SOMBRERO)-relatedprotein].Thesesequenceshaveaconservedtranscriptionalregulation
andcellularfunctionbetweenmossandArabidopsiswater-conductingcells.Ppat23(PpVNS7)
canregulatemanyputativeorthologoustotheVND/NST/SNDdirecttargets,suchasMYB46/
83/103;transcriptionalactivatorsforsecondarywallformation;CesA,acellulosesynthasesub-
unit;IRX7/FRA8,aglucuronoxylanbiosynthesisprotein;4CL,aligninbiosynthesis,andXCP,
apapain-likecysteinepeptidase.Accordingtoouranalysis,thePpat23(PpVNS7)sequenceof
P.patens,whichwasspecifictoeudicotplants,isorthologoustotheVvin25protein,whichin
turnisorthologoustotheVND1sequenceinArabidopsis.TheOGPpVNS7-Vvin25appears
tobethebasalgroupoftheVND-relatedproteins,suggestingtheexpansionofthisgroup
throughconcertedevolution.WehypothesizethatVvin25,Vvin16andVvin28(groupI-A)
weretheprincipalNACVNDsequencesthatexpandedthroughangiospermlineage.We
foundthreeVNDgenesforpapayatree,tomato,andcucumber,sevengenesforArabidopsis,
and15VNDgenesforPoplar.Inpreviouswork,itwasdeterminedthatonlytwoVNDgenes
arepresentinP.abies(Gymnosperm)comparedwithsevengenesinArabidopsis[78].
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