Genome-wide analysis of the AP2/ERF superfamily in apple and transcriptional evidence of ERF involvement in scab pathogenesis

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Genome-wide analysis of the AP2/ERF superfamily in apple and transcriptional evidence of ERF involvement

in scab pathogenesis

Cesar Luis Girardi, César Valmor Rombaldi, Joceani Dal Cero, Paula Macedo Nobile, François Laurent, Mondher Bouzayen, Vera Quecini

To cite this version:

Cesar Luis Girardi, César Valmor Rombaldi, Joceani Dal Cero, Paula Macedo Nobile, François Lau-

rent, et al.. Genome-wide analysis of the AP2/ERF superfamily in apple and transcriptional evidence

of ERF involvement in scab pathogenesis. Scientia Horticulturae, Elsevier, 2013, vol. 151, pp. 112-

121. �10.1016/j.scienta.2012.12.017�. �hal-01071932�

To link to this article : DOI:10.1016/j.scienta.2012.12.017 URL: http://dx.doi.org/10.1016/j.scienta.2012.12.017

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Girardi, Cesar Luis and Rombaldi, César Valmor and Dal Cero, Joceani and Nobile, Paula M. and Laurent, François and Bouzayen, Mondher and Quecini, Vera Genome-wide analysis of the AP2/ERF superfamily in apple and transcriptional evidence of ERF involvement in scab pathogenesis. (2013) Scientia Horticulturae, vol. 151. pp. 112-121. ISSN 0304-4238

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Genome-wide analysis of the AP2/ERF superfamily in apple and transcriptional evidence of ERF involvement in scab pathogenesis

César Luís Girardi

, César Valmor Rombaldi

, Joceani Dal Cero

, Paula M. Nobile

, Franc¸ ois Laurens

, Mondher Bouzayen

, Vera Quecini

aEmbrapaUvaeVinho,CaixaPostal130,95700-000BentoGonc¸alves,RS,Brazil

bUniversidadeFederaldePelotas,FaculdadedeAgronomiaEliseuMaciel,DepartamentodeCiênciaeTecnologiaAgroindustrial,s/n^◦caixapostal354,96010-900Pelotas,RS,Brazil

cUMR1259GénétiqueetHorticulture(GenHort),Angers,France

dUMR990,GénomiqueetBiotechnologiedesFruits,INP,ENSAT,INRA,F-31320CastanetTolosan,France

Keywords:

Apple Ethylene Fungalpathogen Fruitripening Physiologicalconditions Venturiainequalis

a b s t r a c t

TheAPETALA2(AP2)/ETHYLENERESPONSEFACTOR(ERF)superfamilyoftranscriptionalregulatorsis involvedinseveralgrowth,developmentandstressresponsesprocessesinhigherplants.Currently,the availableinformationonthebiologicalrolesofAP2/ERFgenesisderivedfromArabidopsisthaliana.Inthe presentwork,wehaveinvestigatedgenomicandtranscriptionalaspectsofAP2/ERFgenesintheeco- nomicallyimportantperennialspecies,Malus×domestica.Wehaveidentified259sequencescontaining atleastoneERFdomaininapplegenome.Thevastmajorityoftheputativeproteinsdisplaypredicted nuclearlocalization,compatiblewithabiologicalroleintranscriptionregulation.TheAP2andERFfami- liesaregreatlyexpandedinapple.Whole-genomeanalysesinotherplantspecieshaveidentifiedasingle genomicsequencewithdivergentERF,whereasinapplesevensoloistsarepresent.Intheapplegenome, themostnoteworthyexpansionoccurredinsub-groupsV,VIIIandIXoftheERFfamily.Expressionpro- filinganalyseshaverevealedtheassociationofripening-involvedERFgenestoscab(Venturiainequalis) pathogenesisinthesusceptibleGalacultivar,indicatingthatgeneexpansionprocesseswereaccompa- niedbyfunctionaldivergence.ThepresentedanalysesofAP2/ERFgenesinappleprovideevidencesof sharedethylene-mediatedsignalingpathwaysinripeninganddiseaseresponses.

1. Introduction

Hormonesareresponsibleforthecontrolofplantgrowthand development and are also involved in mediating responses to severalbioticandabioticstresses.Besidesitsimportantroleinreg- ulatingdistinctphysiologicalprocesses;suchasseedgermination, cell elongation,wounding, ripening, senescence and abscission, ethylenealsoregulatestheplantresponsestobioticandabiotic stresses(Chenetal.,2002).Ethyleneexertsitsactionthroughcom- plexregulatory stepsof its biosynthesis, perception and signal transduction,leadingtodramaticchangesingeneexpression(Chen etal.,2002;KendrickandChang,2008).Thepromotersequences of severalgenes induced by ethylene were foundto contain a cis-regulatoryelementknownastheethylene-responsiveelement

∗ Correspondingauthorat:CNPUV(NationalCenterforGrapevineandWine Research),Embrapa (BrazilianAgriculturalCorporation),Rua Livramento,515, 95700-000BentoGonc¸alves,RS,Brazil.Tel.:+555434558000;

fax:+555434512792.

E-mailaddress:vera.quecini@embrapa.br(V.Quecini).

(ERE)(Broglieetal.,1989).SequenceanalysisofvariousEREregions identifiedashortmotif,richinG/Cnucleotides,labeledtheGCC- box,essentialforethylene-mediatedresponses.TheEREmotifis recognizedbyafamilyoftranscription factors:theEREbinding factors(ERF)(Fujimotoetal.,2000).

The ERF is a large gene family of transcription factors that constitutea sub-groupoftheAPETALA2(AP2)/ERF superfamily, whichalsocontainstheAP2andRAVfamilies(Riechmannetal., 2000).ThesuperfamilyisdefinedbythepresenceoftheAP2/ERF domain,aconservedDNA-bindingsequenceconsistingofapproxi- mately60–70aminoacids(Weigel,1995).TheAP2familyconsists inproteinswithtwoAP2/ERFdomains,whereastheERFfamilyis constitutedofproteinscontainingasingleAP2/ERFdomain,and the RAV family consistsof proteinscharacterized by thepres- enceofaB3domain,aDNA-bindingdomainconservedinother plant-specifictranscriptionfactors,inadditiontoasingleAP2/ERF domain.Previousstudieshaveproposedasub-divisionoftheERF family intotwo major sub-families;the ERFsubfamilyand the CBF/DREBsubfamily(Sakumaetal.,2002).Morerecentdatasug- gest the existence of ten groups containing distinct conserved motifswithinthefamilyratherthandistinctsub-families(Nakano etal.,2006).

The role of the ERF proteins in stress-related, important agronomicalresponses(Xuetal.,2011)andtheaccumulationof genomicand expressedsequencedatahave promptedin-depth bioinformatic investigation of the ERF sub-family in several plantspecies, includingArabidopsis, rice,Populus,soybean and grapevine(Nakanoetal.,2006;Licausietal.,2010;Zhangetal., 2008;Zhuangetal.,2008).Infruitspecies,ERFsareassociatedto flavorbiosynthesisandtexturemodificationduringripening(Alba etal.,2005;daSilvaetal.,2005;Janssenetal.,2008;Licausietal., 2010; Ziliotto et al., 2008). In apple (Malus×domestica Borkh.

cv.GoldenDelicious),twoERFtranscripts,MdERF1andMdERF2, demonstrated to be predominantly and exclusively expressed inripeningfruits,respectively,wereisolatedfromripeningfruit (Wanget al.,2007).Similarly,Newcombet al.(2006)identified expressedsequencesofethyleneresponsefactorsinthetagcol- lectionandthepipelineannotationofthedraftapplegenomehas found274sequencescorrespondingtoAP2/ERFfactors(Velasco etal.,2010).

Duetoitsimportanceinplantreproductivedevelopmentand abioticandbioticstressresponses,AP2/ERFtranscriptionfactors representinterestinggenepoolstobeinvestigatedforbreedingand geneticengineeringpurposes(Xuetal.,2011).Thus,wehaveper- formedanin-depthinvestigationofAP2/ERFsuperfamilyinapple, demonstratingthepresenceof259sequencescontainingatleast asingleAP2domain.TheAP2,ERFandArabidopsissoloistfami- lieshaveundergonedifferentialexpansioninapple.GroupsV,VIII andIXoftheERFfamilywerepreferentiallyexpanded.Inapple, ERFgenesareexpressedinvegetativeandreproductivetissuesand anERFgene,previouslyassociatedtoripening,hasbeendemon- stratedtobeinducedin Venturiainequalis-infectedtissue.Thus, suggestingaroleforethyleneandethyleneresponsegenesinfungal pathogenesisinapple.

2. Materialsandmethods

2.1. Databasesearchesandalignments

SequencesofArabidopsisthalianaAP2/ERFgeneswereretrieved fromtheTAIRwebsiteandtheaminoacidsequenceofthemembers describedbyNakanoetal.(2006)wereusedasbaitstosearchthe applegenomeattheGenomeDatabaseforRosaceae(GDR)using theBatchBLASTsearch(Altschuletal.,1997).Inordertoretrieve allAP2-containingsequences,theconsensusaminoaciddomain sequencewasalsoemployedtosearchtheNCBIdatabaseforMalus.

TherecoveredsequenceswerecheckedbyreverseBLASTanalyses againsttheArabidopsisgenomeandsequencesfailingtoretrieve theoriginalbaitwereeliminated.Thepresenceandthesignificance ofAP2domainsintherecoveredapplesequencesweredetermined bySMARTanalyses(Letunicetal.,2008).

2.2. Phylogeneticanalysis

ThefunctionalityofMalusgenesincomparisontotheirAra- bidopsis counterparts was assessed by genetic distance and phylogenetic studies. Amino acid sequence alignments were performed using ClustalX (Thompson et al., 1997).When nec- essary, alignments were manually adjusted using Lasergene MegAlign(DNASTAR, Madison, WI,USA). Phylogenetic analyses were performedusing distanceand parsimonymethods in the softwarePAUP*4.0b10(http://paup.csit.fsu.edu/),usingthesoft- waredefaultparameters.Resamplingbootstraptreescontaining 1000 random samples were constructed using PSIGNFIT soft- ware (http://www.bootstrap-software.org/). Modular functional domainswereemployedforgeneticdistancestudiesforgenespre- viouslycharacterizedashavingdivergentregionsandconserved blocks.

2.3. Motifanalysisandinsilicocharacterization

Conservedmotifswerefurtherinvestigatedbymultiplealign- ment analyses using ClustalX and the MEME version 4.7 suite (BaileyandElkan,1994).Thepresenceandsequenceconservation ofrecognizablefunctionaldomainswasstudiedemployingpro- tein analysisandgenefunction toolsfromdatabases(European BioinformaticsInstitute-EuropeanMolecularBiologyLaboratory– EMBL-EBI;ExpertProteinAnalysisSystem–ExPaSyfromSwiss InstituteofBioinformatics–SIB;GeneOntologydatabase–GO;

ProteinFamiliesdatabase–Pfam).

2.4. Predictionofcellularandmolecularparameters

The physical characteristics; isoelectric point (IP) and pre- dictedmolecularweight,ofappleERFsequenceswerecalculated from the deduced amino acid using default parameter of the LasergeneMegAlignsoftware(DNASTAR,Madison,WI,USA).The proteinsfolding state waspredictedby theFoldIndex program (http://bioportal.weizmann.ac.il/fldbin/findex). Theprediction of thesub-cellularcompartmentlocationoftheappleERF-likepro- teins wasperformedusing theplantalgorithm of thesoftware WoLFPSORT(http://wolfpsort.org/)(Hortonetal.,2007).

2.5. Geneexpressionanalysis

Qualitative gene expression profiling was performed by in silicoanalysesoftheMalusESTdatabasesusingvirtualnorthern blotanalyses. The gene ofinterest wasused inqueries against reference sequence databases, generating an alignment of the input gene to its orthologs. The resulting alignment was used to find sequences in the entire mRNA input that are specific tothegene (probe). The resultingalignments werecollectively usedtoquerytheESTdatabaseagainusingBLAST.Thisheuristic was critical to avoid false-positives, or ESTs from a paralog of the input gene rather than the gene itself. The identity num- bers of the ESTs matching the probes wererecovered and the databases were used to find the names of the libraries from which thoseESTswerederived. Thedescriptionofthelibraries used is available at The Malus×domestica Gene Index, at the ComputationalBiologyandFunctionalGenomicsLaboratory(Dana Farber Cancer Institute and Harvard School of Public Health, http://compbio.dfci.harvard.edu/tgi/gi/mdgi/searching/xpress search.html) and in Supplementary Table IV. The frequency of reads of each EST contigin a given librarywas calculated and normalized according to the total number of reads from the investigatedlibraryandthetotalnumberofreadsinalllibraries.

AcorrelationmatrixbetweenESTcontigsandlibrarieswasthen generatedandgeneexpressionpatternsamongESTsandlibraries wereobtainedbyhierarchicalclusteringbasedonSpearmanRank correlation matrix using Cluster v.2.11 software (Eisen et al., 1998), by substituting theclusters by theiraverageexpression pattern. Graphicoutputs weregenerated using TreeView v.1.6 software(http://rana.lbl.gov/EisenSoftware.htm)andpresentedin colorscale.

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.

scienta.2012.12.017.

2.6. PlantmaterialandV.inequalisinoculation

Lateral shoots of Malus×domestica ‘RoyalGala’ were prop- agated in MS medium (Murashige and Skoog, 1962) pH 5.8 supplemented with 8.8mM of 6-benzylaminopurine, 30gL⁻¹ sucroseandsolidifiedwith6gL⁻¹ agar,under16-hphotoperiod and 22±2^◦C temperature. After four weeks of culture, plants

Table1

SummaryoftheAP2/ERFsuperfamilyofMalus×domesticaandmodelspeciesArabidopsisthaliana,Vitisvinifera,PopulustrichocarpaandOryzasativa.ThetotaldataforAP2, At4g13040,RAVandERFfamiliesarepresentedinboldletters.

Classification Group Species

Arabidopsisthaliana Malus×domestica Vitisvinifera Populustrichocarpa Oryzasativa

AP2family 18 51 20 26 29

SingleAP2/ERF 4 15 0 0 5

DoubleAP2/ERF 14 36 20 26 24

At4g13040 DivergentERF 1 7 1 1 1

RAVfamily AP2/ERFandB3 6 6 6 6 5

ERFfamily 122 195 122 169 145

I 10 10 5 5 9

II 15 13 8 20 16

III 23 22 22 35 27

IV 9 23 5 6 6

V 5 19 11 10 8

VI 8 6 5 11 6

VI–L 4 4 2 4 3

VII 5 8 3 6 15

VIII 15 31 11 17 15

IX 17 45 40 42 18

X 8 12 10 9 12

Xb– L 3 2 0 4 10

Total 147 259 149 202 180

were transferred to rooting medium, containing MS salts and vitamins at pH 5.8 and supplemented with 5mM of indole-3- butyricacid, 15gL⁻¹ sucroseand solidifiedwith6gL⁻¹ agarto inducerooting.Rootedplantsweregraduallyexvitroacclimated and fourweekslater, theplants wereinoculated withthefun- gus.

V.inequalis(applescab)inoculawerepreparedfrommonoconi- dialisolatescultivatedinPotato-Dextrose-Agar(PDA)medium.The plateswereincubatedat16^◦Cundercontinuouslightfor15days and sporulating colonies werescraped for inocula preparation.

Conidiawereresuspendedinsalinesolutionand theconcentra- tionwasadjusted to2.5×10⁵conidia/mLbeforeplantspraying.

Theplantsweresprayedwithapproximately50mLofconidiasus- pensionuntiltheformationof droplets.Negativecontrolplants weresprayedwithsterilesaline solutionwithoutconidia.After inoculation,theplantswerekeptfor30daysunder20^◦C,90–100%

relativehumidityand 12-hphotoperiodandsymptomdevelop- mentwasvisuallyevaluated.Leafsampleswereharvestedafter 30days,immediatelyfrozeninliquidnitrogenandstoredat−80^◦C untilprocessed.

Fig.1.PhylogeneticanalysesofAP2/ERFsuperfamilyinMalus×domestica.Neighbor-joiningtreeforfulllengthsequenceswerealignedwithClustalX.Sub-familiesAP2,RAV andtheArabidopsis-likesoloistsequencesinapplegenomearerepresentedbygrayshading.RomannumeralsrepresentERFfamilysub-groups.

Fig.2.ChromosomelocationofERFgenesinapplegenome.M.×domesticachromosomesarerepresentedaccordingtotheevolutionmodelproposedbyVelascoetal.(2010).

2.7. RNAextractionandcDNAsynthesis

TotalRNAwasextractedasdescribedbyBossetal.(1996)and thesamplesweretreatedwith100UofRNase-freeDNase(NEBBio- labs,USA)andfurtherpurifiedwithchlorophormwashandethanol precipitation.ThepurityandconcentrationoftheisolatedRNAs werecheckedon1%(w/v)agarosegelsandbyspectrophotometry.

Reversetranscriptionwascarriedoutusing2mLoftotalRNAand M-MLVReverseTranscriptaseRT-PCR(Promega)andoligod(T)25 primers,asrecommendedbythemanufacturer.Theconcentration ofthecDNAwasdeterminedspectrophotometrically.

2.8. Semi-quantitativePCRanalyses

Polymerase chain reactionswere carried out in a final vol- ume of 25mL, containing 2.5mL of 10× PCR buffer, 2.5mL of MgCl2(25mM),4.0mLofdNTPmix(2.5mM),1mLofeachprimer (10mM),1mLcDNAand2.0mLTaqDNApolymerase.Thesequence oftheprimersusedisasfollows:5^′ATGACCTGGTGGCATATCAG3^′ and 5^′CACCGTAGCAAACAACACAC3^′ for MDP0000128979, 5^′TATGCTGGCAATTGGCGAGC3^′ and 5^′ATGACCAATCCCGCAC- TCAC3^′ forMDP0000226115and 5^′GGCTGGATTTGCTGGTGATG3^′ and5^′TGCTCACTATGCCGTGCTCA3^′ for anormalizergeneMdACT (Wangetal.,2007).Thereactionlinearrangewasdeterminedby serialtestsofcyclelengthsandwasdeterminedtobelinearat27 amplificationcycles.Digitalanalysesofbandintensityonagarose gelswereperformedtoquantifygeneexpression.

3. Results

3.1. Thefamilyofethyleneresponsefactorsinapple

SequencescontainingAP2domainswereextensivelysearched intheapplegenomeand259wereconfirmedbyreverseBLASTand SMARTdomainanalysestobemembersoftheAP2/ERFsuperfamily (Table1,TableSI).Thenumberofidentifiedsequencesissmaller than thatfoundbyautomated annotationoftheapplegenome (Velascoetal.,2010)(274)duetotheeliminationofshortsequences andofthosewithintronslongerthan10kb,whicharelikelyresul- tantfromassemblymistakes.TheAP2familyinappleconsistsof 51 genes,fromwhich 36 containthecharacteristic double AP2 domain.TheRAVfamilyofproteins,containingtheB3domainin additiontothefamilycharacteristicAP2,consistsofsixmembersin appleasobservedinotherinvestigatedplantspeciesanditsmem- bernumberappearstobehighlyconservedthroughoutevolution.

Onehundredandninety-fivegenesencodingproteinswithasin- gleAP2/ERFdomainwereassignedtotheERFfamily.InArabidopsis andotherhigherplantspecies,asinglelocus(At4g13040)codesfor aproteinexhibitingdivergentAP2domainwasidentified,thusit waslabeledthesoloist(Nakanoetal.,2006;Zhuangetal.,2008;

Licausietal.,2010).Inapplegenomethegroupconsistsofseven closelyrelatedproteinscontainingthedivergentAP2domain,sim- ilartothatofAt4g13040(Figs.1and2).AppleERFproteinswere classifiedinto11groupsaccordingtotheirsimilaritytoArabidop- sisERFsequencesandconservedmotifsoutsidetheDNA-binding

Fig.3. PhylogeneticrelationshipsamongtheArabidopsisERFgenes,fromgroupIV(D)inArabidopsisandMalusERFfamilies.Bootstrapvaluesfrom1000replicatesare shownabovethebranches.ThephylogenetictreeandaschematicdiagramoftheproteinstructuresofgroupsI–XareshownasSupplementaryFigs.S1–S10.Eachcolored boxrepresentstheAP2/ERFdomainandconservedmotifs,asindicatedinlegendsbelowthetrees.Theaminoacidsequencesoftheconservedmotifsaresummarizedin SupplementaryTableIV.ClassificationbyNakanoetal.(2006)isshownaboveeachtreeandbySakumaetal.(2002),indicatedinparentheses.(Forinterpretationofthe referencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthearticle.)

AP2 domainwereidentified(Figs.1 and 2,TableSII).Although thevastmajorityoftheArabidopsismotifswerepresentinapple sequences,specificconservedregionswereidentifiedinthepro- teinsidentifiedinMalus×domestica(Figs.S1–S10).TheappleERF family(195members)isexpandedincomparisontotheArabidop- sis(147),poplar(202)andrice(180)families.Theexpansionofthe applefamilyissignificantforERFproteinsclusteredingroupV,VIII andIX(Table1,Figs.S1–S10).Interestingly,groupsVandIXarealso expandedingrapevineandaretranscriptionallyassociatedtoberry ripeningprocesses(Licausietal.,2010).Inpoplar,ERFgroupsVand IXarealsoexpandedalthoughnofunctionalsignificancehasbeen attributedtothosegroups(Zhuangetal.,2008).TheXb-likegroup ofERFproteinsisabsentfromthegrapevinegenome,however,in appleitconsistsoftwoproteins,similartothenumbersfoundin Arabidopsis(3)andPopulus(4)(Nakanoetal.,2006;Zhuangetal., 2008;Licausietal.,2010).

Supplementary data associated with this article can be found,intheonlineversion,athttp://dx.doi.org/10.1016/j.scienta.

2012.12.017.

3.2. AccuracyofproteinAP2/ERFpredictions

Genomedatabasesemployabinitioproteinpredictionmeth- ods,whicharepronetoerrors.Inordertoconfirmtheexistenceof theidentifiedAP2/ERFproteins,wehavecheckedintronjunctions

incomparisontothehomologsinArabidopsisandpoplar.Subse- quently,thepredictedmRNAsofallidentifiedproteinswereused toquery apple EST databases. We foundno unrealistic introns andESTsequencesmatching(e-valuelowerthan1·e⁻²⁵)allpre- dictedmRNAwerefoundinthedatabases,exceptforgenemodel MDP0000876858.

3.3. CellularandmolecularcharacteristicsofappleAP2/ERF proteins

Wehaveemployedbioinformatictoolstodeterminethephys- icalpropertiesoftheERF-likesequencesfromapple,suchasthe molecular weight (MW), pHvalue of isoelectric point (pI) and foldingindex.ThevastmajorityoftheappleERF-likesequences werepredictedtohavelowmolecularweight,rangingfrom1.5to 4.48kDaandanaverageof233aminoacids,goingfrom111to413 aminoacids(TableSIII).Inapple,mostofthesequencessharing sequencesimilaritytoArabidopsisERFproteins(74.2%,49)were predictedtobebasictoneutral,whereasonly25.8%(17)displayed predominanceofacidicaminoacids(TableSIII).

Supplementary data associated with this article can be found,intheonlineversion,athttp://dx.doi.org/10.1016/j.scienta.

2012.12.017.

Thefolding statesof ERFfamily proteinsin applewerepre- dictedbyFoldIndexprogram(Priluskyetal.,2005).Themajority

Fig.4. ExpressionprofileofERFgenesfromgroupsIV,VIIandVinleaf,flower,fruitandscab-infectedtissues.ApplegenemodelsarelistedasavailableatGenomeDatabase forRosaceae(GDR).HierarchicalclusteringisbasedonSpearmanRankcorrelation.Graphicoutputsarerepresentedascolorscale.(Forinterpretationofthereferencesto colorinthisfigurelegend,thereaderisreferredtothewebversionofthearticle.)

oftheidentifiedapplesequencessharingsequence similarityto ERFswerepredictedtobeunfolded(59.1%,39)underphysiolog- icalconditions(TableSIII).Thepredictedsub-cellularlocalization oftheERFproteinsidentifiedinMaluswasinvestigatedusingthe softwareWoLFpSORT(Hortonetal.,2007).MostoftheERF-like sequencesfromapple(83.3%,55)waspredictedtobeexclusively nuclearorwithambiguoussub-cellularlocationforthenucleusand organelleorcytoplasm(TableSIII)(Fig.3).

3.4. ExpressionprofilingofERFgenes

The expression profile of 195 ERF genes was preliminarily investigated by in silico analyses for reproductive and veg- etative libraries. Moreover, the relative expression of the 10 groupsconstitutingthesub-familywasalsoinvestigatedinscab infected tissues. The expression patterns of the genes encod- ing for members of the ERF family, presented as groups, are shown in Figs. 4 and 5. Transcripts corresponding to the vast majorityoftheidentifiedgeneswereexpressedinappleleaves, flowers and fruits, except for gene models MDP0000451365 (groupIII),groupVsequencesMDP000031419,MDP0000525933, MDP0000276536,MDP0000551969,MDP0000246184,MDP0000- 127054,MDP0000297646andMDP0000243375,MDP0000467753 and MDP0000778140 (group VIII) and group IX sequences MDP0000447570,MDP0000133854,MDP0000829925,MDP0000- 155543, MDP0000789227, MDP0000880063, MDP0000187369 thatwereexpressedinbuds,rootsandvascularvesseltissues.The expressionprofileofmostERFgenesisdevelopmentallyand/or

environmentallycontrolledin apple(Figs.4 and5).WithinERF groups,theexpressionpatternswerealsodivergent,especiallyfor groupsI,VIL,VII,VIIIandIX(Figs.4and5).Transcriptscorrespond- ingtothesequencesMDP0000127123andMDP0000316843,from groupXb,wereexclusivelypresentinfruits(Figs.4and5).Most ERFgenesfromgroupsVIIandIXexpressedinthefruitswerealso highly expressedin V. inequalis infectedtissues (Figs. 4 and5).

Thus,we havefurtherinvestigatedthetranscriptionalprofileof ripening-associatedERFsinscabpathogenesisusingmonosporic V.inequalisculturesinoculatedtoinvitropropagatedplantsand semi-quantitativePCR.

Thirtydaysafterinoculation,plantleavesexhibitedscabsymp- toms in the leaves and stem and a three-fold induction of MDP0000128979 (group VII) expression (Fig. 6). Phylogenetic analysesdemonstratedthatMDP0000128979correspondtofruit- ripeningassociatedMdERF1(Wangetal.,2007).Insilicoanalyses suggestedtheassociationoftheinducedtranscriptionofgroupIX genestoscabresponseandfruitripening(Fig.5),howevertran- scription of MDP0000226115 wasonly slightly altered in scab infectedtissues(Fig.6).

4. Discussion

4.1. IdentificationoftheAP2/ERFfamilytranscriptionfactorsin apple

A comprehensivesearchfor AP2/ERFtranscription factorsin Malus×domestica genome has revealed the presence of 259

Fig.5.ExpressionprofileofERFgenesfromgroupIXinleaf,flower,fruitandscab-infectedtissues.ApplegenemodelsarelistedasavailableatGenomeDatabaseforRosaceae (GDR).HierarchicalclusteringisbasedonSpearmanRankcorrelation.Graphicoutputsarerepresentedascolorscale.(Forinterpretationofthereferencestocolorinthis figurelegend,thereaderisreferredtothewebversionofthearticle.)

AP2-motifcontainingsequences.Thenumberissmallerthanthe 274 AP2/ERF foundby the automated annotationof theapple genome (Velasco et al., 2010) due to the elimination of short sequencesandofthosecontainingunrealisticintrons.Theapple superfamily is largerthantherice and Populusones,with180 and202genes,respectively(Table1).Thelargersizeofthesuper- familyinappleismainlyduetoexpansionoftheAP2, ERFand At4g13040-likefamilies,sincethenumberofgenesintheRAVfam- ilyisconservedthroughoutevolution(Table1).Inapplegenome, recentgeneduplicationappearstobeinvolvedintheexpansion oftheAt4g13040-likegenes,since itis representedbya single sequence with divergent AP2 domain in all investigated plant species,whereasinapple,itisrepresentedbysevensequences.The sub-setofAP2proteinscontainingadoubledAP2domaininappleis morethantwotimeslargerthanitisinArabidopsisandhasapprox- imately40%moremembersthantheoneinPopulustrichocarpa.

Interestingly,theexpansionpatternofthefamiliesisconservedin thewoodyperennialspeciesinvestigated;poplar,grapevineand apple(Zhuangetal.,2008;Licausietal.,2010).

TheERFfamilyinappleconsistsof195sequencesphylogeni- callyclassifiedintothetengroups(I–X)identifiedinArabidopsis byNakanoetal.(2006).ThedistributionofappleERFsequencesin groupsissimilartothatofotherinvestigatedspecies.Asobserved inwoodyperennialVitisviniferaandPopulustrichocarpa,groupsV andIXareexpandedinapplegenome,althoughbothalsodisplay alowernumberofgenesingroupI,whichdoesnotoccurinapple.

ArecentstudyinVitisviniferahasproposedthatthelowernumber ofERFgenesingroupIinwoodyperennialislikelytobeduetoits functionalredundancyincontrollingwaxaccumulation(Aharoni etal.,2004)tothegenesfoundinexpandedgroupV(Licausietal., 2010).Thedifferentialexpressionandtheexpansionofbothgroups

IandVsuggestthatinappleitdoesnotoccur.Incontrast,thelow levelsofexpressionofgroupIVgenesinapplemayindicatefunc- tionalredundancytootherERFgenegroup.ThegroupXbinapple isexclusivelyexpressedinfruitsandhasanumberofgenessimilar tothatfoundinArabidopsisandpoplar.Thegroupisabsentfrom grapevine.ThelargestERFgroupinappleisgroupIX,asobservedin poplarandgrapegenomes,itisalmosttwicethesizeofthegroup inArabidopsisandrice(Table1).Asobservedingrapevine(Licausi etal.,2010),appleERF-IXsequencesexhibitspecies-specificpro- teindomains(SupplementaryTableSIII)anddistinctexpression patterns.Interestingly,membersofgroupIXdisplayedaparticular clusteredorganizationalongchromosomes7and16(Fig.2),similar totheoneobservedinthepoplarandgrapevinegenome(Zhuang etal.,2008;Licausietal.,2010),althoughinthisspeciesthetan- demrepetitiondoesnotexceedfourgenes.Thecodingsequences ofproteinsinvolvedinsignaltransductionandtranscriptionalreg- ulationarefrequentlyduplicatedinhigherplantgenomes,dueto theirroleinplantfitnessresponses(Patersonetal.,2010).

Supplementary data associated with this article can be found,intheonlineversion,athttp://dx.doi.org/10.1016/j.scienta.

2012.12.017.

Transcriptionfactorsoften exhibit regionsoutside the DNA- bindingdomainthatareinvolvedinregulationofthetranscriptio- nalactivity,protein-proteininteractions,andnuclearlocalization (Liu etal.,1998).Thesemotifs,frequentlyassociatedtospecific functionsorregulationpatterns,arecharacteristicoflargetran- scriptionfactorfamiliesinplants,suchasMYB,WRKY,NAC,Dof, GATA,andGRAS(Eulgemetal.,2000;Kranzetal.,1998;Lijavetzky etal.,2003;Ookaetal.,2003;Reyesetal.,2004;Tianetal.,2004).

Thevast majority of theERF-likesequences identified inapple sharedoneormoremotifsoutsidetheAP2/ERFdomainwiththeir

Fig.6. ExpressionprofileofappleERFsequencesfromgroupsVIIandIXinVenturiainequalis-infectedvegetativetissue.(A)Mock-inoculatedcontrol;(B)plantsprayedwith scabconidia;(C)scabsymptomsonleavesandpetioles30daysafterinoculationand(D)relativeexpressionofMDP0000128979(groupVII)andMDP0000226115(group IX)infungus-infectedandcontrolplants.Thebarsrepresentpercentagestandarderror.

Arabidopsiscounterparts,asobservedinrice,Populus,soybeanand grapevine(Nakanoetal.,2006;Licausietal.,2010;Zhangetal., 2008; Zhuangetal., 2008).The functionallycharacterizedgene MdERF1(A1LM1),associatedtofruitripeninginapple(Wangetal., 2007)clusteredinthemostabundantgroupVII,whereasMdERF2 (A1LM2),whichisexclusivelyexpressedinripeningfruitswasclus- teredingroupIX.

4.2. CellularandmolecularcharacteristicsofappleERF-like proteins

Proteinsbiochemical andphysical propertiesareresponsible forseveralcharacteristicsassociatedtotheirbiologicalfunction, suchasmolecularstructure,ligandbindingandsubcellularloca- tion.SimilartoArabidopsis,rice,Populus,soybeanandgrapevine (Nakanoetal.,2006;Licausietal.,2010;Zhangetal.,2008;Zhuang et al.,2008), appleERF-likeproteinsare predictedtohave low molecularweight.Inapple,mostoftheERF-likesequenceswere predictedtobebasictoneutral,asobservedforArabidopsisand Populus, although the proteins from the annual and perennial modelplantsexhibitslightlylowerpIvalues.Theseobservations are consistent with the presence of a basic region mediating sequence-specific DNA-binding in families of transcriptional

regulators from evolutionarily distant organisms (Miller et al., 2003;Panneetal.,2004).Thus,thepredictedchemicalnatureof theproteinsidentified inappleis compatiblewitha functional roleintranscriptionalcontrol.

Thepredictedsub-cellularlocalizationoftheERFproteinsiden- tifiedinMaluswasinvestigatedusingthesoftwareWoLFpSORT (Hortonetal.,2007).Thesub-cellularlocationisanimportantclue forthebiologicalfunctionofidentifiedsequences,especiallyfor thosehypothesizedtobeinvolvedingeneexpressionregulation.

ThemajorityoftheERF-likesequencesfromapplewerepredicted tobeexclusivelynuclearorexhibitingambiguoussub-cellularloca- tionforthenucleusandorganelleorcytoplasm.Atthispoint,the incompletenatureofsomeofthesequencesandthelimitationsof bioinformaticanalysespreventusfromestablishinghowaccurate thepredictionsareinvivo.Furtherfunctionalanalyseswillbenec- essarytodeterminethesub-cellularlocationoftheappleERF-like sequences.

4.3. ExpressionanalysesofERFgenes

TheexpressionprofilingofERFgenesinapplealongwiththe knowledgeontheirmolecularfunctioninmodelplantspeciesisan importanttooltouncoverthebiologicalroleofthesetranscription

factors in developmental and stress-response processes. Tradi- tionally,the expressionof ERF genes hasbeenassociated with themolecularresponsetoethylene(Dietzetal.,2010).However, genome-widetranscriptionalprofilingofAP2/ERFgenesinseveral plantspecieshasdemonstratedthattheyareregulatedbyanum- berofphysical-chemicalstimuli.Asub-setof ERFgenesexhibit aclassicalethyleneresponsivepatternandhave beenshownto beinvolvedintheripeningprocessinclimactericfruits.Although severalof theinvestigated ERFgenes werehighly expressedin fruits,onlythosefromgroupXbwereexclusivelyfoundinfruits.

InArabidopsis,NicotianatabacumandSolanumlycopersicumtrans- criptsofgroupIIandIIIERFshavebeenassociatedtoabioticstress responses(Gilmouretal.,1998;Tsutsuietal.,2009),whereasin Vitisvinifera,theirexpressionisconstitutivelyhigh,suggestinga role ina default defensesystem(Licausiet al.,2010).Inapple, genesfromthesegroupsexhibitanexpressionpatternmoreclosely relatedtostressresponses,asobservedinArabidopsis,tobaccoand tomato.ThegenesfromERFgroupIX,thelargergroupinapple, exhibitdistinct regulationpatterns,witha sub-setbeinghighly inducedinfruitsandinscabinfectedtissues,thusindicatingthat theripening and the V. inequalis-induced stresspathway share commontranscriptionalregulatorsbutwhetherthetargetsofthese transcriptionfactorsisalsosharedorindependentremainstobe elucidated.Inapreviousstudytocharacterizeripening-associated genesinapple,theexpressionofMDP0000128979(MdERF1)and MDP0000226115(MdERF2),analyzedbynorthernblotandRT-PCR, wasdemonstratedtooccurexclusivelyinfruittissues(Wangetal., 2007).

OurresultsindicatethatERFgenesarealsoexpressedinveg- etativetissueandthatMDP0000128979(MdERF1)transcriptionis inducedinplantsexhibitingscabsymptoms,confirmingthetrans- criptionalassociationbetweentheripeningfactorandV.inequalis pathogenesisin apple. Theseresultsare inaccordance toother reportsdemonstratingaroleforethyleneinbioticstressresponses inapple(Akagietal.,2011)andotherplantspecies(Guttersonand Reuber,2004;Broekaertetal.,2006).Ingeneral,analysesofERF expressionpatternssuggestsomeextentoffunctionalspecializa- tioninapple.

Acknowledgments

Theauthorswouldliketoacknowledgetheexcellenttechnical assistanceofDanielaDalBoscoandIraciSinskiinplantpropagation andacclimation.WealsothankRenataGavaforfunguscultiva- tionandplantinoculation.ThisresearchisfundedbyanEmbrapa grantnumber02.07.05.001.00Macroprograma2–Agrofuturoto C.L.G.VQisrecipientofaproductivityresearchgrantfromCNPq (307031/2010-1).

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