Genome-Wide Characterization and Expression Analysis of Major Intrinsic Proteins during Abiotic

17 Pages·2015·1.24 MB·English

RESEARCHARTICLE
Genome-Wide Characterization and
Expression Analysis of Major Intrinsic
Proteins during Abiotic and Biotic Stresses in
Citrus sinensis
Sweet Orange ( L. Osb.)
CristinadePaulaSantosMartins1,2,AndresaMunizPedrosa1,DongliangDu2,Luana
PereiraGonçalves1,QibinYu2,FrederickG.Gmitter,Jr.2,MarcioGilberto
CardosoCosta1*
1 CenterforBiotechnologyandGenetics,BiologicalSciencesDepartment,StateUniversityofSantaCruz,
Ilhéus,Bahia,Brazil,2 CitrusResearchandEducationCenter,DepartmentofHorticulturalSciences,
UniversityofFlorida,LakeAlfred,Florida,UnitedStatesofAmerica
*[email protected]
Abstract
OPENACCESS
Thefamilyofaquaporins(AQPs),ormajorintrinsicproteins(MIPs),includesintegralmem-
Citation:dePaulaSantosMartinsC,PedrosaAM, braneproteinsthatfunctionastransmembranechannelsforwaterandothersmallmole-
DuD,GonçalvesLP,YuQ,GmitterFG,Jr.,etal.
culesofphysiologicalsignificance.MIPsareclassifiedintofivesubfamiliesinhigherplants,
(2015)Genome-WideCharacterizationand
ExpressionAnalysisofMajorIntrinsicProteinsduring includingplasmamembrane(PIPs),tonoplast(TIPs),NOD26-like(NIPs),smallbasic
AbioticandBioticStressesinSweetOrange(Citrus (SIPs)andunclassifiedX(XIPs)intrinsicproteins.Thisstudyreportsagenome-widesurvey
sinensisL.Osb.).PLoSONE10(9):e0138786. ofMIPencodinggenesinsweetorange(CitrussinensisL.Osb.),themostwidelycultivated
doi:10.1371/journal.pone.0138786
Citrusspp.Atotalof34differentgenesencodingC.sinensisMIPs(CsMIPs)wereidentified
Editor:DavidDFang,USDA-ARS-SRRC,UNITED
andassignedintofivesubfamilies(CsPIPs,CsTIPs,CsNIPs,CsSIPsandCsXIPs)based
STATES
onsequenceanalysisandalsoontheirphylogeneticrelationshipswithclearlyclassified
Received:May1,2015 MIPsofArabidopsisthaliana.Analysisofkeyaminoacidresiduesallowedtheassessment
Accepted:September3,2015 ofthesubstratespecificityofeachCsMIP.Genestructureanalysisrevealedthatthe
Published:September23,2015 CsMIPspossessanexon-intronorganizationthatishighlyconservedwithineachsubfam-
ily.CsMIPlociwerepreciselymappedoneverysweetorangechromosome,indicatinga
Copyright:©2015dePaulaSantosMartinsetal.
Thisisanopenaccessarticledistributedunderthe widedistributionofthegenefamilyinthesweetorangegenome.Investigationoftheir
termsoftheCreativeCommonsAttributionLicense, expressionpatternsindifferenttissuesandupondroughtandsaltstresstreatments,aswell
whichpermitsunrestricteduse,distribution,and aswith‘CandidatusLiberibacterasiaticus’infection,revealedatissue-specificandcoordi-
reproductioninanymedium,providedtheoriginal
natedregulationofthedifferentCsMIPisoforms,consistentwiththeorganizationofthe
authorandsourcearecredited.
stress-responsivecis-actingregulatoryelementsobservedintheirpromoterregions.Aspe-
DataAvailabilityStatement:Allrelevantdataare
cialroleinregulatingtheflowofwaterandnutrientsisproposedforCsTIPsandCsXIPsdur-
withinthepaperanditsSupportingInformationfiles.
ingdroughtstress,andformostCsMIPsduringsaltstressandthedevelopmentofHLB
Funding:Thisworkwassupportedinpartby
disease.TheseresultsprovideavaluablereferenceforfurtherexplorationoftheCsMIPs
researchgrantsfromCNPq(Brasília,Brazil),CAPES
(Brasília,Brazil),FAPESP(SãoPaulo,Brazil)and functionsandapplicationstothegeneticimprovementofbothabioticandbioticstresstoler-
FloridaCitrusResearchandDevelopment anceincitrus.
Foundation(USA).Thefundershadnoroleinstudy
design,datacollectionandanalysis,decisionto
publish,orpreparationofthemanuscript.
PLOSONE|DOI:10.1371/journal.pone.0138786 September23,2015 1/17
Genome-WideCharacterizationofSweetOrangeMIPs
CompetingInterests:Theauthorshavedeclared Introduction
thatnocompetinginterestsexist.
Aquaporins(AQPs)areintegralmembraneproteinsthatassisttherapidmovementofwateras
wellasotherlowmolecularweightmoleculesacrosscellularmembranes[1–3].AQPsbelongto
theancientfamilyofmajorintrinsicproteins(MIPs)foundinmicroorganisms,plantsandani-
mals.WhileasmallnumberofdifferentAQPshavebeenidentified(2inE.coli,9inS.cerevisiae,
11inC.elegansand13inmammals[4]),asurprisinglylargenumberofMIPhomologueshave
beenfoundinplants;forexample,35AQPswerefoundinArabidopsis[5],36inZeamays[6],
33inOryzasativa[7],28inVitisvinifera[8],55inPopulustrichocarpa[9],71inGossypium
hirsutum[10],47inSolanumlycopersicum[11]and66inGlycinemax[12].Theseobservations
highlightamajorroleforplantMIPsaskeyregulatorsoftheintricateflowsofwaterandsolutes
requiredforgrowthandadaptiveresponsestotheever-changingenvironment.
PlantMIPswereoriginallycategorizedintofoursubfamiliesonthebasisofsequencehomol-
ogiesandsubcellularlocalization:plasmamembrane(PIP),tonoplast(TIP),nodulin-like(NIP)
andsmallbasic(SIP)intrinsicproteins[2,13].Morerecently,studiesinthemossPhyscomitrella
patensrevealedthepresenceofnovelAQPisoformsinadditiontothefourconservedplant
AQPsubfamilies:ahomologueoftheEscherichiacoliintrinsicproteinGlpF(GIPs),intrinsic
hybridproteins(HIPs)andunclassifiedXintrinsicproteins(XIPs)[9,13–17].XIPhomologues
havealsobeenidentifiedinsomehigherplants,suchasSolanumlycopersicum,Populustricho-
carpaandGlycinemax[9,11,12,15].ThesefindingssuggestthatthefamilyofplantMIPsis
largerandmuchmorecomplexthanpreviouslyanticipatedand,hence,mayplaycriticalroles
inawiderangeofbiologicalprocessesthatgofarbeyondthecurrentknowledge.
AQP-mediatedwatertransportinplantshasbeenimplicatedtoplayacentralregulatorystep
indiversebiologicalprocesses,includingcellelongation,seedgerminationandosmoregulation
[18].Inaddition,AQPsfacilitatethetransportofsmallunchargedmoleculesofphysiological
significancelikeglycerol,urea,boricacid,silicicacid,hydrogenperoxide(H O ),ammonia
2 2
(NH )andcarbondioxide(CO )throughtheplantcellmembranes[1,2]andalsoregulate
3 2
phloemsaploadingandunloading,stomatalandleafmovement,andcytoplasmichomeostasis
[1,2,13,19].Therefore,itisnotsurprisingthattheirexpressionandbiologicalactivitieshave
beenshowntobedevelopmentallyanddifferentiallyregulatedinacell-specificmanner,viaphy-
tohormonessuchasabscisicacid(ABA),gibberellinsandpossiblybrassinosteroids,andbyenvi-
ronmentalsignalssuchaslight,waterstress,nematodeinfection,lowtemperature,andsalinity
[4].However,ageneralexpressionpatternamongthedistinctMIPisoformscannotbedistin-
guished,astheyareeitherup-ordownregulateddependingonthestimulusand/orthecell-type
studied[4,19,20].ThisdifferenceintranscriptionalregulationsuggeststhateachMIPisoform
mayplayadistinctroleinplantgrowth,developmentandstressresponse[4].
Asamajorhorticulturalcrop,thecultivatedCitrusspp.faceconstantbioticandabioticcon-
straintsinthemainregionsofproduction,includingdrought,salinity,extremetemperatures
andseriousdiseaseslikeHuanglongbing(HLB,orcitrusgreening),whicharepredictedto
increaseinintensity,frequency,andgeographicextentasaconsequenceofglobalclimate
change.DespitethehighlightedimportanceofAQPs,thereareonlyafewstudiestodateoncit-
rusMIPsandtheirpredictedroleinthetransportofwaterandsolutesrequiredforplant
growth,developmentandadaptiveresponsestotheenvironment.TheexpressionoftwoMIP
genes,PIP1andPIP2,hasbeeninvestigatedinrootsofPoncirustrifoliata(L.)Raf.,Cleopatra
mandarin(C.reshniHortexTan.)andoneoftheirhybrids,subjectedtomoderatewaterdeficit
[21],andinrootsofP.trifoliata,CleopatramandarinandCarrizocitrange(C.sinensis[L.]
Osb.×P.trifoliata[L.]Raf.),subjectedtosalttreatment[22].PIP1andPIP2geneexpressiondif-
ferenceswerecorrelatedwithalterationsinroothydraulicconductance(Kr)andchloride(Cl-)
exclusionfromleavesand,hence,tolerancetowaterandsaltstresses,respectively.Withthe
PLOSONE|DOI:10.1371/journal.pone.0138786 September23,2015 2/17
Genome-WideCharacterizationofSweetOrangeMIPs
recentcompletionandpublicationofthegenomesequencesofsweetorange[23–25],itisnow
possibletoidentifyandcharacterizethecompleterepertoireofMIPsincitrus,aswellastocarry
outcomparativegenomeanalysisinordertounderstandtheirevolutionaryhistory.Therefore,
theobjectiveofthepresentstudywastoidentifysweetorangeMIPgenesthroughagenome-
wideanalysisandtocharacterizetheirsequences,evolutionaryrelationships,putativefunctions
andexpressionpatternsinvarioustissuesandinresponsetoabioticandbioticstresses.Thisis
thefirstcomprehensivestudyoftheMIPgenefamilyinsweetorange,providingvaluableinfor-
mationforfurtherexplorationofthefunctionsofthisimportantgenefamilyincitrus.
MaterialsandMethods
Plantmaterialsandstresstreatments
Two-year-oldsweetorangeplantsgraftedonRangpurlime(C.limoniaOsbeck),arootstock
highlyresistanttodrought,wereusedinthedroughtstressexperiment.Plantswerefirst
prunedandacclimatizedtogreenhouseconditions(25±4°C,16hoflightandrelativehumidity
oscillatingbetween80and95%)during90daystoobtainadequaterootdevelopmentanduni-
formleafflushes.Duringacclimatization,plantsweregrowninplasticpotsof45-L,containing
amixtureofsoilandsand(ratio3:1)andmicronutrientmixFTE(50gperpot),irrigatedwith
tapwatertwiceaweek,andfertilizedweeklywith1literofthefollowingnutrientsolution:1.0g
l-1Ca(NO ) ,0.4gl-1KNO ,0.6gl-1MgSO and0.4gl-1NH H P0 (MAP).Thereafter,the
3 2 3 4 4 2 4
potswereclosedwithaluminumfoiltopreventwaterlossbyevaporation,andasetof10plants
wasrandomizedovertheexperimentalareaandsubjectedtothefollowingtreatments:(i)5
plantsincontrol,inwhichplantsweremaintainedatleafpredawnwaterpotentialvaluesof
-0.2to-0.4MPabydailyirrigationand(ii)5plantsindrought,inwhichtheplantswere
exposedtoaprogressivesoilwaterdeficituntiltheirleavesreachpredawnwaterpotentialval-
uesof-1.5MPa.Theleafpredawnwaterpotentialwasrecordedonthethirdfullyexpanded
matureleaffromtheapexofeachplant,between2AMand4AM,usingaScholander-type
pressurepump(m670,PmsInstrumentCo.,Albany,USA).
Forsalttreatment,sweetorangeseedsweregerminatedinvitroasdescribedbydeOliveira
etal.[26].Twenty-day-oldseedlingsofnucellaroriginwereselectedbasedontheiruniformity,
andtransferredtoMSmediumalone(control)orcontaining150mMNaCl(Merck,Darm-
stadt,Germany).Eachtreatmentconsistedof15nucellarplants(biologicalreplicates).Leaves
androotswereharvested20daysafterthetreatmentsandimmediatelyfrozeninliquidnitro-
genandstoredat-80°C.
Plantswereinfectedwith‘CandidatusLiberibacterasiaticus’asdescribedinFanetal.[27].
Briefly,two-year-oldseedlingsofroughlemon(C.jambhiriLush.)andsweetorange(C.sinen-
sisL.Osbeck)weregraft-inoculatedwithbudwoodfrom‘Ca.L.asiaticus’infected‘Carrizo’
citrangetreeskeptundergreenhouseconditions.Forcontrols,theplantsweregraftedwithbud
woodfromhealthyCarrizotrees.AlltheseplantswerekeptinaUnitedStatesDepartmentof
AgricultureAnimalandPlantHealthInspectionServiceandCenterforDiseaseControl-
approvedandsecuredgreenhouseattheUniversityofFlorida,CitrusResearchandEducation
Center,LakeAlfred.Threebiologicalreplicateswereproducedforeachcitrusspeciesineach
treatment.Quantitativereal-timePCRwasperformedtoconfirmthepresenceof‘Ca.L.asiati-
cus’intheinoculumsourceandinoculatedplantsasdescribedinLietal.[28].Fourfully
expandedleavesweresampledseparatelyfrom‘Ca.L.asiaticus’inoculatedplantsandmock-
inoculatedplants(usedascontrols)ofeachcitrusspeciesat0,7,17,and34weeksafterinocula-
tion(WAI).Leaveswereimmediatelyfrozeninliquidnitrogenandstoredat-80°Cuntiluse.
Threebiologicalreplicateswereproducedforeachcondition.Intotal,12plantswith48leaf
sampleswerecollected(2speciesx2treatmentsx3replicatesx4timepoints).
PLOSONE|DOI:10.1371/journal.pone.0138786 September23,2015 3/17
Genome-WideCharacterizationofSweetOrangeMIPs
IdentificationandclassificationofCsMIPs
TheHiddenMarkovModelprofileofthePFAM(http://pfam.sanger.ac.uk/)[29]motif
PF00230(majorintrinsicprotein)wasusedasakeywordtosearchthesweetorangegenome
sequencedatabase(http://www.phytozome.org/citrus/)[25].TheKEGGOrthology(KO)
termsK09872(aquaporinPIP),K09873(aquaporinTIP),K09874(aquaporinNIP)and
K09875(aquaporinSIP)werealsousedaskeywordstosearchthesweetorangegenome
sequenceatPhytozome.Toavoidthedeficienciesoftheautomaticannotation,the35Arabi-
dopsisthalianaMIPproteinsequenceswereretrievedfromTAIR(http://www.arabidopsis.org/
),accordingtopreviousreports[5,30],andalsousedtoalignwiththesweetorangegenome
sequenceassemblyavailableatPhytozomeusingtheTBLASTNtool.Aftermergingtheresults
fromallthesestrategies,uniqueentries(withuniquelocusID)wereidentifiedtoremovethe
redundancy.Theresultingsequencesweremanuallyinspectedforthepresenceofcharacteris-
ticandfunctionallyimportantMIPaminoacidsandmotifs.
ThesweetorangeMIPswereclassifiedindifferentisoformsbasedonsequenceanalysisof
themultiplealignmentsandontheirphylogeneticrelationshipwiththoseclearlyclassified
MIPsofArabidopsisthaliana,RicinuscommunisandNicotianabenthamiana,downloaded
fromtheTAIRandNCBIdatabases.Multiplesequencealignmentsofthededucedaminoacid
sequencesofCsMIPsandthoseofA.thaliana,R.communisandN.benthamianawereper-
formedusingthedefaultparametersofClustalW[31].Thedendrogramwasgeneratedbythe
MEGA6program[32]usingtheNeighbor-Joining(NJ)method[33]andbootstrapanalysis
(1,000replications).
AnalysisofCsMIPproteinpropertiesandconservedaminoacid
residues
Informationaboutcodingsequence(CDS),full-lengthsequenceandpredictedaminoacid
sequencewasobtainedforeachsweetorangeMIPgenefromthePhytozomedatabase.The
GRAVY(grandaverageofhydropathy),molecularweightandisoelectricpoint(pI)ofthe
deducedaminoacidsequenceswerepredictedbythePROTPARAMtoolavailableonthe
ExpertProteinAnalysisSystem(ExPASy)proteomicsserver(www.expasy.ch/tools/protparam.
html).ThesubcellularlocalizationofMIPproteinswaspredictedusingtheWoLFPSORTtool
availableathttp://www.genscript.com/psort/wolf_psort.html.Carefulvisualinspectionof
aminoacidsequencealignmentswereperformedtoidentifythecharacteristicMIPamino
acidsandmotifsandtheresiduesinsevenkeypositionsthathavebeenreportedtobespecific
foreachfunctionalsubgroup[12,30,34,35].
AnalysisofpromotersequencesandchromosomallocationsofCsMIPs
Onekbupstreamregionfromthetranslationstartsitewasextractedfromallthesweetorange
MIPgenesandsubsequentlyanalyzedinthePLACEdatabase(http://www.dna.affrc.go.jp/
PLACE/signalscan.html)toidentifythepresenceofthestress-responsivecis-actingregulatory
elementsABRE(ABA-responsiveelement;ACGTG),DRE/CRT(dehydrationresponsiveele-
ment/C-repeat;G/ACCGAC),MYBS(MYBbindingsite;TAACTG)andLTRE(low-tempera-
ture-responsiveelement;CCGAC)intheirpromoters.ThephysicallocationsofCsMIPswere
determinedbyconfirmingthestartingpositionofallgenesoneachchromosome,using
BLASTNsearchingagainstthelocaldatabaseoftheCitrussinensisAnnotationProject(CAP;
http://citrus.hzau.edu.cn/orange/).MapChartsoftwarewasusedtoplotthegenelocionthe
sweetorangechromosomes[36].
PLOSONE|DOI:10.1371/journal.pone.0138786 September23,2015 4/17
Genome-WideCharacterizationofSweetOrangeMIPs
ExpressionanalysisofCsMIPs
TotalRNAisolation,cDNAsynthesisandquantitativereal-timeRT-PCR(qPCR)analysis
wereperformedasdescribedpreviously[26].qPCRprimersweredesignedappropriatelyto
avoidtheconservedregions.PrimersequencesareshownindetailinS1Table.Glyceralde-
hyde-3-phosphatedehydrogenaseC2(GAPC2)wasusedasaninternalreferencegenetonor-
malizeexpressionamongthedifferentsamples[37].Datawereobtainedfromapoolofthree
biologicalreplicatesthatwereindividuallyvalidated.
RNA-seqdataweredownloadedfromCAP[24]andusedtoanalyzetheexpressionpatterns
ofCsMIPsindifferenttissues,namelycallus(C),flower(Fl),leaf(L),fruit(Fr),andmixedtis-
suesfromfruitatthreedevelopmentalstages(Mix.1,Mix.2,andMix.3).Theheatmapwasgen-
eratedusingR3.1.0software.
ResultsandDiscussion
MIPencodinggenesinthesweetorangegenome
SearchesinthesweetorangegenomesequencedatabaseatPhytozomeusingannotationinfor-
mation,aswellasthe35proteinsequencesofthecompletesetofA.thalianaMIPs(AtMIPs)
asquerysequences,resultedintheidentificationof34differentgenesencodingC.sinensis
MIPs(CsMIPs)(Table1).Theretrievedsequencesweremanuallyinspectedforthepresenceof
characteristicandfunctionallyimportantMIPdomainsandmotifs,suchasthehighlycon-
servedNPA(Asn-Pro-Ala)motifs,andconsideredtobecorrect.ThenumberofMIPgenes
describedinthisstudyissimilartothatfoundinthegenomesofArabidopsis[5],maize[6],
rice[7]andgrape[8],butsignificantlylowerthanthatidentifiedinthegenomesofpoplar[9],
cotton[10],tomato[11]andsoybean[12].Theabsenceofrecentwhole-genomeduplication
(WGD)eventsinthesweetorangegenome,asdescribedbyXuetal.[23],couldaccountfor
therelativelysmallsizeoftheMIPfamilyinthecitrusgenome.
TheCsMIPswereclassifiedinfivedifferentsubfamilies,PIPs,TIPs,NIPs,SIPsandXIPs,
basedonanalysisoftheaminoacidresidueslocatedinsevenkeypositions(P1toP7)thatwere
previouslyproposed[12,34,35]todiscriminatethedifferentsubfamilies(S2Table),aswellas
ontheirphylogeneticrelationshipswiththewellclassifiedMIPsofA.thalianaandXIPsofR.
communisandN.benthamiana(S1Fig).Ouranalysisrevealedthepresenceof8PIPs,11TIPs,
9NIPs,3SIPsand3XIPsinthesweetorangegenome(Table1).TheCsMIPswerenamed
accordingtothenomenclatureproposedinclassificationoftheMIPsofA.thaliana.This
nomenclaturewasbasedonphylogeneticanalysesandwherethenamesinasystematicway
reflectdistinctcladesthatareevolutionarilystable[5].PIPs,TIPs,NIPsandSIPsfromsweet
orangegroupedwiththeirrespectiveArabidopsiscounterparts,indicatingthelargeextentof
conservationbetweenthesweetorangeandArabidopsisMIPgenefamilies(S1Fig).Theonly
exceptionwasCsTIP6;1,whichwasfoundtoencodeaN-andC-terminallytruncatedTIPpro-
teincomparedtotherestofthesubfamily.
ToexaminewhetherthenumberofMIPgenesfoundinthediploidsweetorangeiscompa-
rabletothatofthedihaploidsweetorangeandhaploidClementinemandarin,wealsoper-
formedhomologysearchesagainstthedihaploidsweetorangedraftgenomeavailableatthe
CitrussinensisAnnotationProject(CAP)andthereferencehaploidClementinemandarin(C.
clementina)genomeavailableatPhytozome(S3Table).AlthoughthetotalnumberofMIP
geneswasroughlysimilaramongthedifferentcitrusgenomes,significantdifferenceswere
observedinthenumberofmemberswithinthesubfamilies(Table2).BLASTsimilarityanaly-
sisrevealedthatthedihaploidsweetorangeandhaploidClementinecontainedtwoadditional
PIPisoformscloselyrelatedtotheCsPIP2;1(S3Table).Clementinealsocontainedone
PLOSONE|DOI:10.1371/journal.pone.0138786 September23,2015 5/17
Genome-WideCharacterizationofSweetOrangeMIPs
Table1. GenesandencodedMIPproteinsinsweetorange.
Gene Locus Chromosomelocation Group Kegg Polypeptide pI GRAVY Predictedsubcellular
name OrthID length(MW) localization
CsPIP1;1 orange1.1g018895m chr7:31,253,722...31,256,103 PIP K09872 349(37.56kDa) 9.39 0.397 plasmamembrane
CsPIP1;2 orange1.1g023021m chr7:31,247,369...31,248,902 288(30.67kDa) 7.71 0.414 plasmamembrane
CsPIP1;3 orange1.1g023107m chr5:1,804,896...1,807,677 287(30.70kDa) 8.97 0.344 plasmamembrane
CsPIP1;4 orange1.1g023069m chr6:9,907,825...9,909,541 287(30.82kDa) 8.96 0.343 plasmamembrane
CsPIP2;1 orange1.1g023108m chr6:12,833,884...12,835,042 287(30.67kDa) 8.74 0.376 plasmamembrane
CsPIP2;2 orange1.1g022966m chr8:19,657,287...19,659,497 289(31.05kDa) 7.62 0.392 plasmamembrane
CsPIP2;3 orange1.1g019681m chr7:26,202,220...26,205,546 337(36.34KDa) 9.77 0.403 plasmamembrane
CsPIP2;4 orange1.1g023370m chr8:981,841...983,571 283(30.14kDa) 8.99 0.431 plasmamembrane
CsTIP1;1 orange1.1g025548m chrUn:46,663,407...46,665,011 TIP K09873 251(26.06kDa) 6.12 0.675 vacuole
CsTIP1;2 orange1.1g025600m chr8:20,659,157...20,660,437 250(25.65kDa) 5.32 0.841 cytosol
CsTIP1;3 orange1.1g037978m chr8:20,659,157...20,660,437 124(12.92kDa) 4.37 0.734 cytosol
CsTIP1;4 orange1.1g025464m chr7:29,135,182...29,136,531 252(26.01kDa) 5,69 0,786 vacuole
CsTIP2;1 orange1.1g025817m chr1:18,627,617...18,629,472 247(25.15kDa) 5.59 0.894 vacuole
CsTIP2;2 orange1.1g025865m chr1:18,627,617...18,629,472 247(25.10kDa) 5.59 0.902 vacuole
CsTIP2;3 orange1.1g038895m chr5:5,749,487...5,750,939 206(20.55kDa) 4.72 0.979 vacuole
CsTIP3;1 orange1.1g025197m chr5:16,938,542...16,940,192 256(26.99kDa) 7.07 0.626 cytosol
CsTIP4;1 orange1.1g025864m chr4:19,032,254...19,033,990 247(16.27kDa) 6.27 0.825 vacuole
CsTIP5;1 orange1.1g046726m chr9:14,144,215...14,145,199 161(16.86kDa) 9.00 0.770 cytosol
CsTIP6;1 orange1.1g042738m chr9:14,144,215...14,145,199 107(11.11kDa) 4.54 0.636 secreted
CsNIP1;1 orange1.1g023184m chr2:2,151,220...2,153,387 NIP K09874 286(30.45kDa) 8.64 0.434 vacuole
CsNIP2;1 orange1.1g036721m chr6:18,134,848...18,136,228 223(23.70kDa) 9.69 0.889 cytosol
CsNIP2;2 orange1.1g040981m chr6:18,134,848...18,136,228 211(22.32kDa) 9.39 0.967 cytosol
CsNIP2;3 orange1.1g040755m chr2:13,464,261...13,465,771 275(29.39kDa) 8.88 0.628 plasmamembrane
CsNIP3;1 orange1.1g023102m chr6:20,482,599...20,486,038 287(30.30kDa) 8.40 0.387 plasmamembrane
CsNIP4;1 orange1.1g046511m chr3:23,770,831...23,774,009 282(29.28kDa) 8.84 0.372 vacuole
CsNIP5;1 orange1.1g035030m chr1:13,680,675...13,682,992 75(7.75kDa) 8.98 0.291 chloroplast
CsNIP5;2 orange1.1g027840m chr1:13,678,241...13,681,090 218(22.51kDa) 7.75 0.462 plasmamembrane
CsNIP6;1 orange1.1g039196m chr9:3,798,017...3,800,780 288(30.20kDa) 7.53 0.718 plasmamembrane
CsSIP1;1 orange1.1g026039m chr5:28,968,880...28,972,401 SIP K09875 244(25.92kDa) 9.35 0.727 plasmamembrane
CsSIP1;2 orange1.1g026082m chr3:1,234,876...1,236,556 244(26.17kDa) 9.83 0.749 plasmamembrane
CsSIP2;1 orange1.1g026600m chr6:17,078,102...17,081,323 236(25.57kDa) 9.70 0.600 chloroplast
CsXIP1;1 orange1.1g036381m chr8:7,139,938...7,141,114 XIP - 235(25.09kDa) 8.70 0.821 plasmamembrane
CsXIP1;2 orange1.1g040654m chr8:7,131,064...7,132,799 302(32.68kDa) 8.74 0.573 plasmamembrane
CsXIP2;1 orange1.1g045670m chr8:7,128,184...7,129,448 319(34.58kDa) 8.32 0.681 plasmamembrane
doi:10.1371/journal.pone.0138786.t001
additionalPIPisoformcloselyrelatedtotheCsPIP1;2andonePIP,TIPandNIPisoformwith-
outhomologytoanyMIPsequencefromthediploidsweetorange(S3Table).CsTIP1;2and
CsTIP1;3,CsTIP2;1andCsTIP2;2,CsTIP5;1andCsTIP6;1,andCsNIP2;1andCsNIP2;2exhib-
itedsignificanthitstothesameMIPisoformsofthedihaploidsweetorangeandhaploidClem-
entine(S3Table).TheseobservedvariationsinthesizeoftheMIPsubfamiliesmaybea
consequenceofthedifferentsequencingdepthandassemblyqualitybetweenthediploidand
dihaploidsweetorangegenomes[23,25]andtheevolutionaryoriginofClementine,whichisa
hybridofWillowleafmandarinandsweet-orange[25]and,thereby,itcontainsmoreC.reticu-
latahaplotyperegionsthanfoundinsweetorange.
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Genome-WideCharacterizationofSweetOrangeMIPs
Table2. ComparisonofthenumberofthedifferentMIPfamilymembersindiploid(Phytozome)anddihaploid(CAP)sweetoranges(C.sinensis)
andhaploidClementine(C.clementina).
Subfamily DiploidC.sinensis DihaploidC.sinensis C.clementina
PIP 8 10 11
TIP 11 8 9
NIP 9 8 8
SIP 3 3 3
XIP 3 3 2
Total 34 32 33
doi:10.1371/journal.pone.0138786.t002
CsMIPproteinpropertiesandconservedaminoacidresidues
TheCsMIPsencodeproteinsrangingfrom75(7.7kDa)to349(37.6kDa)aminoacidsin
length,andpIvaluesrangingfrom4.37to9.77(Table1).TheaverageproteinlengthofPIPs,
TIPs,NIPs,SIPsandXIPswere300.8(32.2kDa),213.4(21.1kDa),238.3(25.1kDa),241.3
(25.9kDa)and285.3(30.8kDa)aminoacids,respectively.TheaveragepIofPIPs,TIPs,NIPs,
SIPsandXIPswere8.77,5.86,8.68,9.63,and8.59,respectively.ThesedatarevealthatCsTIPs
arenotonlysmaller,butmostofthemarealsomuchmoreacidicthantheotherCsMIPs,as
reportedforArabidopsisMIPs[38].ThecauseoftheselargedifferencesinTIPshasbeenattrib-
utedtothesmalleramountofbasicresiduesfoundatthecarboxylterminiofTIPscompared
withtheotherMIPs[5].AlltheCsMIPshadapositiveGRAVYscore(Table1),suggestingthat
theyarehydrophobicproteins,whichisanecessarycharacteristicforAQPs[1].Analysisofthe
predictedsubcellularlocalizationshowedthatallCsPIPsandCsXIPswerelocalizedtoplasma
membrane(Table1).ThepredictedlocalizationofCsTIPs,CsNIPsandCsSIPswasmore
diverse,includingvacuole(CsTIP1;1,CsTIP1;4,CsTIP2;1,CsTIP2;2,CsTIP2;3,CsTIP4;1,
CsNIP1;1andCsNIP4;1),cytosol(CsTIP1;2,CsTIP1;3,CsTIP3;1,CsTIP5;1,CsNIP2;1and
CsNIP2;2),plasmamembrane(CsNIP2;3,CsNIP3;1,CsNIP5;2,CsNIP6;1,CsSIP1;1and
CsSIP1;2),chloroplast(CsNIP5;1andCsSIP2;1)andsecreted(CsTIP6;1)(Table1).These
resultsseemtobeinagreementwiththeexperimentallydeterminedlocalizationsofMIPs
reportedintheliterature[13,39].
MIPfoldingischaracterizedbysixtransmembraneα-helices(H1toH6)thatareconnected
byfiveloops(loopsA-E),forminganaqueoustransmembraneporethatconstitutesthefunc-
tionalcoreofMIPs[35].LoopsB(LB)andE(LE)containtwohighlyconservedNPA(Asn-
Pro-Ala)motifsthatareconsideredtobecriticalforthesubstrateselectivityofMIPs[40,41].
Anothersetoffourconservedresiduesformsthearomatic/Arginineselectivityfilter(ar/Rfil-
ter),whichhasbeenproposedtoactasasizeexclusionbarrierforsubstratemolecules[42].
ThefirsttworesiduesarelocatedinH2andH5,whilethelattertwoarefoundinLE(LE1and
LE2).Finally,sevenkeyaminoacidresidues(namedpositionsP1toP7)havebeenproposedto
discriminatethefivesubfamilies[12,34].P1islocatedintheterminalpartofH3,whileP2and
P3arelocatedinLE,justbehindthesecondNPAmotif(2ndand6thresiduesafter2ndNPA,
respectively).P4andP5correspondtotwoconsecutiveaminoacidslocatedinH6,whileP6
andP7alsocorrespondtotwoconsecutiveaminoacidslocatedinH3.Themultiplesequence
alignmentswerecarefullyanalyzedandalltheseconservedmotifsandaminoacidresidues
wereidentifiedinmostCsMIPs,indicatingthattheyarefunctionalchannelsforwaterand
othersolutes(S2Table).AlltheCsPIPsshowedthedualtypicalNPAmotifsandanar/Rfilter
configurationcharacteristicforawater-transportingMIP(F,H,T,R).Additionalpresenceof
theS-A-F-WresiduesatP2-P5positions,asobservedinallCsPIPs,exceptforCsPIP1;1,has
beeninterpretedasasignatureofCO transporterPIPs[35].AlltheCsTIPsalsohadthetwo
2
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Genome-WideCharacterizationofSweetOrangeMIPs
canonicalNPAmotifs,exceptCsTIP1;3,CsTIP5;1andCsTIP6;1,whichwerefoundtoencode
truncatedproteinslackingeitherthefirst(CsTIP1;3)orthesecondNPAmotif(CsTIP5;1and
CsTIP6;1),aswellasotherconservedaminoacidresiduesofthear/Rfilterregion(S2Table).
TIPscontainingtheH-I-A-VorH-I-G-Rresiduesinthear/RfilterandT-A-A-Y-Wor
T-S-A-Y-WresiduesatP1-P5positions,likeCsTIP1s,CsTIP2sandCsTIP3,havebeenshown
totransportureaandH O [35].TheCsNIP1;1,CsNIP2;1,CsNIP2;2andCsNIP2;3showedan
2 2
ar/RfilterconfigurationidenticaltothatofsoybeanNodulin26,indicatingthattheyarealso
abletofacilitatewaterandsolutetransportcapability[30].TheresidueattheH5positionof
thear/RfilterofAtNIP5;1wasshowntoplayakeyroleinthemembranepermeabilityto
water,silicicacid(Si)andboricacid(B)[42].AtNIP5;1withAIGRresiduesforthear/Rfilter
wasshowntotransportwater,Bandarsenite(As),butnotSi[42].CsNIP3;1,withGSGRresi-
duesforar/Rfilter,canbeexpectedtotransportwater,SiandB,whileCsNIP4;1(AIGRresi-
dues)maytransportwater,BandAs.TheCsSIPsshowedalessconservedfirstNPAmotif,
whilethesecondNPAmotifwasperfectlyconservedinallmembers(S2Table).AtSIP1iso-
forms,butnotAtSIP2;1,wereshowntobefunctionalwaterchannels[43],suggestingthatthe
lattermaybeinvolvedinthetransportofsolutes.However,SIPtransportfunctionandstruc-
turalorganizationstillawaitbiochemicalcharacterization.TheCsXIPsalsoshowedamodified
firstNPAmotif,NPL(CsXIP1s)orSPV(CsXIP2),andaconservedsecondNPAmotif(S2
Table).Thefourpositionsinthear/Rfilterregioncontainedaminoacidresiduesthatwere
strictlyconservedamongtheCsXIPs.CsXIP1;1wasobservedtocontainaninternaldeletionof
13aminoacidresiduesintheH2regionthatabolishedtheconservedaminoacidVatposition
H2ofthear/Rselectivefilter.Sincethefirstthreeaminoacidofthear/Rfilterhaverather
hydrophobicresidues(VVARorVVVR),theCsXIPsmightbeinvolvedinthetransportof
moleculesotherthanwater[35].Infact,arecentstudyhasindicatedthattheSolanaceaeXIPs
areplasmamembraneaquaporinsinvolvedinthetransportofmanyunchargedsubstrates,
suchasurea,H O andB[44].
2 2
GenomicorganizationofCsMIPs
Theexon-intronstructureofall34CsMIPgeneswasanalyzedusingthesweetorangegenemod-
elsannotatedinPhytozome.Withafewexceptions,thenumberandsizeoftheexons,butnot
oftheintrons,wereobservedtobeconservedwithineachCsMIPsubfamily(S2Fig).Allthe
CsPIPspresentedthreeintronsandfourexons,asreportedforallArabidopsis[5],poplar[9],
tomato[11]andsoybeanPIPs[12].ThemajorityofCsTIPscontainedtwointronsandthree
exons,withexceptionofCsTIP1;1,CsTIP2;3andCsTIP6;1,whichshowedoneintronandtwo
exons,andthetruncatedCsTIP1;3(oneexon)gene.Suchamorevariedpatternofexon-intron
structurehasbeenalsoobservedinArabidopsis[5],poplar[9],tomato[11]andsoybeanTIPs
[12].MostCsNIPscontainedfourintronsandfiveexons,likeallArabidopsis[5]andmostpop-
lar[9],tomato[11]andsoybeanNIPs[12].TheexceptionswereCsNIP2;2(threeintronsand
fourexons),CsNIP5;2(twointronsandthreeexons)andthetruncatedCsNIP5;1(oneintron
andtwoexons)genes.TheCsSIPsfeaturedtwointronsandthreeexons,exceptCsSIP1;2(one
exonandoneintroninthe3’-UTRregion).Similarpatternsofexon-intronstructurewerealso
reportedfortomatoSIPs[11],whilealltheArabidopsis[5]andpoplarSIPs[9]hadtwointrons
andthreeexons.ThegenestructureofCsXIPsvariedamongallmembers,whichcontainedtwo
intronsandthreeexons(CsXIP1;2),eitheroneintronandtwoexons(CsXIP2;1)oronlyone
exon(CsXIP1;1).Thepatternoftwointronandthreeexonshasbeenreportedformostpoplar
[9]andtomatoXIPs[11],whileallsoybeanXIPscontainedasingleintronandtwoexons[12].
Thepositionsofall34CsMIPsweremappedonthesweetorangechromosomesbyhomol-
ogysearchesagainstthefull-lengthsweetorangegenomeassemblyavailableattheCAP
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Genome-WideCharacterizationofSweetOrangeMIPs
database(Table1andS3Table).ExceptforCsTIP1;1,whichwasnotexactlylocatedonany
chromosomebecauseofanincompletephysicalmapforsweetorange,alltheCsMIPlociwere
preciselymappedoneverysweetorangechromosome,indicatingawidedistributionofthe
genefamilyinthesweetorangegenome(Table1andS3Fig).ThecloselyrelatedCsMIPiso-
formsCsTIP1;2andCsTIP1;3,CsTIP2;1andCsTIP2;2,CsTIP5;1andCsTIP6;1,andCsNIP2;1
andCsNIP2;2wererespectivelymappedonidenticalchromosomepositionssincetheyshowed
significanthitstothesamegenesintheCAPdatabase(S3Table).SevenCsMIPswerefoundto
betandemduplicatedgenesaccordingtothecriteriaofHanadaetal.[45],whichdefinedtan-
demduplicatesasgenesinanygenepair,T andT ,that(1)belongtothesamegenefamily,
1 2
(2)arelocatedwithin100kbeachother,and(3)areseparatedby10orfewernonhomologous
spacergenes.ThesewereCsNIP5;1andCsNIP5;2onchromosome1,CsPIP1;1andCsPIP1;2on
chromosome7,andCsXIP1;1,CsXIP1;2andCsXIP2;1onchromosome8(Table1andS3Fig).
TheseresultssuggestthatalltheseCsMIPsmayhaveevolvedfromtandemduplicationevents,
asalsorecentlyproposedforthetomatoXIPs[11].
Analysisofpreviouslymappedtraitsrevealedthatthe282-kbregionsurroundingtheCitrus
TristezaVirusresistance(Ctv)locusisphysicallylinked(~40-kb)totheCsNIP5;1and
CsNIP5;2genes,onthechromosome1[46].ThisregionwasalsoreportedtocontainTyr1,the
majorlocuscontrollingcitrusnematode(Tylenchulussemipenetrans)resistance[47,48].
ExpressionpatternsofCsMIPgenesindifferenttissues
ToinvestigatetheexpressionpatternsofCsMIPsindifferenttissues,RNA-seqdataweredown-
loadedfromCAP[24].Theheatmapgeneratedshowedadifferentialtranscriptabundanceof
the34CsMIPsinfourmajortissues,namelycallus,flower,leaf,fruit,andmixedfruittissuesat
threedevelopmentalstages(S4Fig).SomeoftheCsMIPs(CsPIP1;1,CsPIP1;3,CsPIP2;2,
CsPIP2;3andCsSIP1;1)showedhigherexpressioninalltheseventissues,indicatingarolein
constitutivetransportprocessesthroughouttheplant.Othersgeneswerefoundtohavealow
expressioninallthetissues(CsTIP1;1,CsTIP1;2,CsTIP1;3,CsTIP2;3,CsTIP5;1,CsTIP6;1,
CsNIP2;1,CsNIP2;2,CsNIP2;3,CsNIP5;1,CsNIP5;2,CsXIP1;1andCsXIP2;1).Theputativetan-
demduplicatedCsMIPgeneswereobservedtohavedivergentexpressionprofiles,whichprob-
ablyhascontributedtotheirmaintenancethroughregulatorysubfunctionalizationand
neofunctionalization[49].CsPIP1;1showedahigherexpressionthanCsPIP1;2inalltheseven
tissuesanalyzed.CsXIP1;1andCsXIP2;1showedlowexpressioninalltheseventissuesana-
lyzed,whileCsXIP1;2exhibitedahighexpressioninflower,leafandmixedfruittissue(Mix.3).
Thecelltypelocalizationofaquaporinexpressioncanalsoprovidecluesabouttheirphysio-
logicalroles.Forinstance,expressionofPIPaquaporinsisgenerallylocalizedinorgansandtis-
suescharacterizedbylargefluxesofwater,suchasvasculartissues,guardcells,flowersand
fruits[4].Theirexpressioninrootsandleaveshasbeenalsocorrelatedwiththepresenceof
apoplasticbarriers,theexodermisandendodermisinrootsorinsuberizedbundlesheathcells
inleaves,suggestingtheiressentialroleinthetransmembranewaterdiffusionwhenitsmove-
mentishindered[19,20,50–55].ExceptforCsPIP2;1,alltheCsPIPswerefoundtobehighly
expressedinflower,leaf,fruitandmixedfruittissues(S4Fig),supportingtheiractiverolein
thetransportofwaterandsolutesacrossthesetissues.Bycontrast,theexpressionofTIPiso-
formshasbeenmorerelatedtodevelopmentalstagesand/ororganspecificity[56].For
instance,theexpressionofAtTIP2;1isespeciallyhighinthevascularsystemoftheshootbutis
barelydetectableintheroot[57].AtTIP3;1ishighlyexpressedincotyledonsandassociated
withthemembraneofproteinstoragevacuoles[4].TIPsarealsodifferentiallyexpresseddur-
ingfruitmaturation,e.g.,theTIP1;1homologinpearishighlyexpressedintheyoungfruit,
whereasTIPproteinslevelsingrapegraduallyincreasealongwithripening[58,59].Vacuoles
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Genome-WideCharacterizationofSweetOrangeMIPs
participateincellexpansionand,thus,theirenlargementbywatercompartmentationisessen-
tialtoprovoketherapidfruitgrowththatischaracteristicoftheripeningprocess.Thelackof
specificregulationobservedalongfruitripeningforPIPsisoformspointsouttheessentialrole
ofTIPsinthisprocess[60].ThedifferentialexpressionofCsTIP1;3,CsTIP1;4,CsTIP2;1,
CsTIP2;2,CsTIP3;1,CsTIP4;1andCsTIP5;1highlightsthefunctionalimportanceofthese
CsMIPsoneachtissueandstageoffruitdevelopmentanalyzed(S4Fig).Theoveralllevelof
NIPexpressionisusuallylowerthantheexpressionofPIPsandTIPs,anditisusuallyassoci-
atedwithspecializedorgansandcells[10].Forinstance,AtNIP2;1isspecificallyexpressedin
theendoplasmicreticulum(ER)ofroots,whereasAtNIP5;1isaplasmamembraneMIPmainly
expressedinrootelongationzones[39,61].OuranalysisshowedthattheCsNIPshadpreferen-
tialexpressioneitherinflower(CsNIP6;1),leaf(CsNIP2;2andCsNIP3;1)ormixedfruittissues
atdifferentdevelopmentalstages(CsNIP1;1,CsNIP2;2,CsNIP3;1,CsNIP4;1andCsNIP6;1)(S4
Fig).SIPsseemtobeexpressedinarangeoftissuesinArabidopsis,includingyoungroots,flow-
ersandpollen[62].ItisremarkablethatSIPsarealsostronglyexpressedinsuspensioncultured
cellscomparedtootherMIPs[62].CsSIP1;1wasobservedtobeconstitutivelyexpressedinall
theseventissuesanalyzes,whiletheotherswerepreferentiallyexpressedinflowerandfruittis-
sues(S4Fig).XIPswerereportedtobeexpressedinalmostallthepoplartissues[9].Bycon-
trast,CsXIPsshowedalowexpressioninalltissuesanalyzed,exceptCsXIP1;2,whichshowa
relativelyhighexpressioninflower,leafandmixedfruittissuesatthirddevelopmentalstage
(Mix.3)(S4Fig).
ExpressionpatternsofCsMIPgenesunderabioticandbioticstresses
ToidentifyCsMIPswithapotentialroleinabioticandbioticstressresponseofsweetorange,
theexpressionpatternsofallthe34sweetorangeMIPswereinvestigatedinplantsexposedto
drought,highsalinityand‘Ca.L.asiaticus’(HLB)infection,byqPCR.Consideringthelog
2
foldchange(LFC)of(cid:1)1.00or(cid:3)-1.00ascutoffthresholdbetweenstressedandcontrolplants,
theqPCRanalysesshowedthatalltheCsMIPsweredifferentiallyexpressedinatleastone
stressconditionandtissueanalyzed(Figs1–3).TwelveCsMIPs(CsPIP1;1,CsPIP2;4,CsTIP1;3,
CsTIP2;1,CsTIP2;2,CsTIP3;1,CsTIP4;1,CsNIP1;1,CsSIP1;2,CsXIP1;1,CsXIP1;2and
CsXIP2;1)wereobservedtobedifferentiallyexpressedinresponsetoallthethreestresscondi-
tionsanalyzed,inatleastonetissuestudied.Interestingly,onlyCsTIP1;1showeddifferential
expressionexclusivelyinresponsetotheabioticstresstreatments,whilesixCsMIPs(CsPIP1;2,
CsPIP2;2,CsNIP2;2,CsNIP5;2,CsNIP6;1andCsSIP1;1)weredifferentiallyexpressedexclu-
sivelyunderthebioticstresstreatment.Theseresultsseemtobeconsistentwiththerespective
organizationofthestress-responsivecis-actingregulatoryelementsobservedintheCsMIPs
promoters(S5Fig).CsTIP1;1wasobservedtocontainthehighestnumberofABREcopiesin
thepromotorregionamongthesweetorangeMIPgenes,whilenooralownumberofABRE
(lessthan2copies)andotherstress-responsivecis-actingregulatoryelementswasdetectedin
thepromoterregionsoftheCsMIPsthatwerenotinducedbytheabioticstresstreatments(S5
Fig).AsinglecopyofDRE/CRThasbeenobservedtobesufficientforABA-independent
stress-responsivegeneexpression,whilemorethantwoABREsequencesareusuallyrequired
fortheABA-responsivetranscription[63].
AlltheCsTIPsandCsXIPswereupregulatedinleavesbutdownregulatedinrootsbydrought
treatment,whileonlytwoCsPIPs(CsPIP1;1andCsPIP2;4)andoneCsNIP(CsNIP1;1)and
CsSIP(CsSIP1;2)weredifferentiallyupregulatedbydroughttreatmentinleaves(Fig1).Most
CsMIPswereupregulatedbysalttreatmentinroots,andeitherupregulatedordownregulated
bythistreatmentinleaves,dependingontheisoform(Fig2).Acoordinatedup-anddownregu-
lation,dependingontheMIPgeneandorganexamined,hasbeendescribedasageneralpattern
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