The Small-RNA Profiles of Almond

24 Pages·2016·7.95 MB·English

RESEARCHARTICLE
The Small-RNA Profiles of Almond (Prunus
dulcis Mill.) Reproductive Tissues in Response
to Cold Stress
MarziehKarimi1☯,FarahnazGhazanfari1☯,AdelehFadaei1☯,LalehAhmadi1☯,
BehrouzShiran1,2*,MohammadRabei1,HosseinFallahi3,4*
1 DepartmentofPlantBreedingandBiotechnology,FacultyofAgriculture,ShahrekordUniversity,
Shahrekord,P.O.Box115,Iran,2 InstituteofBiotechnology,ShahrekordUniversity,Shahrekord,P.O.
Box115,Iran,3 DepartmentofBiology,SchoolofSciences,RaziUniversity,Bagh-e-Abrisham
Kermanshah,Iran,4 MedicalBiologyResearchCenter,KermanshahUniversityofMedicalSciences,
Kermanshah,Iran
☯Theseauthorscontributedequallytothiswork.
a11111
*[email protected](BS);[email protected](HF)
Abstract
SpringfrostisanimportantenvironmentalstressthatthreatenstheproductionofPrunus
OPENACCESS trees.However,littleinformationisavailableregardingmolecularresponseoftheseplants
tothefroststress.Usinghighthroughputsequencing,thisstudywasconductedtoidentify
Citation:KarimiM,GhazanfariF,FadaeiA,Ahmadi
L,ShiranB,RabeiM,etal.(2016)TheSmall-RNA differentiallyexpressedmiRNAs,boththeconservedandthenon-conservedones,inthe
ProfilesofAlmond(PrunusdulcisMill.)Reproductive reproductivetissuesofalmondtolerantHgenotypeundercoldstress.Analysisof50to58
TissuesinResponsetoColdStress.PLoSONE
millionrawreadsledtoidentificationof174uniqueconservedand59novelmicroRNAs
11(6):e0156519.doi:10.1371/journal.pone.0156519
(miRNAs).Differentialexpressionpatternanalysisshowedthat50miRNAfamilieswere
Editor:MuhammadBarozai,Universityof
expresseddifferentiallyinoneorbothofalmondreproductivetissues(antherandovary).
Balochistan,PAKISTAN
Outofthese50miRNAfamilies,12and15displayedup-regulationanddown-regulation,
Received:February1,2016
respectively.ThedistributionofconservedmiRNAfamiliesindicatedthatmiR482fharbor
Accepted:May16,2016 thehighestnumberofmembers.ConfirmationofmiRNAsexpressionpatternsbyquantita-
Published:June2,2016 tivereal-timePCR(qPCR)wasperformedincoldtolerant(Hgenotype)alongsideasensi-
tivevariety(Sh12genotype).Ouranalysisrevealeddifferentialexpressionfor9miRNAsin
Copyright:©2016Karimietal.Thisisanopen
accessarticledistributedunderthetermsofthe antherand3miRNAsinovarybetweenthesetwovarieties.TargetpredictionofmiRNAsfol-
CreativeCommonsAttributionLicense,whichpermits lowedbydifferentialexpressionanalysisresultedinidentificationof83targetgenes,mostly
unrestricteduse,distribution,andreproductioninany
transcriptionfactors.ThisstudycomprehensivelycataloguedexpressedmiRNAsunderdif-
medium,providedtheoriginalauthorandsourceare
ferenttemperaturesintworeproductivetissues(antherandovary).Resultsofcurrentstudy
credited.
andthepreviousRNA-seqstudy,whichwasconductedinthesametissuesbyourgroup,
DataAvailabilityStatement:Theauthorsconfirm
provideauniqueopportunitytounderstandthemolecularbasisofresponsesofalmondto
thatalldataunderlyingthefindingsarefullyavailable
withoutrestriction.Thedatahavebeenuploadedat coldstress.Theresultscanalsoenhancethepossibilityforgenemanipulationtodevelop
SRApartofNCBIhttp://www.ncbi.nlm.nih.gov/sra coldtolerantplants.
[accessionnumberPRJNA245549].
Funding:Theauthorsreceivednospecificfunding
forthiswork.
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist.
PLOSONE|DOI:10.1371/journal.pone.0156519 June2,2016 1/24
SmallRNA-SeqinAlmondunderColdStress
Introduction
AlmondisaperennialspeciesbelongingtoPrunoideae,asubfamilyofRosaceae.Almondis
predominantlyself-incompatible.Ithassixteen(2n=16)smallchromosomesandasmall
diploidgenomeofapproximately300Mbp[1].Springfrostisoneofthemostimportant
environmentalfactors,whichlimitsalmond’sproduction.Duringwinter,budsaredormant
andhard.Buttheyswellandexpandintoblossomswhenthetemperaturerises.Asaresult,
theybecomelessresistanttothecold.Frostcandamage10%to90%ofthealmondtreeindif-
ferentphonologicalstagesofflowering[2].Followingourpreviousstudyontranscriptomeof
almondreproductivetissuesinresponsetospringfrost[3],wehavedecidedtofurther
expandourunderstandingofthemolecularbasisofalmondresponsestocoldstressbycon-
ductingsmallRNA-Seqanalysisinthesametissues.AsmiRNAsaremegabio-regulatorsin
plants[4]andareinvolvedindifferentstressresponses[5,6],wethoughtthatchangesin
geneexpressioninresponsetocoldwouldalsoreflectasimilarpatterninmiRNAsexpression
pattern.
PlantsinvolvementofmiRNAsandtheirtargetgenesinresponsetostressconditionsis
welldocumentedinseveralstudiessuchascoldsignalingregulation[7,8]andsaltand
nutrientdeficiency[9–11].Forinstance,Barakatetal.[12]haveidentified157conserved
and230non-conservedmiRNAssequencesinPrunuspersicainresponsetocoldstress.
InMedicagotruncatula,283and293miRNAswereidentifiedfromcontrolanddrought
stresslibraries,respectively[13].Zhangetal.[14]reported106conservedmiRNAsfromtea
leavestreatedwithcold(Camelliasinensis).Sunandcolleagues[15]identified136known
and68novelmiRNAsinRaphanussativusinasalinitystressexperiment.AmongthesemiR-
NAs,severalwerecharacterizedasstressresponsive.ManystudieshaveshownthatmiR156/
157,miR159/319,miR165/166,miR169,miR172,miR393,miR394,miR396,miR397and
miR398areup-regulatedinresponsetocoldstress[8,16,17].Someofthestudies,moreover,
haveshownthedown-regulationofmiRNAssuchasmiR156g-j,miR475a,bandmiR476a
undercoldstressinPopulus[18].AlthoughinformationregardingmiRNAsexpressionin
manyspeciesisbuildingupquickly,wedonothaveanyreportsregardingthenumberof
miRNAsandtheirexpressionpatterninplantssuchasalmond(Prunus.dulcis(Mill.)D.A.
Webb).
Differentapproachesincludingforwardgenetics,directcloningandbioinformaticspredic-
tionfollowedbyexperimentalvalidationcanidentifymiRNAs[6,19].However,these
approacheshavelimitationssuchastime,costandtheversatilityofthemethods.Inrecent
years,nextgenerationsequencingplatformslikeGenomeAnalyzer(IlluminaInc.)orGenome
SequencerTMFLX(454LifeScienceTMandRocheAppliedScience)havebeenusedtodetect
smallRNAmoleculesanddeterminetheexpressionlevelsofbothconservedandnovelmiR-
NAs[19–22].Inmanyplantspecies,thismethodhasbeensuccessfullyappliedtodiscover
miRNAsresponsestodifferentabioticstress.Someoftheexamplesofsuchapplicationsarethe
identificationofdroughtstressresponsivemiRNAsinPopulustrichocarpa[23],saltstress
responsivemiRNAsinSaccharumspontaneum[24]andheavymetalstressresponsivemiRNAs
inMedicagotruncatula[25].
Untilnow,thereisnoreportontheresponsesofalmond’smiRNAstocoldstress,espe-
ciallyinthereproductivetissues,wherethecoldstresswouldhavethemostdevastating
effects.Asthewholegenomesequencingofalmondisnotavailable,theresultsofourtwo
studiescouldbemergedtouncoverthemolecularresponsesofreproductivetissuesofalmond
tocoldstress.
PLOSONE|DOI:10.1371/journal.pone.0156519 June2,2016 2/24
SmallRNA-SeqinAlmondunderColdStress
Results
Constructionandhigh-throughputsequencingofsmallRNAslibrariesin
almond
cDNAlibrariesofsmallRNAswereconstructedfromantherandovaryundercoldstressand
controlconditionsinordertoidentifymiRNAsinalmond.Oursamplecollectionconsistedof
anthersamplesunderstress(HSA)andundercontrol(HCA);andovarysamplesunderstress
(HSO)andundercontrol(HCO).AllsampleswereobtainedfromHgentoptypeofalmond,
whichisbeleivedtobetoleranttocoldstress.Wehaveobtained50to58millionrawreads,
usingIlluminasequencingplatformwithaveragereadlengthof51nucleotidesandthequality
of39forallsamples.GCcontentsforallsampleswere52%,whileitwas51%intheHSA.
HCAsamplesyieldedthehighestnumberofsmallRNAreadsofabout58,664,982,while
HSOsamplescontained50,245,161reads.85.74%(12.77%uniquereads)fromHCA,78.33%
(13.45%uniquereads)fromHSA,91.03%(16.09%uniquereads)fromHCOand88.85%
(17.77%uniquereads)fromHSOwereobtainedafterfiltering.Theadaptorsandallcontami-
nantedsmallRNAsincludingtRNA,snRNA,snoRNAandrRNAwereremovedandresidual
readswerequeriedagainstknownmiRNAsinmiRBase(version20).2,137,645,1,626,754,
2,767,683and2,197,689readsfromHCA,HSA,HCOandHSOlibrarieswereidentifiedas
conservedmiRNAs.Therestofthesequenceswereconsideredasnon-conservedsRNA
sequences.
IdentificationofconservedmiRNAsinalmond
AfterperformingablastagainstmiRBasedatabase(version20),sequencesimilarityreleaved
thepresenceof131,122,123and119miRNAsintheHCA,HCO,HSA,andHSOlibraries,
where94miRNAswereunique.Outofthese94uniqulyidentifiedconservedmiRNAfamilies
infourlibrarires,26werehighlyconservedand68wereconservedinsomeplantspecies(S1
Table).ThehighlyconservedmiRNAfamiliescontained1–10membersinourdataset.
miR482ffamilyhadthehighestnumberofmembers(10members).Thiswasfollowedby
miRNA171family,having8members(Fig1).Ontheotherhand,amongtheconserved
miRNAsfamily,miR6267,miR6291,miR7122with3membershadthehighestnumberof
Fig1.ThedistributionofhighlyconservedmiRNAsfamilysizeinprunusdulcis.
doi:10.1371/journal.pone.0156519.g001
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SmallRNA-SeqinAlmondunderColdStress
Fig2.ThedistributionofconservedmiRNAsfamilyinprunusdulcis.
doi:10.1371/journal.pone.0156519.g002
members,whilethemostoftheotherconservedmiRNAfamiliesconatinedonlyonemember
(Fig2).
AcomparisonbetweenmiRNAfamilymembersinthesefourlibrariesshowedthat32
miRNAfamilieshadtissues-specificexpressionpattern.AmongthesemiRNAs,21were
expressedonlyintheantherand11intheovary(Fig3A).Interstingley,10miRNAsfoundto
beexpressedsolelyundercold-stressconditions.NineteenmiRNAswerespecificallyexpressed
undercontrolconditionsandtherfeore,theirexpressionwasnotdetectableunderstresscondi-
tions(Fig3B).AlsothesizedistributionofmiRNAfamiliesbetweenthefourlibrariesrevealed
62commonmiRNAs,thoughthenumberofmembersweredifferentineachlibrary(Fig4A
and4B).
IdentificationofnovelmiRNAsinalmond
Inourstudy,wefoundmanyreadswithoutanyhitsintheRNAsdatasets.Theywere289,302,
245,497,151,928and177,393readsintheHCA,HSA,HCOandHSOlibraries,respectively.
Therefore,thesereadswereminedtoidentifynon-conservedmiRNAs.FollowingmiRNA
annotationcriteriasetbyMeyerandcollaborators[26],wesuccessfullyidentified59novel
miRNAssequences.Thestem-loopstructuresofthesemiRNAprecursorswereobtainedby
Mfold[27]andarepresentedinFig5.ThemauturelengthofthesemiRNAsvariedbetween18
and22nt.Themostabundantlengthwas21nt.Theprecursorlengthswerefoundtobe
between45to147nt.Theminimumfreeenergy(MFE)variedfrom-71.8to-8.5kcal/mol(S2
Table).TheMFEindex(MFEI)fortheseprecursers,asthemostimportantcriterionforpredic-
tionofstem-loopstructureofmiRNA[28],werecalculatedaccordingtoZhangetal.[14].
Notabley,thematuresequencesof34novelmiRNAswerelocatedon3'armoftheirprecursers
andtherestwerelocatedon5'arm.
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SmallRNA-SeqinAlmondunderColdStress
Fig3.CommonandspecificmiRNAfamiliesidentifiedbyhighthroughputsequencing.A:CommonandspecificmiRNAsin
reproductivetissuesofalmond;B:CommonandspecificidentifiedmiRNAsundercontrolandcoldstress.
doi:10.1371/journal.pone.0156519.g003
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SmallRNA-SeqinAlmondunderColdStress
Fig4.ThedistributionofknownmiRNAfamiliesinHCA,HSA,HCOandHSOlibraries.A:Highlyconserved;B:Conservedinsomespecies.
doi:10.1371/journal.pone.0156519.g004
ExpressionpatternanalysisofknownmiRNAs
ExpressionanalysisofconservedmiRNAsindicatedthatmiR159,miR166,miR482and
miR1511familieshadthehighestexpressioninthefourlibraries.However,distinctexpression
variationwasobservedwithinthemembersofsomefamilies.Forinstance,inHCAlibrary
miR482fhad109668copies,whereasthereadnumberformiR482b-5pwasonly10.The
expressionpatternofmiRNAsinantherindicatedthat53miRNAshadhigherexpressionin
controlconditionscomparedtoovary.Incontrast,48miRNAswereup-regulatedunderstress
conditionsinanthercomparedtoovary.Moreover,41miRNAsshowedsimilarpatterninboth
tissuesexposedtocoldstress.Therefore,wesuggestthatthesemiRNAsshouldbeconsidered
ascoldstressresponsivemiRNAs.FromthesemiRNAs,22weredown-regulatedand19were
up-regulated.AmongdifferntiallyexpressedmiRNAs,miR482d-3pwasup-regulatedin
anther,whileitwasdown-regulatedinovary.Incontrast,miR172a-5pandmiR1511-3pwere
up-regulatedinovaryanddown-regulatedinanther(S3Table).Comparativeanalysisof
expressionpatterninthesetworeproductivetissuesrevealed11and9up-regulatedmiRNAsin
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SmallRNA-SeqinAlmondunderColdStress
Fig5.ThestructureofcandidatenovelmiRNAsprecursor.
doi:10.1371/journal.pone.0156519.g005
antherandovary,respectively(Fig6A).Additionally,20and13down-regulatedmiRNAswere
foundinthesetissuesrespectively(Fig6B).Futhermore,theanalysisidentified5up-regulated
and11down-regulatedmiRNAsinantherwithnosignificantchangesinovary.However,we
found8repressedmiRNAsinanther,whiletheirexpressionwasiducedinovarytissues.
ExpressionpatternvalidationofmiRNAsinreproductivetissuesof
almond
TovalidatetheexpressionpatternsdetectedformiRNAsinthehighthroughputsequencing,16
differentiallyexpressedmiRNAswereselectedandanalyzedwithqPCR.Inaddition,tofurther
investigatethemiRNAresponsestocoldstress,weincludedarathercold-sensitivegenotype
(Sh12)inouranalysis.ExpressionpatterncomparisonbetweensmallRNAsequencingdata
andqPCRresultsindicatedverysimilarpatternsofexpressionfor11and7miRNAsinanther
andovarysamplesunder-2°Ctreatment,respectively.Inanther,furthermore,miR162,
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SmallRNA-SeqinAlmondunderColdStress
Fig6.RelativedifferentiallyexpressedconservedmiRNAinreproductivetissues.A:Anther;B:Ovary.
doi:10.1371/journal.pone.0156519.g006
miR166d,miR168,miR171a,miR398a-3p,miR403,miR482f,miR6285andmiR8123-5p
wereexpresseddifferentiallyinHgenotypeandSh12varieties.However,inovary,miR403,
miR1511-3pandmiR7122a-3pdisplayeddifferentialexpressioninthesetwogenotypes.Onthe
otherhand,miR7122a-3pinantherandmiR162,miR166d,miR398b,miR398a-3p,miR160a,
miR319a,miR168,miR6285andmiR8123-5pinovaryhadsimilarexpressionpatterninboth
tolerantandsensitivevarieties.Unsurprisingly,somemiRNAsdisplayeddifferentmodeof
expressioninantherandovarysamplesofthesetwoalmondverities.Forexample,miR162,
miR477a-3pandmiR482finHgenotypeandmiR168,miR403,miR6285andmiR8123-5pin
Sh12showeddifferentexpressionbetweenthesestudiedtissues.(Figs7,8Aand8B).Amongall
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SmallRNA-SeqinAlmondunderColdStress
Fig7.qPCRanalysisforvalidationofdifferentiallyexpressedmiRNAsexpressioninreproductivetissuesofalmond.Each
barrepresentedthemean±SE.
doi:10.1371/journal.pone.0156519.g007
16differentiallyexpressedmiRNAsstudiedbyqPCR,coldstressrepressedtheexpressionof10
miRNAsinanthertissueofHgenotypeandhadnoeffectsontheexpressionofmiR160a,
miR319a,miR394b,miR398b,miR1511-3pandmiR7122a-5p.Incontrast,miR162,miR171a,
miR319a,miR394b,miR398bwereup-regulatedinSh12undercoldstress.Inaddition,itwas
foundthat-2°CcoldtreatmentinducedtheexpressionofmiR398b,miR477a-3p,miR394b,
miR7122a-3p,miR162andmiR166dinbothovaryandanthertissuesofSh12variety.Different
expressionpatternweredetectedforsomemiRNAsincludingmiR1511-3p,miR398a-3p,
miR166dandmiR7122a-5pinthesevarietiestreatedwithtwotemperatures.Forinstancethe
expressionofmiR1511-3p,miR398a-3pandmiR166dwaslowerin0°Ccomparedtothatof
-2°C.TranscriptofmiR394bandmiR398bwasnotalteredintheanthertissueofHgenotypein
-2°C.ButthetwomiRNAsweredown-regulatedin0°C.AmongthetestedmiRNAs,miR477a-
3p,whichincreased8.6-foldintheovarytissueofHgenotypeunder0°C,andmiR166d,with
8.27-foldincreaseinSh12under-2°C,showedthemostchangesintheirexpressionpattern.In
anthertissueofHgenotype,miR166dandmiR160ahadthegreatestreductionofexpression
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SmallRNA-SeqinAlmondunderColdStress
Fig8.qPCRanalysisofcoldstress-responsivemiRNAsandtheirtargetsexpressionprofiles.A:Anther;B:Ovary.Eachbarrepresentedthe
mean±SE.
doi:10.1371/journal.pone.0156519.g008
under0and-2°C,respectively.MiR319awastheonlymiRNA,whichwasup-regulatedinboth
varietiesunderbothstressconditions.Incontrast,miR160awastheonlymiRNAthatwas
down-regulatedundercoldstressinbothvarieties.InotherstudiesthismiRNAwasintroduced
asacold-inducedmiRNA[29].
Inthisstudy,amongthepredictedtargetsforcold-responsivemiRNAs(S4Table),the
expressionpatternof6miRNA'stargetsnamelyDUF1639(miR398btarget),ABCtransporter
andAAE7(miR477a-3ptargets),FBX(miR394btarget),BHLG041(miR7122a-3ptarget),
DCL1(miR162target)andATHB-15andABA-insensitive5(miR166dtargets)wereexamined
inthelab.NegativecorrelationbetweensomeofthesemiRNAsandrelatedtargetswasdetected
(Table1).Forinstance,cold-stressrepressedmiR477a-3pexpressionwasnegativelycorrelated
withtheconverselyup-regulatedexpressionofbothABCtransporterandAAE7genesin
anthertissueofHgenotype.Ontheotherhand,theup-regulationofmiR394bwasfollowedby
thedown-regulationoftheexpressionofFBXgeneinSh12.Amongthestudiedtargets,ABC
transportergene,oneofthemiR477a-3ptargets,showedthehighestnumberoffoldchangesin
anthertissuesofcold-tolerantgenotypeH.
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