Identification of 14-3-3 Family in Common Bean and Their Response to Abiotic Stress

18 Pages·2015·4.52 MB·English

RESEARCHARTICLE
Identification of 14-3-3 Family in Common
Bean and Their Response to Abiotic Stress
RuihuaLi1☯,XiaotongJiang1☯,DonghaoJin1☯,SangeetaDhaubhadel2,ShaominBian1*,
XuyanLi1*
1 CollegeofPlantScience,JilinUniversity,Changchun,China,2 AgricultureandAgri-FoodCanada,
SouthernCropProtectionandFoodResearchCentre,London,Ontario,Canada
☯Theseauthorscontributedequallytothiswork.
*[email protected](SB);[email protected](XL)
Abstract
14-3-3sareaclassofconservedregulatoryproteinsubiquitouslyfoundineukaryotes,
whichplayimportantrolesinavarietyofcellularprocessesincludingresponsetodiverse
stresses.Althoughmuchhasbeenlearnedabout14-3-3sinseveralplantspecies,it
remainsunknownincommonbean.Inthisstudy,9commonbean14-3-3s(PvGF14s)were
identifiedbyexhaustivedataminingagainstthepubliclyavailablecommonbeangenomic
OPENACCESS database.AphylogeneticanalysisrevealedthateachpredictedPvGF14wasclusteredwith
twoGmSGF14paralogsfromsoybean.Bothepsilon-likeandnon-epsilonclassesof
Citation:LiR,JiangX,JinD,DhaubhadelS,BianS,
LiX(2015)Identificationof14-3-3FamilyinCommon PvGF14swerefoundincommonbean,andthePvGF14sbelongingtoeachclassexhibited
BeanandTheirResponsetoAbioticStress.PLoS similargenestructure.Among9PvGF14s,only8aretranscribedincommonbean.Expres-
ONE10(11):e0143280.doi:10.1371/journal.
sionpatternsofPvGF14svarieddependingontissuetype,developmentalstageandexpo-
pone.0143280
sureofplantstostress.Aprotein-proteininteractionstudyrevealedthatPvGF14aforms
Editor:ZhulongChan,ChineseAcademyof
dimerwithitselfandwithotherPvGF14isoforms.Thisstudyprovidesafirstcomprehensive
Sciences,CHINA
lookatcommonbean14-3-3proteins,afamilyofproteinswithdiversefunctionsinmany
Received:July15,2015
cellularprocesses,especiallyinresponsetostresses.
Accepted:November3,2015
Published:November23,2015
Copyright:©2015Lietal.Thisisanopenaccess
articledistributedunderthetermsoftheCreative
CommonsAttributionLicense,whichpermits Introduction
unrestricteduse,distribution,andreproductioninany
14-3-3proteinsareagroupofconservedregulatorymoleculesthatubiquitouslyexistinall
medium,providedtheoriginalauthorandsourceare
credited. eukaryotes.Generally,14-3-3proteinsactashomo-orheterodimerstofunctionthroughtheir
abilitytobindwiththeirphosphorylatedproteinclients.Thisprocessresultsinalterationin
DataAvailabilityStatement:Allrelevantdataare
stability,activity,intracellularlocalizationorinteractioncapabilityoftheirclientproteins[1–
withinthepaperanditsSupportingInformationfiles.
3].Ithasbeendemonstratedthat14-3-3proteinsareabletorecognizehighlyconservedbind-
Funding:Thisworkwasfinanciallysupportedbythe
ingmotifwithintheirclientprotein.Sofar,threecanonicalmotifshavebeendefinedfor14-3-
NationalNaturalScienceFoundationofChina(No.
3bindingsuchas(R/K)SX(S/T)PXP,(R/R)XFX(S/T)PXPand(S/T)PX -COOH[4],whereX,
31300253),http://www.nsfc.gov.cn/. 1-2
Fand(S/T)Pindicateanyaminoacid,aromatic/aliphaticaminoacid,andserine/threonine
CompetingInterests:SDisanAcademicEditorfor
thatcouldbepotentiallyphosphorylated,respectively.Nevertheless,14-3-3scanalsobind
PLOSONE.Thisdoesnotaltertheauthors'
someproteinclientsbymeansofnoncanonicalorphosphorylation-independentmotifssuch
adherencetoPLOSONEpoliciesonsharingdata
andmaterials. asWLDLEandGHSL[5,6].
PLOSONE|DOI:10.1371/journal.pone.0143280 November23,2015 1/18
CommonBean14-3-3FamilyandAbioticStress
Plant14-3-3proteinswereidentifiedconcurrentlyfromArabidopsisthaliana,Hordeumvul-
gare,SpinaceaoleraceaandOenotherahookeri[7–9].Sincethen,many14-3-3shavebeeniso-
latedandcharacterizedinseveralotherplantspecies[10–17].Todate,manyeffortshavebeen
madetoelucidatetherolesof14-3-3sinplantdevelopmentandresponsetoabioticstresses
[18–23].Over-expressionorsilencingof14-3-3sinfluencedstresstoleranceinplants.For
example,over-expressionofArabidopsisAtGF14λincreaseddroughttoleranceincotton[24],
whereassilencingofAtGF14μinArabidopsispromoteddroughttolerance[25].Similarly,
over-expressionofTOMATO14-3-3PROTEIN4(TFT4)inArabidopsisincreasedalkaline
stressresponse[26],whiletheknock-outofRCI1A/AtGF14ψenhancedtheconstitutivefreez-
ingtolerance[27].Additionally,14-3-3sthemselvescanbeaffectedbyabioticstresses.For
instance,transcriptionalaccumulationsof14-3-3swerealteredbycold,heat,drought,salinity
andnutritiondeficiency[27–31].14-3-3salsointeractwithcomponentsofstresssignaling
pathwayssuchasABA-responsiveelementbindingfactors,involvedinABA-dependentsignal-
ingpathwayundersalinitystresses[32],H+-ATPase,creatinggradientforstomatalopening
[33],SALTOVERLYSENSITIVE2(SOS2)thatmediatesintracellularsodiumionhomeostasis
andsalttolerance[34].
Comparedtootherorganisms,plantscontainalargenumberof14-3-3isoforms.Forexam-
ple,thereare1314-3-3proteinisoformsinArabidopsis[35],8inrice[16],16insoybean[14],
8infoxtailmillet[36]and10inrubber[15].Theseisoformsareencodedbymulti-genefamily
withsmalldifferenceinsequence.However,emergingevidencesindicatedthat14-3-3sexert
theirregulatoryfunctionsinanisoform-specificmanner.Ithasbeendemonstratedthat14-3-3
isoformsdisplayeddifferentialsubcellularlocalization,distincttissue-specificand/orinducible
expression[14,15,26,37,38],whichimpliedtheirspecificinteractionswithcellularclientsdur-
ingdevelopmentalprocessesorinresponsetodiversestresses.Forinstance,soybean14-3-3
isoformsshoweddifferentbindingaffinitytoGmMYB176(anisoflavonoidregulator)[19],
whilerice14-3-3isoformsdisplayeddifferentialbindingspecificitytowardsACCsynthase
[39].Evidently,14-3-3isoformsplayimportantrolesindeterminingcomplexityandspecificity
ofbiologicalfunctionsinplants.Thus,addressingtheimplicationsof14-3-3familydiversity
becomesanimportantsteptowardselucidatingtheirrolesinplantdevelopmentalprocesses
and/orresistancetostresses.
Commonbean(PhaseolusvulgarisL.)isoneofthemostimportantcroplegumesworld-
wide.Itisadiploidspecieswith11chromosomes(2n=2x=22)[40],andagenomesizeof473
Mb[41].Althoughmuchhasbeenlearnedabout14-3-3sinseveralplantspecies,no14-3-3has
beenidentifiedincommonbean.Availabilityofthewholegenomesequenceofcommonbean
facilitatestosystematicallyanalyzegenefamilymembersandtheirpossiblerolesincommon
bean.Inthisstudy,dataminingwasconductedagainstpubliclyavailablecommonbeangeno-
micdatabase,andatotalof914-3-3s(PvGF14s)wereidentified.ThePvGF14isoformsshowed
highsequenceconservationwithSGF14sfromsoybean.Furthermore,PvGF14sdisplayedtis-
sue-specificexpressionpatterns,andtheirtranscriptionalactivitieswerealteredwhensub-
jectedtocold,droughtandsalinitystress.Thesefindingsprovideafoundationforelucidating
therolesofPvGF14sincommonbeanduringdevelopmentorinresponsetoabioticstress.
MaterialsandMethods
Plantmaterialsandtreatments
Commonbean(PhaseolusvulgarisL.)cvDongbeixiaoyoudouisalocalcultivarinthenortheast
ofChina.Plants(Dongbeixiaoyoudou)weregrownatexperimentalstationinJilinUniversity
(Changchun,JilinProvince,China),in2013,andseedswerecollectedforthefollowing
experiments.
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CommonBean14-3-3FamilyandAbioticStress
Seedsofcommonbeancultivar"Dongbeixiaoyoudou"weresurfacesterilizedbyusing10%
(w/v)sodiumhypochloritefor20min,andthenwashedthoroughlywithsteriledistilledwater.
Thesesterilizedseedswereallowedtogerminatein150mmdiameterplatewithwetfilterpaper
understerileconditions.Subsequently,sixwell-germinatedseedswerechosenandsownon
eachpotfilledwith65gvermiculite.Alltheseedlingsweregrownundera14hlightand10h
darkphotoperiodat25°C(light)and20°C(dark)inachamberandregularlywateredwith
Hoaglandliquidmedium.Ten-day-oldseedlingsweresubjectedtothefollowingtreatments
andsixpotsofseedlingswereusedforeachtreatment:(1)Forcoldstress,seedlingsweretrans-
ferredto4°Candsampleswerecollectedat0,1,3,6,12and24haftercoldtreatment;(2)For
droughtstress,watersupplywaswithheldandsampleswerecollectedat0,1,3,5,7and9days
ofwaterstress;(3)Forsalinitystress,200mMNaClsolutionwasappliedtoseedlingsandsam-
pleswerecollectedat0,3,6,12,33,48and72haftersalttreatment.Theabove-groundparts
werecollectedandfrozeninliquidnitrogenandstoredat-80°C.
IdentificationofPvGF14sincommonbean
Theknown14-3-3proteinsequencesfromsoybean,Arabidopsisandricewereobtainedfrom
NCBIdatabase,whichwereusedasqueriestoconductBLASTsearchagainstthepublicgeno-
micdatabase(http://phytozome.jgi.doe.gov/pz/portal.html).Theaccessionnumbersof14-3-3
proteinsfromArabidopsis,soybeanandricewerelistedinS1Table.Additionally,toidentify
allPvGF14s,akeywordsearchusingtheword"14-3-3"wasconductedagainstthecommon
beanwholegenomedatabase(http://phytozome.jgi.doe.gov/pz/portal.html#!search?show=
KEYWORD&method=Org_Pvulgaris).AlltheputativePvGF14sweresearchedfor14-3-
3-specificdomainandsignatureusingPROSITE(http://prosite.expasy.org/),Pfam(http://
pfam.xfam.org/search)andSMART(http://smart.embl-heidelberg.de/)programs.Theirsub-
cellularlocalizationwaspredictedusingPSORTalgorithmswithdefaultparameters(http://
www.psort.org/).
Multiplesequencealignment,phylogenetictreeconstructionandgene
structure
MultiplesequencealignmentofallputativePvGF14proteinswereperformedbyClustalX,and
phylogenetictreeswereconstructedbytheNeighbor-joining(NJ)methodusingMEGA5soft-
ware[42].Bootstrapvalueswerecalculatedusing1000replicates.GenestructuresofPvGF14s
werebuiltusingSIM4(http://pbil.univ-lyon1.fr/members/duret/cours/inserm210604/
exercise4/sim4.html).
CalculationofKa/Ksvalues
TheDnaSPprogramversion5.10.1wasusedtocalculatetheratiosofnon-synonymous(Ka)
versussynonymous(Ks)substitutionrate(Ka/Ks)fororthologousgenepairsof14-3-3s[43].
Generally,Ka/Ks=1referstoneutralselection,Ka/Ks>1referstopositiveselectiontoacceler-
ateevolution,andKa/Ks<1referstopurifyingselectionduringevolution[44].
Chromosomallocalizationandgeneduplication
TodeterminethelocationofputativePvGF14sincommonbeanchromosomes,coordinateof
individualgeneandchromosomelengthwereobtainedfromPhytozomedatabase.PvGF14sin
duplicatedgenomicregionsandKa/KsvaluesforeachduplicatedPvGF14wereretrievedfrom
batchdownloadoptionofPlantGenomeDuplicationDatabase(http://chibba.agtec.uga.edu/).
Tandemduplicationsweredefinedastwoparalogsseparatedbylessthanfivegenesinthe
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CommonBean14-3-3FamilyandAbioticStress
samechromosome[45],whilesegmentalduplicationsreferredtothosehomologousgenesdis-
tributedonduplicatedchromosomalblocksfromthesamegenomelineage.
ExpressionanalysisofPvGF14s
TotalRNAwasisolatedfromcommonbeantissuesusingRNAprepPurePlantKit(Tiangen
Inc,China),accordingtothemanufacture’sinstructionfollowedbyRNase-FreeDNaseI(NEB
Inc,NewEngland)treatmenttoremoveDNA.TotalRNA(2μg)wasusedtosynthesizefirst-
strandcDNAusingpRimeScriptRTreagentkitwithgDNAEraser(TakaraInc,Japan).
RT-PCRwasperformedbyusingthePvGF14gene-specificprimers(S2Table).qPCRwascon-
ductedusingABI7500real-timePCRdetectionsystemandSYBRPremixExTaq(TakaraInc,
Japan).DatawereanalyzedbyABI7500softwarev.2.0.6,usingACTIN11astheinternalrefer-
ence.Theexpressionlevelsofthecontrolsforeachtypeofstresstreatmentsweresetas1,and
relativeexpressionlevelofeachPvGF14foreachtreatmentwasnormalizedaccordingly.The
primersequencesusedinthestudyarelistedinS3Table.Statisticalsignificanceofthedatawas
analyzedbyone-wayANOVAwithLSDtest,andpvalue<0.05wasconsideredtobestatisti-
callysignificant.
ToanalyzetheexpressionsofPvGF14sindifferenttissues,fragmentsperkilobaseoftran-
scriptpermillionmappedreads(FPKM)valuesforeachPvGF14wereextractedfromPhyto-
zomedatabasebytrackingcommonbeangene-levelexpression(http://www.phytozome.net).
TheheatmapforPvGF14geneswasgeneratedinRusingtheheatmap.2functionfromthe
gplotsCRANlibrary(http://CRAN.R-project.org/package=gplots).ToconfirmtheRNA-seq
datafromthepublicdatabase,RT-PCRwasperformedbyusingthePvGF14gene-specific
primers(S3Table).Forpromoteranalysis,1500bpupstreamregionofthetranscriptionalstart
siteofeachPvGF14genewereanalyzedinPlantCAREdatabase(http://bioinformatics.psb.
ugent.be/webtools/plantcare/html/).
Yeasttwo-hybridassay
Toconductyeasttwo-hybrid(Y2H)assay,thegateway-compatiblevectorspGBKT7-DEST
andpGADT7-DESTwereutilizedtopreparethebaitandtheprey,respectively[46].Full-
lengthPvGF14acDNAwasclonedintopGBKT7-DESTasabaitandself-activationwas
checked.Thefull-lengthcDNAsofallPvGF14sexceptPvGF14qwereclonedseparatelyinto
thepGADT7-DESTvectoraspreys.Thebaitvectorandeachofthepreyvectorscontaining
PvGF14genewereco-transformedintoyeaststrainAH109,andgrownonsyntheticdefined
(SD)/-Leu/-Trpselectiveagarmedium.Selectedindividualyeasttransformantsweregrownon
liquidmedium,and5μLofyeastsuspensionculturewithaseriesof10Xdilutionswasspotted
ontoSD/-Leu/-TrpandSD/-Ade/-His/-Leu/-Trpplates,andgrownfor5daysat30°C.Empty
vectorswereco-transformedasnegativecontrols.
Results
Identificationofcommonbean14-3-3genefamily
Toidentify14-3-3proteingenesincommonbean,14-3-3proteinsequencesfromsoybean,
ArabidopsisandricewereusedasqueriestoconductBLASTsearchagainstthecommonbean
genomicdatabase(http://www.phytozome.net/).Inaddition,akeywordsearchusingtheword
‘14-3-3’wasalsoconductedagainsttheabovedatabase.Thisprocessidentifiedatotalof9puta-
tive14-3-3genes(PvGF14a-PvGF14e,PvGF14g,PvGF14h,PvGF14nandPvGF14q),which
werenamedintermof14-3-3nomenclatureinsoybean.Table1providesdetailinformation
onallputativePvGF14s.Thededuced14-3-3proteinscontain248to263aminoacidsresidues
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CommonBean14-3-3FamilyandAbioticStress
Table1. CharacteristicsofPvGF14sgenesincommonbean.
Gene Locusname Chromosomallocationa Protein Predicted CorrespondingESTc
name subcellularlocationb
aa Mw pI
(kDa)
PvGF14n Phvul.005G066600 Chr05:10288469–10295349 263 30.21 4.75 Cyto,chl,per CV539470
(+strand)
PvGF14d Phvul.005G095500 Chr05:28501915–28505383 261 29.56 4.71 Cyto,chl,per GW892341
(+strand)
PvGF14c Phvul.002G238300 Chr02:40393090..40396629 258 29.25 4.77 Cyto,chl,per CV530328
(-strand)
PvGF14q Phvul.002g102500 Chr02:20470454..20474678 258 29.45 4.75 Cyto,mito,per none
(-strand)
PvGF14e Phvul.003G043200 Chr03:4827912..4831544(-strand) 259 29.51 4.83 Cyto,chl,per HS103842
PvGF14b Phvul.009G143400 Chr09:20974074..20976460 248 28.19 4.85 Cyto,chl,per FE704892
(-strand)
PvGF14a Phvul.008G004300 Chr08:482680..485463(-strand) 256 28.98 4.59 Cyto,chl,per CV530850
PvGF14g Phvul.008G162500 Chr08:41741118..41743699 261 29.30 4.57 Cyto,chl,per GW901700
(+strand)
PvGF14h Phvul.009G032500 Chr09:7120999..7123241(+strand) 259 29.16 4.56 Cyto,chl,per GW904281
aChromosomallocationindicatesthepositionofeachgeneinchromosome
bcyto,chl,mitoandperrefertocytoplasm,chloroplast,mitochondrialandperoxisome,respectively
cEST(ExpressedSequenceTags)accessionwiththehighesthomologytocorrespondingPvGF14gene;aa,aminoacid;pI,isoelectricpoint;Mw,
molecularweight.
doi:10.1371/journal.pone.0143280.t001
withthecalculatedmolecularweightsfrom28.19to30.21kDa,andtheestimatedisoelectric
pointsfrom4.56to4.85.AsshowninFig1,allthepredictedPvGF14scontaina14-3-3con-
serveddomainfeaturedbyoneortwo14-3-3proteinsignatures,andtheywerepredictedto
localizeincytoplasm,peroxisome,chloroplastand/ormitochondria(Table1).
AnalignmentofdeducedaminoacidsequencesofPvGF14switheachotherindicatedthat
theisoformsexhibithighsequenceconservationwiththeidentityrangingfrom63.0%to94.2%
ataminoacidlevel(Fig1andS4Table).Thesequencediversificationmainlyoccurredatthe
N-terminalandtheC-terminalregions,suggestingthatthoseregionsarepossiblyresponsible
forisoformspecificity[47].Additionally,sequenceconservationataminoacidlevel(57.6%to
96.9%)wasalsoobservedbetweensoybeanandcommonbean14-3-3proteins.EachPvGF14
showedmorethan92%sequenceidentitywithitsorthologsinsoybean(S5Table).
Evolutionaryrelationshipandgenestructureof14-3-3genefamilyin
commonbean
Toexaminetheevolutionaryrelationshipof14-3-3proteinsfromcommonbeanandother
plantspecies(soybean,Arabidopsisandrice),aphylogeneticanalysiswasconductedatboth
nucleotideandproteinlevels.Inboththecases,treeswithsimilartopologieswereobtained
(Fig2).PvGF14n,PvGF14d,PvGF14c,PvGF14eandPvGF14qwereclusteredintotheepsilon-
likeclass,whilePvGF14a,PvGF14g,PvGF14handPvGF14bweregroupedtogetherwithnon-
epsilonisoformsof14-3-3proteinsfromArabidopsis,riceandsoybean.EachPvGF14was
clusteredtogetherwithtwoGmSGF14orthologs(Fig2andS5Table).Theanalysisrevealed
thatPvGF14sareevolutionarilyclosertosoybean14-3-3scomparedtoArabidopsisandrice,
whichisconsistentwiththespeciesevolutionaryhistory[40].The14-3-3sfromArabidopsis
andriceformedseparatecladesorbranchesinthephylogenetictreesuchasAtGF14pi,
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CommonBean14-3-3FamilyandAbioticStress
Fig1.SequencealignmentofcandidatePvGF14proteins.Identicalaminoacidresiduesareshowninblack.Theα-helixsofPvGF14sareshownasaline
and14-3-3signaturesofPvGF14sareshowninrectangularboxes.
doi:10.1371/journal.pone.0143280.g001
AtGF14epsilon,AtGF14omicronandOsGF14h(Fig2),suggestingthattheseepsilon-like14-3-
3geneswerelostincommonbeanandsoybeanduringevolutionorevolvedanewfunction.
Tobetterunderstandtheevolutionaryrelationshipbetween14-3-3s,theratiosofKa/Ksfor
14-3-3pairsfromcommonbean,soybean,Arabidopsisandricewereestimated(S6Table).As
aresult,theKa/Ksvaluesrangedfrom0to0.347withanaverageof0.083.Allthe14-3-3s
appeartobeunderpurifyingselectionduringevolution,astheirKa/Ksratioswereestimated
<1.SinceeachofPvGF14sanditstwoclosely-relatedorthologsinsoybeanwereclusteredinto
samediscretecladeinthephylogenetictree,theKa/Ksratioswerefurtherobserved.TheKa/Ks
ratiosfortheclosestorthologpairsvariedfrom0to0.153withanaverageof0.060,suggesting
thattheorthologpairsamonglegumestendtohavelessevolutionarydiversification.
Toinvestigatetheexon-intronorganizationinPvGF14s,genestructuresweremappedon
thebasisofthegenomicandcodingregionsequences.Commonbean14-3-3genestructure
comprisedof4exonsinnon-epsilonclass,and6–7exonsinepsilon-likeclass(Fig3).The
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CommonBean14-3-3FamilyandAbioticStress
Fig2.PhylogeneticanalysisofPvGF14sandotherGF14sfromdifferentspecies.PhylogenetictreeswerecalculatedbasedonCDSmatrix(A)and
proteinmatrix(B)fromcommonbean(PvGF14),soybean(GmSGF14),Arabidopsis(AtGF14)andrice(OsG14F),andthetreewasclassifiedintoepsilon
andnon-epsilongroups.EachPvGF14/PvGF14isindicatedbyastar.
doi:10.1371/journal.pone.0143280.g002
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CommonBean14-3-3FamilyandAbioticStress
Fig3.GenestructuresandchromosomallocalizationofPvGF14sincommonbean.TheleftpanelisthephylogenetictreeofPvGF14s;themiddlepanel
istheintron-exonstructureswheretheexonsareshownbyrectangular,andtheintronsarerepresentedbythinlines;therightpaneldisplaysthe
chromosomallocalizationofPvGF14s.
doi:10.1371/journal.pone.0143280.g003
PvGF14sbelongingtothesameclasscontainedsimilarsizeofexons,suchasPvGF14qand
PvGF14e,PvGF14nandPvGF14d,PvGF14gandPvGF14h(Figs2and3).Evidently,conserved
genestructureofPvGF14sstronglysupportsthereliabilityofphylogenetictree.
Chromosomaldistributionandduplicationsof14-3-3sincommonbean
TheninePvGF14sarelocatedonfivedifferentchromosomes(chromosomes2,3,5,8and9)in
commonbean.EachchromosomecontainstwoPvGF14sexceptchromosome3(Fig3).Gene
familycanarisefromthesegmentalduplicationortandemamplificationofchromosomal
regions[48].Generally,tandemamplificationwasdefinedastwoparalogsseparatedbyless
thanfivegenesinthesamechromosome.ThePvGF14sinthesamechromosomeweredistrib-
utedfarfromeachother(Table1andFig3),suggestingthatcommonbean14-3-3genefamily
waslikelyderivedfromsegmentalduplicationratherthantandemamplificationofchromo-
somalregions.Furthermore,weinvestigatedwhethertraceablegenomeduplicationscontribu-
tetotheexpansionofthe14-3-3genefamilyincommonbean.Theresultsrevealedthatthesets
ofPvGF14s(PvGF14candPvGF14d,PvGF14qandPvGF14e)weremappedontheduplicated
block120andblock93,respectively,suggestingthatthesetwopairswerepossiblyderivedfrom
segmentalduplicationeventsduringtheevolutionaryprocess.Notraceableduplicationevent
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CommonBean14-3-3FamilyandAbioticStress
Fig4.ExpressionanalysisofPvGF14genesinvarioustissues.Thetranscriptomedataofcommonbeanacrossdifferenttissuesanddevelopmental
stageswereextractedfromthepublicly-availablePhytozomedatabase(http://www.phytozome.net)forheatmapgeneration.Thecolorscaleabovetheheat
mapindicatesgeneexpressionlevels,lowtranscriptabundanceindicatedbygreencolorandhightranscriptabundanceindicatedbyredcolor.Maximum
FPKMvalueforeachPvGF14isshown.
doi:10.1371/journal.pone.0143280.g004
wasobservedforotherPvGF14s.ToinvestigatetheselectiveevolutionarypressureonPvGF14
genedivergenceafterduplication,thenon-synonymous/synonymoussubstitutionratio(Ka/
Ks)wasretrievedforthetwoduplicationpairsof14-3-3genes.Consequently,Ka/Ksvalueof
thegeneduplicationpairs,PvGF14candPvGF14daswellasPvGF14qandPvGF14ewere0.058
and0.073,respectively,suggestingthatthesegenespossiblyhaveundergoneapurifyingselec-
tionwithlimitedfunctionaldivergenceafterduplication.
ExpressionanalysisofPvGF14sincommonbeantissues
ToinvestigateexpressionpatternsofPvGF14s,weutilizedthepubliclyavailablegenome-wide
transcriptprofilingdataofcommonbeantissuesfromPhytozomedatabase(http://www.
phytozome.net),whichcontainsRNAseqreadsfromvegetativetissues(trifoliates,nodule,
root,stem,leaf)andproductivetissues(flowerbud,flower,pod).Allthecommonbean14-3-3
genesshowedtissue-specificexpressionpatterns(S7Table).Overall,PvGF14transcriptscan
becategorizedinto3groupsbasedontheirexpressionpatterns(Fig4).Group1comprisedof
PvGF14g,PvGF14handPvGF14a,whichweremainlyexpressedinstems,flowerbudsand/or
root_10.Group2containedPvGF14d,PvGF14candPvGF14bwithhighexpressioninflowers,
flowerbuds,stemsand/orpods,whilegroup3consistedofPvGF14n,PvGF14eandPvGF14q
withtranscriptabundanceeitherinstemsorrootsorflowerbuds.Also,fourrepresentative
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CommonBean14-3-3FamilyandAbioticStress
PvGF14s(PvGF14candPvGF14dfromepsilon-likeclass,PvGF14aandPvGF14bfromnon-
epsilonclass),werechosentoconfirmthepubliclyavailabletranscriptprofilingdatausing
RT-PCRapproach,andsimilarexpressionpatternswereobservedinstem,leaf,flowerandpod
ofcommonbean(S1Fig).
Themaximumfragmentsperkilobaseoftranscriptpermillionmappedreads(FPKM)for
PvGF14qwaslow(3.69)comparedtothereadsforotherPvGF14s(40.02to593.92).Addition-
ally,blastsearchagainstExpressedSequenceTags(ESTs)inNCBIdatabasedidnotfindany
ESTcorrespondingtoPvGF14q(Table1).OurattemptstoamplifyPvGF14susingcDNAsyn-
thesizedfromRNAisolatedfromseveraldifferenttissuesofcommonbeanyieldedsuccessful
resultsforallthePvGF14sexceptPvGF14q.Theseresultsindicatedthatcommonbeancontain
only8putative14-3-3genesthataretranscribed.
EffectofabioticstressesontheexpressionofPvGF14s
Severalstudieshavedocumentedaroleofplant14-3-3proteinsinabioticstressresponse[24–
31].ToinvestigateifPvGF14salsohavesimilarrolesincommonbean,weexaminedthe
expressionpatternsofPvGF14sinresponsetocold,droughtandsalinitystress.
Ten-day-oldcommonbeanseedlingswereexposedtocoldstressat4°Cfor0,1,3,6,12or
24h,andexpressionofPvGF14sweremonitored.Theresultsrevealedthatcoldstressaltered
theexpressionsofPvGF14sthatcouldbegroupedinto2categories.AsindicatedinFig5A,cat-
egory1containedgenesthatshowedgradualincreaseintranscriptaccumulationasthestress
prolonged.Forexample,PvGF14n,PvGF14d,PvGF14e,PvGF14gandPvGF14htranscriptlevels
increasedto1.9,2.1,2.6,2.8and1.8fold,respectivelyascomparedtocontrol.Allthesefive
genefamilymemberswereexpressedtotheirhighestleveleitherat12or24haftercoldstress.
Thecategory2comprisedthegenes(PvGF14a,PvGF14bandPvGF14c)whosetranscriptlevels
increasedwithcoldstresstreatmentfollowedbygradualdecreaseasthestresscontinued.
PvGF14candPvGF14atranscriptsreachedtotheirmaximumlevelat12hcoldstresswith3.58
and1.85foldincrease,respectivelyascomparedtocontrol,whilePvGF14bwasexpressedto
thehighestlevelat3hwith3.73foldincreaseascomparedtocontrol.
Whencommonbeanseedlingswereexposedtodroughtstress,theexpressionofPvGF14e,
PvGF14gandPvGF14hwasdecreasedto5.3,13.0and2.9foldon9,7and5daysaftertreatment,
respectively(Fig5B).Onthecontrary,adistinctincreaseintranscriptaccumulationon9days
ofdroughttreatmentwasobservedforPvGF14nandPvGF14cwith2.82and1.86foldchanges,
respectively.AdramaticincreaseinPvGF14atranscript(5.32fold)ascomparedtocontrolwas
observedon5daysofdroughttreatment.
Whenyoungseedlingsweresubjectedtosaltstress,theexpressionsofPvGF14n,PvGF14e,
PvGF14gandPvGF14hweregraduallydecreasedasthesaltstressdurationincreased,andtheir
maximumfoldchangeswereupto3.54,1.97,1.83and1.67,respectively(Fig5C).Onthecon-
trary,theexpressionsofPvGF14d,PvGF14bandPvGF14awerepronouncedatoneormore
stresstimepointsby1.54,1.42and1.47fold,respectivelyascomparedtonon-salinitycontrol.
PvGF14qtranscriptwasnotdetectedforanyofthestresstreatmentusedinthisstudy.
TobetterunderstandtheroleofPvGF14sinabioticstressresponse,promoteranalysiswas
alsoconducted.ItwaspredictedthatpromoterregionsofPvGF14eandPvGF14gcontainLTR
cis-actingelementinvolvedinlow-temperatureresponsiveness,supporting2.6and2.8fold
increaseintheexpressionofPvGF14eandPvGF14gundercoldstress.PvGF14q,PvGF14nand
PvGF14dcontainABREcis-actingelementintheirpromoter,whichisimplicatedinABA
response.HSE,acis-actingelementrelatedtoheatstressresponse,wasfoundinthepromoters
ofPvGF14q,PvGF14c,PvGF14n,PvGF14gandPvGF14h.Allthe14-3-3genesexceptPvGF14c
containTC-richrepeat,acis-actingelementinvolvedindefenseandstressresponse.In
PLOSONE|DOI:10.1371/journal.pone.0143280 November23,2015 10/18