Rubisco Activase Is Also a Multiple Responder to Abiotic Stresses in Rice

16 Pages·2015·3.51 MB·English

RESEARCHARTICLE
Rubisco Activase Is Also a Multiple Responder
to Abiotic Stresses in Rice
YueChen,Xiao-ManWang,LiZhou,YiHe,DunWang,Yan-HuaQi,De-AnJiang*
StateKeyLaboratoryofPlantPhysiologyandBiochemistry,CollegeofLifeSciences,ZhejiangUniversity,
Hangzhou,310058,China
*[email protected]
a11111 Abstract
Ribulose-1,5-bisphosphatecarboxylase/oxygenaseactivase(RCA)isanucleargenethat
encodesachloroplastproteinthatplaysanimportantroleinphotosynthesis.Somereports
haveindicatedthatitmayplayaroleinacclimationtodifferentabioticstresses.Inthis
paper,weanalyzedthestress-responsiveelementsinthe2.0kb5’-upstreamregionsofthe
OPENACCESS
RCAgenepromoterandtheprimary,secondaryandtertiarystructureoftheprotein.We
Citation:ChenY,WangX-M,ZhouL,HeY,WangD, identifiedsomecis-elementsofmultiplestress-relatedcomponentsintheRCApromoter.
QiY-H,etal.(2015)RubiscoActivaseIsAlsoa AminoacidandevolutionanalysesshowedthattheRCAproteinhadconservedregions
MultipleRespondertoAbioticStressesinRice.PLoS
betweendifferentspecies;however,thesizeandtypevaried.Thesecondarystructures,
ONE10(10):e0140934.doi:10.1371/journal.
pone.0140934 bindingsitesandtertiarystructuresoftheRCAproteinswerealsodifferent.Thismight
reflectthedifferencesinthetranscriptionandtranslationlevelsofthetwoRCAisoformsdur-
Editor:NiranjanBaisakh,LouisianaStateUniversity
AgriculturalCenter,UNITEDSTATES ingadaptationtodifferentabioticstresses.Althoughboththetranscriptionandtranslation
levelsofRCAisoformsinthericeleavesincreasedundervariousstresses,thelargeiso-
Received:June1,2015
formwasincreasedmoresignificantlyinthechloroplaststromaandthylakoid.Itcanbecon-
Accepted:September30,2015
cludedthatRCA,especiallyRCA ,isalsoamultiplerespondertoabioticstressesinrice,
L
Published:October19,2015 whichprovidesnewinsightsintoRCAfunctions.
Copyright:©2015Chenetal.Thisisanopen
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare
Introduction
credited.
Therearedifferencesinthegenesandbis-phosphatecarboxylase/oxygenaseactivase(RCA)
DataAvailabilityStatement:Allrelevantdataare
isoformsamongplantspecies.Inmanyplants,therearetwoRCAforms:alarge45–46kDiso-
withinthepaperanditsSupportingInformationfiles.
formandasmall41–43kDisoform.GenomicanalyseshaveidentifiedoneRCAgeneinspin-
Funding:Theauthorshavenosupportorfundingto
ach,Arabidopsis,riceandwheat[1–3],inwhichalternativesplicingoftheRCAtranscript
report.
resultsintwoRCAisoforms[2,4,5].TwoRCAgenesencodetwoRCAisoformsinbarleyand
CompetingInterests:Theauthorshavedeclared
cotton[5,6].InadditiontoonealternativelysplicedRCAgene(rcaA)thatproducestwoRCA
thatnocompetinginterestsexist.
isoforms,asecondgene(rcaB)encodesonlythesmallisoformofRCAinbarley[5].Although
Abbreviations:AAs,aminoacids;AAA+,ATPase morethanthreeRCAgeneshavebeenfoundintobacco[7]andsoybean[8],theyonlyproduce
associatedwithavarietyofcellularactivities;ELISA,
thesmallRCAisoform[9].ThelargestdifferencebetweenthetwoRCAformsisatthecarboxyl
enzyme-linkedimmunesorbentassay;RCA,Rubisco
terminus[9].Comparedwiththesmallisoform,thelargeisoformhasacarboxy-terminal
activase;RCA,RCAlargeisoform;RCA ,RCA
L S
extensionthatcontainsredox-sensitivecysteine(Cys)residues[6,9,10].Boththelargeand
smallisoform;Rubisco,RuBPcarboxylase/
oxygenase. smallisoformscanactivateRubisco;however,theyexhibitslightdifferencesintheirmaximal
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activity[11].Notably,lightmodulationofRubiscoinArabidopsisrequiresredoxregulationof
thelargeisoformviathioredoxin-f[10,12,13].
RCAmaybeimportantintheacclimationofphotosynthesis[14]andthedeactivationof
Rubisco[15]tohightemperaturebecausetheisolatedspinachRCAisveryheatlabile[16].
Spinach[17]butnotArabidopsis[18]suggestsspeciesspecificityfortheisoformtemperature
stability.Inrice,thelargeisoformmayplayanimportantroleinphotosyntheticacclimationto
heatstress,whereasthesmallisoformplaysamajorroleinmaintainingtheinitialactivityof
Rubisco[19].Recently,atotalof2,171saltresponsiveproteinspotshavebeenidentifiedinpro-
teomicsstudiesin34plantspecies[20].RCAisoformshavebeenidentifiedamongthesespots.
Proteomicanalysishasalsoidentifiedproteinspotsthataredifferentiallyregulatedinresponse
todrought,containingRCAisoformsinbarley[21],mulberry[22]andrice[23,24].Inaddi-
tion,RCAmayrespondtoheavymetalstressintobaccoplants[25].
AlthoughadditionalproteomicsresearchhaveshownthattheRCAproteinrespondstovar-
iousabioticstresstreatments,itremainsunclearwhetherthatresponseoccursatthepromoter
levelandwhetherRCAisoformsareregulatedbyabioticstresses.Therefore,itisimportantto
distinguishtheirdifferencesingeneexpressionandproteincontentundervariousconditions.
Basedonbioinformaticsanalysis,wepredicttheenvironmentallyresponsiveelementsandpro-
teinstructureanddeterminethechangeintranscriptionandtranslocationofthetwoisoforms
inriceseedlings.Theresultsshowthatthetranscriptionandtranslationlevelsareactually
inducedbyheat,salt,cold,andpolyethyleneglycol(PEG).Thelargeisoforminboththechlo-
roplaststromaandthylakoidrespondmoresignificantlytothestressesthandothesmalliso-
form.Therefore,weconcludethatRCAinriceisnotonlytheactivatingenzymeofRubiscobut
alsoamultiplerespondertostresses.
MaterialsandMethods
Plantmaterialandstresstreatments
AsreportedbyWangetal.[19],thegerminatedrice(Oryzasativa)seedsweregrowninInter-
nationalRiceResearchInstitute(IRRI)ricenutrientsolutionandriceseedlingsweregrownin
agreenhouseunderaphotosyntheticphotonfluxdensity(PPFD)of500μmolphotonsm-2s−1
controlledataday/nighttemperatureregimenof30/22°C.ThesolutionwasadjustedtopHof
5.0–5.5every2daysandwasrenewedonceinaweek.Whentheseedlingsgrewto5-leafstage,
thetreatmentswereconducted.Thenutrientsolutionwasaddedto200mMNaClforsalt[26,
27],20%PEG6000fordroughttreatments[28],respectively.Theseedlingswereincubatedat
10°Cor40°Cforcoldorheattreatments[19,29,30].Theleafbladeswereharvestedaftervari-
oustreatmentsfor24h,respectively.Theuntreatedseedlingswereusedascontrols.Aftersam-
pling,theleafblades(10seedlingspertreatment)werefrozeninliquidnitrogenandstoredat
-80°Cforlateruse.Alltreatmentwereperformedintriplicate.
IsolationofRNAandcDNApreparation
TotalRNAwasisolatedfrom100mgofstresstreatedanduntreatedriceleafbladesusingthe
RNAprepPureplantkit(TransGenBiotech,China).GenomicDNAcontaminationwas
removedwithDNaseItreatmentaccordingtothemanufacturer’sinstructions.ThecDNAwas
synthesizedusing10μgtotalRNAforeachtemplate.ThefirststrandcDNAwassynthesized
from10μgtotalRNAusingthePrimeScriptonestepRT-PCRkit(TAKARA,Japan)usingthe
oligo(dT)18primeraccordingtothemanufacturer’sinstructions.Threebiologicalreplicates
wereconductedofthestresstreatmentsforRNAandcDNA.
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CloningandsequenceanalysisoftheRCAgenefromrice
ThericeRCALandRCASgenewereamplifiedbyPCRusingFS(5’-
ATGGCTGCTGCCTTCTCCTCC-3’),R (5’-TCAGCTGGATGGCGCAGAACC-3’),F (5’-
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ATGGCTGCTGCCTTCTCCT-3’),andR (5’-TTAAAAGGTGTAAAGGCAGCTGC-3’).In
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thePCRreactions,weusedthefirststrandcDNAsfromNipponbarericeasatemplateto
amplifytheDNA.ThefulllengthriceRCAgeneswereclonedintothepMD19Tsimplevector
(TAKARA).TheputativerecombinantcoloniesofE.coliDH5αwiththedesiredamplification
wereusedtoisolatetheplasmidDNAusingaplasmidkit(TianGen).TheplasmidDNAwas
verifiedforgeneinsertionbyrestrictiondigestionusingtheKpnIandXbaIenzymes.Restric-
tiondigestionconfirmedthattheputativepositiveclonecontainedtheRCAinsert,anditwas
thensubjectedtonucleotidesequencing.
AnalysisofpromotersandgenesequenceofRCA andRCA
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ABLASTsearchofthericegenomeannotationprojectdatabase(http://rice.plantbiology.msu.
edu/)wasusedtoidentifyRCAgenomicDNAsequences,includingthe5’and3’-UTR,exon
andintronsequences[31].Toidentifytheputativecis-regulatoryelementsinthepromoter
regionsofRCA,weuseda2.0kbgenomicsequenceupstreamofthetranslationinitiation
codonoftheRCAgeneusingthePLACEandPlantCAREcis-elementdatabase(http://
bioinformatics.psb.ugent.be/webtools/plantcare/html/)[32].
TertiarystructurepredictionofriceRCA
Anonlineservicesystem,I-TASSERserver,wasusedtodeterminethestructureandfunction
predictionsoftheRCAprotein.ThethreedimensionalstructureofRCAwaspredictedbyan
onlineservicesystemiterativethreadingassemblyrefinementalgorithm(I-TASSER)stand-
alonepackage(version1.1).Atertiarymodelwasbuiltbasedonmultiplethreadingalignments
byLOMETSanditerativeTASSERassemblysimulations[33].Finally,theywereadjusted
usingthePyMOLsoftwarepackage.
Validationsandstructuralmotifanalysis
ThebackboneconformationsofthepredictedmodelswereinspectedbythePhi/PsiRama-
chandranplotobtainedfromPROCHECKserver(http://www.ebi.ac.uk/thornton-srv/
databases/pdbsum/Generate.html).ThequalityofthepredictedproteinmodelofRCAwas
estimatedusingthequalitativemodelenergyanalysis(QMEAN)server(http://
swissmodelexpasy.org/qmean/cgi/index.cgi)[34].ThePDBfilesofthemodeledRCAprotein
weresubjectedtothePDBsumserver(http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/
Generate.html)forstructuralmotifanalysis.
AnalysisofRCAproteinsandphylogenetictree
ThededucedaminoacidsequenceofRCAwascomparedwiththerespectivesubunitsof
monocots,suchasJaponicarice,maize,barley,sorghum,andwheat,usingmultipleaminoacid
sequencealignmentwiththeClustalWmegaprogram(http://www.ebi.ac.uk/).Thepairwise
aminoacidsequenceidentityofRCAamongdifferentspeciesandtherespectivesubunitsof
theplantsnotedabovewerecalculatedusingtheDNAstarsoftware.TheDNAstaraligned
aminoacidsequencesoftheRCAisoformswereusedtoinfertheevolutionaryrelationship
usingtheneighbor-joiningmethod.TheevolutionarydistanceswerecomputedusingthePois-
soncorrectionmethodandareintheunitsofthenumberofaminoacidsubstitutionspersite.
ThesephylogeneticanalyseswereperformedwiththeMEGA5software[35]andiTOLonline
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service[36].IntheparametersusedforanalysisofMEGA5,pairwisealignmentaregapopen-
ingpenaltywith10andgapextensionpenaltywith0.1.Andmultiplealignmentaregapopen-
ingpenaltywith10andgapextensionpenaltywith0.2.Besides,thetestofphylogenyis
accordingtobootstrapmethodwith1000ofbootstrapreplications.Thefunctionalmotifs,pat-
ternsandbiologicallysignificantsitesintheRCAaminoacidsequencewerelocatedwiththe
ExPASyProteomicsServerScanProsite(http://www.expasy.org/tools/scanprosite/).
Quantitativereal-timePCR
ThetranscriptprofileofRCAintheleafbladesunderdifferentstressconditionswasdeter-
minedusingquantitativerealtimePCR.TheqRT-PCRreactionswereperformedasdescribed
previously,usingRCAprimers(F5'-CGTGACGGGCGTATGGAGAAG-3';R5'-GCACGAA
GAGCGCCGAAGAAATC-3')andRCASspecificprimers(F5'-TTCTGCGCCATCCAGCTG
AA-3';R5'-CCTCCTCCTCCTATGCAGG-3').RiceactinandEF-1αgeneswereusedasthe
internalreferences.TheqRT-PCRexperimentwasrepeatedthreetimesindependently,andin
eachexperiment,threetechnicalreplicateswereperformedforeachtreatmenttimepoint.
Semi-quantitativeRT-PCR
Actinwasusedasaninternalstandard(F5'–TCCATCTTGGCATCTCTCAG–3';R5'–GTA
CCCGCATCAGGCATCTG–3').TheRCAgene-specificprimers(F5'–AGCTCGTCGTCCAC
ATCTCCA–3';R5'–CTTGATGATGTCTGCCGCCTC–3')weredesignedforaregionthat
includesthesharedregionoftheRCAsmallisoform(RCAS)andlargeisoform(RCAL)mRNA.
Thethreebiologicalamplificationconditionswereusedforallgenes:95°Cfor5min;followed
by30cycles(95°Cfor30s,60°Cfor30s,72°Cfor60s),andfollowedbyafinalextensionat
72°Cfor10min,andeachbiologicalreplicatewasconductedwiththreetechnicalreplicates.
RubiscoandRCAproteinquantificationbyELISA
ThesampleswerepreparedasdescribedbyWangetal.[19].ThepresenceoftheRubiscosub-
unitandtwoRCAisoformsinthesupernatantandthylakoidweredeterminedbydirectELISA
usingantibodiesproducedbyourlaboratoryagainsttheRubiscolargesubunit(RLS)or
Rubiscosmallsubunit(RSS)andagainstdifferentisoformsofRCA.TheELISAwasmeasured
accordingtothemethoddescribedbyWangetal.[19].
Statisticalanalysis
Thedataabovewereanalyzedbyonewayanalysisofvariance(ANOVA),andsignificantdif-
ferencesbetweentheindividualmeansweredeterminedusingDuncan’spairwisecomparison
testatthe5%and1%(P<0.05andP<0.01)confidencelevels.
Co-IPandSDS-PAGEanalyses
ToinvestigatetheeffectsofabioticstressesontheinteractionbetweenRCA anditsbinding
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protein,thecomplexwasisolatedfromleafextractswithProFound™Co-Immunoprecipitation
Kit(Pierce,Rockford,IL,USA)accordingtothemanufacturer’sinstructions.Antibodiesto
RCA wereimmobilizedonacouplinggeltopulldownthecomplex.Toexcludenon-specific
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bindingbetweentheproteinandthecouplinggelorbetweentheproteinandtheantibodies,a
non-relevantantibodyagainstthe6×Histagwascoupledtotheantibody-couplinggelinapar-
allelassay.Theantibody-coupledgelswereincubatedintheproteinextractionsupernatant
andwashedfivetimeswithwashingbuffer(80mMNaCl,8mMsodiumphosphate,2mM
potassiumphosphateand10mMKCl,pH7.4),andthenthecomplexeswereelutedfromthe
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couplinggel.Theresultingcomplexeswereseparatedon12%SDS—PAGEgelandstained
withsilver.
Ingeldigestionandmassspectrometry
In-geltrypticdigestionofselectedspotswasperformedaccordingtothemethoddescribedby
Wangetal.(2010)[19]withslightmodifications.Selectedproteinspotswereexcisedmanually
fromcolloidalCoomassiestainedgelsanddestainedwith50%acetonitrile(ACN)in25mM
ammoniumbicarbonate(NH HCO )fivetimes.Gelpieceswerethentreatedwith10mM
4 3
DTTin25mMNH HCO andincubatedat56°Cfor1h.Aftercooling,theDTTsolutionwas
4 3
immediatelyreplacedwith55mMiodoacetamidein25mMNH HCO andincubatedfor45
4 3
minatroomtemperature(25±2°C),thenwashedwith25mMNH HCO andACN,driedin
4 3
aspeedvacandrehydratedin20μLof25mMNH HCO solutioncontaining12.5ngμL–1
4 3
trypsin(sequencinggrade,Promega,WI,USA).Aftera10minincubationonice,samples
werekeptat37°Cforovernightdigestion.Afterdigestion,thesupernatantwascollectedin
fresheppendorftubesandthegelpieceswerere-extractedbycontinuousvortexingwith50μL
solutionof1%trifluoroaceticacid(TFA)andACN(1:1v/v)for15min.Supernatantswere
pooledtogether,vacuumdriedusingaspeedvacandthenre-suspendedin5μLof50%ACN
and1%TFA(1:1v/v)solution.Amatrix-assistedlaserdesorption/ionizationtimeofflight
massspectrometry(MALDI-TOFMS)analysiswasconductedwithaMALDI-TOF-TOFmass
spectrometer(BrukerAutoflexIIImartbeam,BrukerDaltonics,Germany).Fromtheabove
preparedsample,2μLwasmixedwithequalvolumesoffreshlyprepareda-cyano-4-hydroxy-
cinnamicacid(CHCA)matrixin50%ACNand1%TFA(1:1v/v),andfinally,1μLofthesam-
plewasspottedonthetargetplate.
MS/MSanalysis
ProteinswereidentifiedfromtheobtainedmonoisotropicpeptidemassesusingtheMASCOT
searchengine(MatrixScience,London,UK;http://www.matrixscience.com)employingBio-
toolssoftware(BrukerDaltonics,Germany).Thefollowingparameterswerefixedfordatabase
searches:taxonomiccategorywassettoViridiplantae(greenplants),modificationsofcarbami-
domethyl(CS),variablemodificationofoxidation(M),enzymetrypsin,peptidechargeof1
+andmonoisotropic.Thesimilaritysearchformassvalueswasperformedwithexistingdigests
andsequenceinformationfromNCBInrandSwissProtdatabase.BasedontheMASCOT
probabilityanalysis(p<0.05),onlysignificanthitswereacceptedforproteinidentification.
Results
PredictionanalysisofRCA andRCA promoters
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TounderstandwhetherthepromoterlevelofRCAisregulatedbyabioticstresses,weanalyzed
thedistributionofregulatorycis-elementsinthe2.0kbupstreampromoterregionofRCAL
andRCASusingstress-responsiveelements(TC-rich),salt-inducedresponsiveelements(GT-1
motif),heatstress-responsiveelements(HSRE),lowtemperature-responsiveelements(LTR)
anddehydration-responsiveelements(MBS,ACGT).TheresultsinFig1Ashowhighfrequent
distributionofstressresponsiveelementsintheRCApromoterregion,including3fordehy-
drationand2fortemperature(Table1).ThisindicatesthatRCAmightberegulatedbyabiotic
stresses.Inaddition,5lightresponseelementswerealsofoundintheregulatorycis-elements
inthe2.0kbupstreampromoterregionofRCA(Table1),whichconfirmsthatRCAisalight-
regulatedgeneandastress-regulatedgene.
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Fig1.AnalysisofRCA’spromoterandgenome.(A)Analysisofstress-responsivecis-regulatoryelements
inthe2.0kb5’-upstreamregionsoftheRCAgene.(B)Theschematicrepresentationofgenomicorganization
(exon-intronorganization)ofthegenomicsequenceofRCAgenesanditsalternativesplicing.Theelements
locatedinthe(+)strandareabovethelines,whereasthoseinthe(-)strandareindicatedbelowtheline.
doi:10.1371/journal.pone.0140934.g001
DifferenceingenomicorganizationandproteinstructureofRCA and
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RCA
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ThecompletecodingsequencesofRCALandRCASwereamplifiedusingPCRwithfirst-strand
cDNAtemplatespreparedfromtotalRNA.Similartoapreviousreport[2],thealignmentof
thegenomicsequencesofRCALandRCASwiththeirrespectivecDNAsequencesidentifiedsix
exons(36,303,187,473,283and119bp)andfiveintrons(99,113,85,92and1032bp)in
RCALandsixexons(36,303,187,473283and19bp)andfiveintrons(99,113,85,92and347
bp)inRCAS.ThenumberofexonsandintronswassimilarbetweenRCALandRCAS,butthe
sizeofRCALwaslarger.(Fig1B)
Theanalysisshowedthattherewere5differentconsecutiveAAs(aminoacids)between
RCA andRCA ,exceptthatRCA hadanadditional33AAsattheC-terminalcomparedwith
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theRCA inrice(Fig2Aand2B).Therefore,RCA had2additionalstrands(436–438,462–
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465)and3additionalexposedregionscomparedwithRCA basedontheREPROFrecand
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PROFAccserverwebsites.ThequantificationofthesedifferencesisshowninFig2C.RCAL
hasmorestrands,intermediateandloopregions,andhasfewerhelixandexposedregions.In
addition,therearemoredisorderregions(22of38AA)inRCA becauseofthedifferentAAs
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accordingtothePROFbval,UconandMDservers.MoredisorderregionsmeanthatRCA
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Table1. ThecisregulatoryelementsfoundinthepromoterregionofRCA.
Function Totalnumber Elementsname Copynumber
Light 5 GT1CONSENSUS 3
INRNTPSADB 1
TBOXATGAPB 1
Dehydration 3 MYCATERD1 1
MYBCORE 1
MBS 1
Temperature 2 MYCCONSENSUSAT 2
hightranscriptionlevels 1 5UTRPY-richstretch 1
doi:10.1371/journal.pone.0140934.t001
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Fig2.ThedifferencesinsecondarystructurebetweenRCA andRCA .(A)ThesecondarystructureanddisorderconfidenceofRCA .(B)The
L S S
secondarystructureanddisorderconfidenceofRCA .(C)The(B)ThequantitativeanalysisofthesecondarystructureofRCA.
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doi:10.1371/journal.pone.0140934.g002
hasmoreregionsthatarepartiallyorwhollyunstructuredanddonotfoldintoastablestatein
three-dimensionstructure.
ThestructureofvarioushomogeneousdomainswassearchedfromthePDBbank,andthe
tertiarystructureofRCAwasidentifiedandanalyzedusingthePhyre2server(http://www.sbg.
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bio.ic.ac.uk/phyre2/html/page.cgi?id=index)andPyMOLsoftware(Fig3).Thetertiarystruc-
tureofRCA wasdifferentfromRCA becauseofthe38additionalAAsattheC-terminal.
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WithinRCA andRCA ,theAAA+domain(labela)wasconserved.However,theC-terminal
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(labelb)inRCA wasdifferentfromthatinRCA .Thisdifferenceresultedinanadditional
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gap(labeld).Furthermore,thedomainneartheC-terminal(labelc)wasalsodifferent.Thedif-
ferencebetweenbothisoformsimpliedthattheyhaddifferentfunctions.Inparticular,the
additionalgapinRCA meansthatitmighthavemorefunctionsassociatedwithprotein-
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binding.
PredictionofactivesitesandinteractionwithRCA andRCA structure
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ThebindingsitesofthetargetproteinwerepredictedusingtheISISmethods[37]andthe
“ImprovementofDNA-andRNA-ProteinBindingPrediction”method.Atotalof21active
bindingsiteswerepredictedinRCA .Amongthem,only4DNA/RNA-bindingsitesare
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showninFig4A.InRCA ,theactivebindingsiteswerereducedto20(Fig4B),whichweresig-
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nificantlyalteredneartheC-terminus(Fig4).TheseresultssuggestedthatthetwoRCAiso-
formsbondedtodifferentproteinsandhaddifferentfunctionsundervaryingconditions.
RCA andRCA phylogenetictree
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ThephylogenetictreeconstructedforRCAclusteredallplantstogether(Fig5)attheamino
acidlevel.Thehypotheticalproteinswithinthefirst100similarproteinsbyblastPofRCA
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proteinwerealsoconsideredinthisstudy.Thisfigureismadeusingthecompletesequence,in
whichtheconservativebetweentwoenzymesishigherthaninthediversitysequence,sothe
RCA ofdifferentspeciesisdispersedinthefigure.Theaminoacidsequencealignmentsofthe
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RCAisoformswiththeircorrespondingsubunitsfromOryzasativa,Brachypodiumdistachyo,
Deschampsiaantarctica,Triticumuartu,andArabidopsisarelistedinFig6andTable2.The
RCA andRCA ofriceshared99%identitywitheachother,andtheC-terminalAAsinRCA
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werealsoconservedaccordingtotheredboxRCA -specificdifferentialfragmentsofacid
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sequencealignmentresult(Fig6).Moreover,theobservedphylogeneticrelatednesssuggested
thattheevolutionofRCAinricewassimilartocloselyrelatedgrassfamilymembers,suchas
BrachypodiumandDeschampsia.TheseresultsindicatedthatthestructureofRCAwasconser-
vative,whereasthedifferentaminoacidsbetweenRCA andRCA werealsoconservative
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withinmanyspecies.ItsuggeststhatthedifferentfunctionofRCA maybetheresultof
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evolution.
ExpressionofRCAisoformsundervariousabioticstresses
ThechangesinthetwoRCAisoformproteinsinriceleavesundervariousabioticstresseswere
assessedusingwesternblotandELISAbasedonspecificmonoclonalantibodiesagainstboth
isoformsandRCA only.TheELISAresultsshowedthatforsolubleRCAinthechloroplast
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stromainrice,RCA was4~5timeshigherthanRCA ,andthetwoRCAisoformsweresignifi-
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cantlyup-regulatedfor24hoursofstress(Fig7A).AstheboundRCAseparatedfromthethy-
lakoidmembrane,bothisoformswereup-regulatedunderallstressedconditions,andRCA
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increasedmorethanRCA ,particularlyfortheheatandNaCltreatments(Fig7B).Western
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blottingalsoconfirmedthesameresults(Fig7C).TheseindicatedthatRCA wasmorehighly
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inducedunderabioticstresses.
TodeterminewhethertheproteinisoformsofRCAwererelevanttomRNAaccumulation,
thequantitativechangesinthetwoRCAisoformmRNAsinriceleavesduringthesameabiotic
stressesweredeterminedusingsemi-RT-PCR(Fig7D)andqRT-PCR(Fig7E).Allofthe
stressessignificantlyincreasedthetotalRCAmRNA(RCAL+S)(Fig7Dand7E).Becausethe
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Fig3.ThedifferencesintertiarystructurebetweenRCA andRCA .(A)and(B)Predictionofthe3D
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structureofRCA andRCA basedonthePDBstructure.(C)and(D)Predictionofthe3DstructureofRCA
S L S
andRCA basedonthePDBstructure.Thelabels(a,b,c,d)indicatethedifferentdomainsbetweenRCA
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andRCA .
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doi:10.1371/journal.pone.0140934.g003
quantificationofRCALexpressionwasimpossibleinourexperiment,wededucedthatthelev-
elsofRCALmRNAwereincreasedbyseveralfoldunderallofthetreatmentconditionsbecause
theincreaseintheRCASmRNAwasnotasmuchasthatoftotalRCAmRNA(RCAL+S)(Fig
7E).ThesefindingswereconsistentwiththeRCAproteininresponsetoabioticstressesand
indicatedthattheincreasedRCAproteinlevelswereattributedtothepromotedtranscription
ofRCALmRNAunderabioticstresses.
Fig4.ThepredictedDNA/RNA-bindingsitesandproteinbindingsitesofRCA andRCA .(A)The
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predictedRCA sites.(B)ThepredictedRCA sites.DotsindicatetheDNA/RNA-bindingsites,andthe
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rhombusindicatestheproteinbindingsites.
doi:10.1371/journal.pone.0140934.g004
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Fig5.ArootedphylogenetictreeconstructedusingMeGa5andiTOLshowingevolutionaryrelatednessofRCAinplants.ReddotsindicateRCA
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withconservativedifferentaminoacidsbetweenRCA andRCA .Thebootstrapvaluesfrom1000replicatesareshownforselectedbranches.
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doi:10.1371/journal.pone.0140934.g005
Discussion
RCAmaybeinvolvedintheresponsetomanyabioticstresses
RCA,anuclear-encodedchloroplastprotein,catalyzesRubiscoactivationduringphotosynthe-
sisbyremovinginhibitorsfromthecatalyticsitesofRubiscoinvivo[9,38,39].However,some
studieshavereportedthatRCAquicklyrespondstostressconditionsandtreatmentwithplant
hormone,whichisquitedifferentfromthefunctionsofRubiscoactivationofRCAacceptedby
mostresearchers.Komatsuetal.[40]reportedthatagibberellin-bindingproteininriceis
homologoustoRCA,andSharmaandKomatsu[41]suggestedthatRCAisassociatedwith
Ca2+-dependentproteinkinasesingibberellinsignaling.Dejimenezetal.[42]firstreported
that45kDRCA isinducedathightemperature(45°C)anddisappearsatnormaltemperature.
L
TheysuggestedthatRCAisapossiblenewmemberofthemolecularchaperonefamily.These
studiessuggestedadditionalrolesforRCAbeyondRubiscoregulation[9].Furthermore,RCA
PLOSONE|DOI:10.1371/journal.pone.0140934 October19,2015 10/16