An evolutionarily conserved P‐subfamily pentatricopeptide repeat protein is required to splice the plastid ndhA transcript in the moss Physcomitrella p Ayaka Ito.pdf

该【An evolutionarily conserved P‐subfamily pentatricopeptide repeat protein is required to splice the plastid ndhA transcript in the moss Physcomitrella p Ayaka Ito 】是由【周瑞】上传分享，文档一共【30】页，该文档可以免费在线阅读，需要了解更多关于【An evolutionarily conserved P‐subfamily pentatricopeptide repeat protein is required to splice the plastid ndhA transcript in the moss Physcomitrella p Ayaka Ito 】的内容，可以使用淘豆网的站内搜索功能，选择自己适合的文档，以下文字是截取该文章内的部分文字，如需要获得完整电子版，请下载此文档到您的设备，方便您编辑和打印。:..Articletype:OriginalArticleAnevolutionarilyconservedP-subfamilypentatricopeptiderepeatproteinisrequiredtospliceitrellapatensandArabidopsisthalianaAyakaIto1,ChiekoSugita1,MizuhoIchinose1,2,YoshinobuKato3,HiroshiYamamoto3,ToshiharuShikanai3,andMamoruSugita1,*eneResearch,NagoyaUniversity,Nagoya,464-8602JapanArticle2InstituteofTransformativeBio-Molecules(WPI-ITbM),NagoyaUniversity,Nagoya,464-8602Japan3DepartmentofBotany,GraduateSchoolofScience,KyotoUniversity,Kyoto,606-0076,Japan(Thefirsttwoauthorsequallycontributedtothiswork)*Correspondingauthor:E-mail.******@-:AplastidndhAintron-specificPPRsplicingfactorKeywords:P-subfamilyPPRprotein,splicing,ndhA,plastid,itrellapatens,Arabidopsisthalianaeptedforpublicationandundergonefullpeerreviewbuthasnotbeenthroughthecopyediting,typesetting,paginationandproofreadingprocess,:.Allrightsreserved.:..AbstractPentatricopeptiderepeat(PPR),,itrellapatensPpPPR_66knockout(KO)-likegrowthphenotypebutshowedaberrantchlorophyllfluorescenceduetodefectsinchloroplastNADHdehydrogenase-like(NDH)-,thetranscriptlevelsof11plastidndhgeneswereanalyzedbyreverse---DNAtaggedlinesofaPPR_66homolog(At2g35130)’,theseresultsindicatethatPpPPR_66actsasaspecificfactortosplicendhApre--encodedpentatricopeptiderepeat(PPR)proteinsaresynthesizedinthecytoplasm,butarethenposttranslationallyimportedintoplastidsormitochondriaorboth,wheretheyfunctioninvariousRNAprocessingsteps(SmallandPeeters,2000,,).ThePPRgenefamilyexistsubiquitouslyineukaryotes,buthasexpandedconsiderablyto450PPRgenesinArabidopsisthalianatoover1,000inthespikemossSelaginellamoellendorffii().(P)motifswhilethePLSsubfamilyconsistsofrepeatedblocksofPandPPR-like(L,S),E/E+,.:..andDYWclasses().Amongthem,-of-anellarfunction,suchasphotosynthesisandrespiration(Schmitz-LinneweberandSmall2008).TheP-subfamilyPPRproteinsarereportedlyinvolvedinintergenicRNAprocessingandtranslation(,,),trans-splicing(Schmitz-)andcis-splicing(,),aswellasthestabilizationofplastidmRNAs(,)andtRNA().Inaddition,P-subfamilyPPRproteinswithasmallMutS-related(Smr)domainwereshowntobeinvolvedinprocessingofchloroplast23S--rRNA(,).Ontheotherhand,thePLS-subfamilyproteinsmostlyfunctionasanRNAeditingsite-recognitionfactorforbothmitochondrialandplastidtranscripts(FujiiandSmall2011,,IchinoseandSugita2017).Besides,somePLSmembersarereportedlyrequiredforRNAsplicingofspecifictranscripts(Chateigner-,,).Thus,bothsubfamiliesbindtoRNAsinagene-specificmannerandcontributetovarioustypesofRNAprocessingsteps(BarkanandSmall2014).However,thefunctionofmostP-~105PPRproteins,over80%ofwhicharemembersofthePsubfamilymember().Todate,thefollowingfourP--mRNA(,HattoriandSugita2009).PpPPR_67and104wererequiredforplastidtRNAmaturation().PpPPR_4wasrecentlyshowntoplayaroleinplastidtRNAIlesplicing().SeveralmossP-subfamilyproteinsareconservedattheaminoacidsequenceleveltofunctionallycharacterizedArabidopsisandmaizePPRproteins().PpPPR_67and104arefunctionallyrelatedtoArabidopsisPRORP1to3().However,-subfamilyproteins,weconstructedaseriesofPPRgeneknockout(KO)-subfamilyPpPPR_66protein,.:..5'-(Pp3c16_5890/Pp1s15_385)encodesapolypeptideof578aminoacids(aa)thatconsistsofanN-posedof11PPRmotifs().TheTargetPprogram(),afusionprotein,posedofitsN-terminal121aaandgreenfluorescentprotein(GFP),().ThePpPPR_66homologs,whichwehererefertoasPPR66L,arefoundinawiderangeoflandplants,includingtheliverwortMarchantiapolymorpha,thespikemossSelaginellamoellendorffii,Arabidopsisthaliana,andZeamays(maize)().ThePpPPR_66geneisinterruptedbyeightintrons().,PpPPR_72(Pp3c6_26210/Pp1s53_63)islikelyaparalogofPpPPR_66().ChloroplastNDHactivityisdefectiveinPpPPR_66KOmutantsForloss-of-functionanalysisofPpPPR_66,wegeneratedPpPPR_66KOlinesbyreplacingitscodingregionwithacassettecarryingthegfpanddrugresistant(hpt)bination().Weconfirmed,bygenomic-PCRanalysis,urredineachofthedesignedtargetedloci().IntheKOmutantlines(?66-2,?66-3),wealsoconfirmed,byRT-PCRanalysis,thatPpPPR_66transcriptwasnotdetected().ThePpPPR_66KOmossesdisplayedawildtype-likegrowthphenotype().Then,,suchasFv/Fm,(.:..ofphotosystemII(PSII))andΦPSII(theeffectivequantumyieldofPSII),werealmostthesameinWTandKOmosses(TableS1).,wemonitoredchlorophyllfluorescenceinthemosseswithapulseamplitudemodulation(PAM)().ItiswellknownthatthischangeinfluorescencerepresentsNDHactivityinchloroplasts(Shikanai2016).Thus,-plexintheKOmutants().Incontrast,plex(Cytf),theβsubunitofH+-ATPsynthase(AtpB),andthePSIIreactioncenterD1protein(PsbA),,NdhB,intheliverwortMarchantiapolymorpha().,includingsplicingofplastidtranscripts(BarkanandSmall2014).plexwasobservedinthePpPPR_66KOmosses,RNAmaturationofplastidndhgenes,suchasRNAstabilityand/orRNAsplicing,,RT-PCRanalysiswasperformedtoinvestigatemRNAlevelsof11ndhgenes,ndhAtondhK,whicharelocatedatfourdifferentpositionsintheplastidgenome().ThisanalysisshowedthatsplicedndhAtranscript(895-bpampliconbyRT-PCR)umulatewhileunsplicedndhAtranscript(1,585-bpamplicon)accumulatedconsiderablyintheKOmosses().Incontrast,,weperformedRNAgelblothybridizationofanndhA-,ndhH,ndhA,ndhI-G-EandpsaCsequences,--kbtranscript,alongertranscript(7kb)wasdetectedintheKOmosses().The7-.:..-kbtranscriptcouldbeproducedfromthe7-,anndhAintron-specificprobe(Int)anda3’exon-specificprobe(Ex),-kbtranscriptwasdetectedintheWTbutnotintheKOmossesandthe7-,-,the7--kband7-kbbandsweresplicedandunsplicedndhAtranscripts,-kbbandwasdetectedinWTbutnotinKOmutants,-,shorterndhAtranscripts,whichcouldbeproducedfromthe3-kbunsplicedndhAprecursors,werenotdetected,,-kbtranscriptwastranscriptionallyorposttranscriptionallyproduced,thestrongsignalthatwasdetectedsuggeststhatpsaCmRNAisextremelystable,unlikendhtranscripts,,weexaminedthepossibilitythatPpPPR_66isinvolvedinsplicingofotherintron--codinggenesandsixtRNAgenes,whichcontainintron(s)().ToassesswhethersplicingofmRNAsandpre-tRNAswasaffectedintheKOmutants,RT-PCRanalysiswascarriedoutusingexon-(s)-containinggenesexceptumulatedtosimilarlevelsasthoseintheWT(,S5).,.:..ToconfirmthatPpPPR_66isessentialforndhAsplicing,wegeneratedmosstransformantsthatexpressedPpPPR_66full-sizedcDNAintheKOmutant?66-().plementationexperimentconfirmedthatthendhAsplicingdefectintheKOmutantswascausedbyaloss-of-%aaidentityand81%similaritytoArabidopsisPPR66L(At2g35130).TheAtPPR66LgeneisinterruptedbysevenintronsandtheirpositionsareidenticaltotheintronpositionsofthePpPPR_66gene().,weanalyzedtheArabidopsisPPR66LArticle(At2g35130)KOnullmutantlines,SALK_043507andSALK_065137().WemeasuredchloroplastNDHactivityinvivobyaPAMchlorophyllfluorometerandfoundthatbothKOmutantsexhibitednochloroplastNDHactivity().However,photosyntheticparameterswerenotaffectedintheKOmutants().Furthermore,bothKOmutantsshowednovisiblephenotypeunderourgrowthconditionsaspreviouslyr