Root Hair

84JOURNALOFBIOSCIENCEANDBIOENGINEERING漏2005,TheSocietyforBiotechnology,JapanVol.99,No.1,84鈥�6.2005DOI:10.1263/jbb.99.084RootHairAbundantGenesLjRH101andLjRH102EncodePeroxidaseandXyloglucanEndotransglycosylaseinLotusjaponicusTAKAKIMAEKAWA,1MAKOTOHAYASHI,1*ANDYOSHIKATSUMUROOKA1DepartmentofBiotechnology,GraduateSchoolofEngineering,OsakaUniversity,2-1Yamada-oka,Suita,Osaka565-0871,Japan1Received26August2004/Accepted21October2004Wehaveidentifiedroothairabundantgenes,LjRH101andLjRH102,fromamodellegumeLotusjaponicusbycDNA-amplifiedfragmentlengthpolymorphism(AFLP).Thesetwogeneswillbesuitablemarkersforthemolecularbiologicalidentificationofroothairs.[Keywords:cDNA-amplifiedfragmentlengthpolymorphism(AFLP),legume,Lotusjaponicus,roothairs]Roothairs,asidefromtheirroleintheuptakeofnutrientsandwater,serveasthefirsthostcellofrhizobialinfection.Rootnoduleorganogenesisistriggeredbytheattachmentofrhizobiatoroothairs.Theapicalcellwallofroothairformsatunnel-likestructurecalledtheinfectionthreadandthisgrowsintothebaseoftheroothaircellinwardlywithrhizo-bia(1).Roothairsarethefirstpointofinteractionwithrhizobia,andbecauseoftheirimportance,molecularinformationre-gardingroothairshasbeenaccumulatedforleguminousplants.Mylonaetal.(2)haveidentifiedapeagene,RH2,encodingapathogen-relatedproteinthatisexpressedattherootepidermisspecifically.Inthisregard,anRH2homologofMedicagotruncatulahasbeenusedasanendogenousmarkertomonitorroothairisolation(3).Inaddition,aroothair-enrichedcDNAlibrarycontainingatotalof899ex-pressedsequencetags(ESTs)wasconstructedforM.trun-catula(4).Lotusjaponicushasbeenproposedasamodellegumeformolecularbiologicalstudiesofsymbioticinteractions(5).RegardingtheLotusESTproject,morethan74,472ESTshavebeenaccumulated(6).However,theseESTsincludelittleinformationaboutroothairs,becauseESTlibrarieshavebeengeneratedfromnoduleprimordia,nodules,wholeyoungplants,flowerbuds,androots,andnotfromroothairsexclusively(6).Moreover,toourknowledge,roothairabundantgeneshavenotbeenidentifiedfromL.japonicus.Roothairabundantgenesofamodellegumewouldbeuse-fulmolecularmarkersforstudiesonearlysymbiosiseventsinroothairs(e.g.,infectionthreadformation).Inthisstudy,wehaveidentifiedLjRH101andLjRH102asroothairabundantgenesusingacDNA-amplifiedfrag-mentlengthpolymorphism(AFLP)methodfromamodellegumeL.japonicus.L.japonicusacc.MG-107鈥楽uita鈥�wasusedfortheisola-tionofroothairsandroots.TheisolationofroothairswasperformedaccordingtoLauteretal.(7)withminormodifi-cation.Surface-sterilizedseedsweregerminatedonsteril-izedfilterpaperon1%agarcontainingB&Dmedium(8)insquareplasticplates(Iwaki,Chiba).Plasticplateswerecov-eredwithdouble-layeredaluminumfoilandincubatedver-ticallyfor4dinacultivationchamber(CFH-305;Tomy,Tokyo)undera16h/8hday/nightcycleataday/nighttemperatureof26
C/23
C.Afterincubation,seedlingswerecollectedanddroppedimmediatelyintoa2-lstainlesssteelbeakerfilledwithliquidnitrogen.Thesampleswerestirredgentlywithaglassrodfor5min.Inthisstep,roothairswerebrokenofffromroots.Inordertoseparateroothairsfromroots,theliquidnitrogencontainingbrokenroothairswasfilteredthroughastainlesstestingsievewithameshapertureof500m(IidaManufacturing,Osaka).Liquidni-trogenwasevaporatedina50-mlsampletubeandroothairswerecollected.Atthesametime,theroothair-strippedrootswereusedforRNApreparation.TotalRNAwasisolatedfromthetissuewiththeRNeasyMiniKit(Qiagen,Tokyo),accordingtothemanufacturer鈥檚protocol.ThecDNA-AFLPreactionwasperformedaccordingtoHabuetal.(9)withminormodifications.TotalRNAfromstrippedrootsorroothairswasusedfordouble-strandcDNAsynthesisusingthecDNASynthesisSystem(Invitrogen,Carlsbad,CA,USA),accordingtothemanufacturer鈥檚pro-tocol.Afterpurification,cDNAwascompletelydigestedwithTaqIandligatedtotheTaqIadaptor.Forpre-selectiveamplification,ligatedproductswereamplifiedwithTaqIAprimerandTaqIT,GorCprimer.Forselectiveamplifica-tion,rhodamine-labeledTaqIAANprimer(NrepresentsAorT)andTaqIT,G,orCprimerwereusedfordetection.Afteramplification,electrophoresisofselectiveamplifica-tionproductswasperformedusingtheGeneGelExcel12.5/24kit(AmershamBiosciences,Uppsala,Sweden)withtheGenePhorDNASeparationSystem(AmershamBiosciences).ThecDNAfragmentsweredetectedwithFMBIOIIMulti-View(Hitachi,Tokyo)andthoseofinterestwerecutout,*Correspondingauthor.e-mail:hayashi@bio.eng.osaka-u.ac.jpphone:+81-(0)6-6879-7417fax:+81-(0)6-6879-7418NOTESVOL.99,200585extractedbywaterwithheating,re-amplifiedbyPCR,andclonedintothepT7Bluevector(Takara).ResultantsequenceswereanalyzedbytheBlastxprogram(NCBI).Forgeneexpressionanalysis,totalRNA(500ng)wasusedforthefirst-strandcDNAsynthesisusingSuperScriptIIRNaseH鈥�ReverseTranscriptase(Invitrogen)andamix-tureofanchoredoligo-dT19N(NrepresentsA,GorC)asaprimerina40-lreaction,accordingtothemanufac-turer鈥檚protocol.Real-timePCRwasperformedwiththeGeneAmp5700SequenceDetectionSystem(AppliedBio-systems,Tokyo)ina20-lreactioncontaining0.1loffirst-strandcDNA,0.2lof100Mforwardprimer,0.2lof100
Mreverseprimer,2
lof10,000-folddilutedSYBRGreen(MolecularProbes,Eugene,OR,USA),1.6lof2.5mMdNTPmixture,2lof10ExTaqbuffer,and0.1lofExTaq.Eachassayincludedsixreplicatesperclone,andPCRwasperformedusingthefollowingprogram:95
Cfor10minand30cyclesofdenaturationat95
Cfor30s,anneal-ingat55
Cfor30sandextensionat72
Cfor30s.Thedis-sociationprogramconsistedof95
Cfor15sand60
Cfor20s.Aputativeconstitutivelyexpressedgene,LjEF1,en-codingelongationfactor1-alphawasusedfortheinternalstandard,andcalculationoftherelativegeneexpressionwasperformedaccordingtoColebatchetal.(10).Primersthatproducedshortampliconslessthan150bpweredesignedbyusingPrimerexpresssoftware(AppliedBiosystems).Primerpairsforeachgenewereasfollows:LjRH101,forward:5-GCTAACGCCATGGTCAAGATG-3,reverse:5-GGAATCACACAGTTTGGCAATG-3;LjRH102,for-ward:5-CGCAGCTCAAACAAAGAAAGC-3,reverse:5-GCTGTAACAATTCCTGCAGAGTTG-3;LjEF1,for-ward:5-GCAGGTCTTTGTGTCAAGTCTT-3,reverse:5-CGATCCAGAACCCAGTTCT-3.Roothairsandrootswereisolatedfrom4-day-oldseed-lingsgrownonfilterpaperonmediumtoavoidroothairsinvadingthemedium.Weconfirmedthatthesamplesiso-latedbyliquidnitrogenwerehighlyenrichedinroothaircellsbymicroscopicobservation(Fig.1).TotalRNAofroothairsamountedto2鈥�gfrom1000seedlings.AftercDNA-AFLPfragmentswereseparatedbynativepolyacrylamidegelelectrophoresisandimagedbyafluo-rescentscanner,twofragmentscorrespondingtoLjRH101andLjRH102amplifiedbytheprimerpairofTaqIAAAandTaqIGwereclonedasabundantgenesinroothairs(Fig.2).Intheothercombinationofprimers,nosignificantdifferencewasdetected.FragmentsofLjRH101andLjRH102includedthese-quenceofAFLPprimers,andwerethereforespecificam-pliconsofcDNA-AFLP.ThefullamplifiedsequencesofLjRH101(640bp)andLjRH102(505bp)weredepositedintheDDBJdatabaseunderaccessionnos.AB188574andAB188575,respectively.Atthenucleotidelevel,LjRH101wasmorethan99%identicaltotwoLotusESTclones,BP083108andAI967610,theclonesofwhichwereiden-tifiedfromtherootlibrary,whereasLjRH102showednoapparenthomologytotheLotusESTdatabase.TherelativeamountsofLjRH101andLjRH102invari-ousplantpartswereanalyzedwithrealtimeRT-PCR.Aconstitutivelyexpressedgene,LjEF1,encodingelongationfactor1-alphawasusedasaninternalstandard(10).Fromtheresultsoftwoindependentexperiments,LjRH101andLjRH102wereexpressedatveryhighlevelsinroothairsincomparisonwithroothair-strippedroots,developedrootsandshoots(Fig.3).TheLjRH101productissimilartoperoxidasethatcata-lyzestheoxidoreductionprocessofhydrogenperoxide.Plantperoxidasesconsistofseveralisoenzymesandhaverolesinlignification,suberization,auxincatabolism,woundhealing,anddefenseagainstpathogeninfection(11).Theplantdefensesystemagainstpathogensprovidesaphysicalbarriervialignificationofcellwallsbyperoxidase.TheLjRH102productissimilartoxyloglucanendotransgly-cosylasethathasrolesinthehypersensitiveresistancere-action(12),fruitripening,floralabscission,andcellelonga-tion(13).Vissenbergetal.(14)havereportedthatxylo-glucanendotransglycosylaseactivityislocalizedattheroothairtipduringroothairinitiationinArabidopsis.Inadditiontoourresults,ithasbeenreportedthatdefense-relatedgenesencodingchitinaseandchalconesynthaseareexpressedinepidermalcellsaswellasRH2(2,15,16).Therefore,ingeneral,aconstitutivedefensesystemwouldbedevelopedatthesurfaceofroots(15,16).FIG.1.Highlyenrichedroothairsseparatedfrom4-day-oldseed-lingsbyliquidnitrogen.Bar:100
m.FIG.2.cDNA-AFLPfingerprintpatternsofroothairsversusroots.Thebandsindicatedbyarrowheadswereextractedfromthegelandcloned.AAAisthe3endoftheselectiveprimersequencelabeledwithrhodamineatthe5end.T,G,orCisthe3endofthenon-labeledselectiveprimersequence.H,Roothairs;R,roots.MAEKAWAETAL.J.BIOSCI.BIOENG.,86Wehavesucceededinidentifyingtworoothairabundantgenes,LjRH101andLjRH102,inthisstudy.Roothairabun-dantgeneswouldbeusefulmolecularmarkersforstudiesonearlysymbiosiseventsinroothairsinamodellegumeL.japonicus.Inadditiontousingendogenousmarkersforroothairisolation,thepromotersofLjRH101andLjRH102wouldbegoodcandidatesforthegeneticengineeringofroothaircells,sinceLjRH101andLjRH102wereexpressedveryhighlevelsinroothaircells.RNAinterferenceagainstsymbiosis-inducedgenesunderthecontroloftheLjRHpro-moterwouldbeausefultooltoinvestigatethefunctionofthesegenesinearlysymbiosisevents.REFERENCES1.Brewin,N.J.:Developmentofthelegumerootnodule.Annu.Rev.Cell.Biol.,7,191鈥�26(1991).2.Mylona,P.,Moerman,M.,Yang,W.C.,Gloudemans,T.,VandeKerckhove,J.,vanKammen,A.,Bisseling,T.,andFranssen,H.J.:Therootepidermis-specificpeageneRH2ishomologoustoapathogenesis-relatedgene.PlantMol.Biol.,26,39鈥�0(1994).3.Ramos,J.andBisseling,T.:AmethodfortheisolationofroothairsfromthemodellegumeMedicagotruncatula.J.Exp.Bot.,54,2245鈥�250(2003).4.Covitz,P.A.,Smith,L.S.,andLong,S.R.:Expressedse-quencetagsfromaroot-hair-enrichedMedicagotruncatulacDNAlibrary.PlantPhysiol.,117,1325鈥�332(1998).5.Handberg,K.andStougaard,J.:Lotusjaponicus,anau-togamous,diploidlegumespeciesforclassicalandmoleculargenetics.PlantJ.,2,487鈥�96(1992).6.Asamizu,E.,Nakamura,Y.,Sato,S.,andTabata,S.:Char-acteristicoftheLotusjaponicusgenerepertoirededucedfromlarge-scaleexpressedsequencetag(EST)analysis.PlantMol.Biol.,54,405鈥�14(2004).7.Lauter,F.R.,Ninnemann,O.,Bucher,M.,Riesmeier,J.W.,andFrommer,W.B.:Preferentialexpressionofanammo-niumtransporterandoftwoputativenitratetransportersinroothairsoftomato.Proc.Natl.Acad.Sci.USA,93,8139鈥�8144(1996).8.Broughton,W.J.andDilworth,M.J.:Controloflegha-emoglobinsynthesisinsnakebeans.Biochem.J.,125,1075鈥�1080(1971).9.Habu,Y.,Fukada-Tanaka,S.,Hisatomi,Y.,andIida,S.:Amplifiedrestrictionfragmentlengthpolymorphism-basedmRNAfingerprintingusingasinglerestrictionenzymethatrecognizesa4-bpsequence.Biochem.Biophys.Res.Com-mun.,234,516鈥�21(1997).10.Colebatch,G.,Kloska,S.,Trevaskis,B.,Freund,S.,Altmann,T.,andUdvardi,M.K.:Novelaspectsofsym-bioticnitrogenfixationuncoveredbytranscriptprofilingwithcDNAarrays.Mol.PlantMicrobeInteract.,15,411鈥�20(2002).11.Hiraga,S.,Sasaki,K.,Ito,H.,Ohashi,Y.,andMatsui,H.:AlargefamilyofclassIIIplantperoxidases.PlantCell.Phys-iol.,42,462鈥�68(2001).12.Cooper,B.:Collateralgeneexpressionchangesinducedbydistinctplantvirusesduringthehypersensitiveresistancere-actioninChenopodiumamaranticolor.PlantJ.,26,339鈥�49(2001).13.Lashbrook,C.C.,Gonzalez-Bosch,C.,andBennett,A.B.:Twodivergentendo-beta-1,4-glucanasegenesexhibitoverlap-pingexpressioninripeningfruitandabscisingflowers.PlantCell,6,1485鈥�493(1994).14.Vissenberg,K.,Fry,S.C.,andVerbelen,J.P.:Roothairinitiationiscoupledtoahighlylocalizedincreaseofxyloglu-canendotransglycosylaseactioninArabidopsisroots.PlantPhysiol.,127,1125鈥�135(2001).15.Samac,D.A.andShah,D.M.:Developmentalandpathogen-inducedactivationoftheArabidopsisacidicchitinasepro-moter.PlantCell,3,1063鈥�072(1991).16.Schmid,J.,Doerner,P.W.,Clouse,S.D.,Dixon,R.A.,andLamb,C.J.:Developmentalandenvironmentalregulationofabeanchalconesynthasepromoterintransgenictobacco.PlantCell,2,619鈥�31(1990).FIG.3.TherelativeamountofLjRH101andLjRH102toLjEF1invariouspartsoftheplant.ThetotalRNAofroothairs(H)andstrippedroots(R1)waspreparedfrom4-day-oldseedlings.ThetotalRNAofdevelopedroots(R2)andshoots(S)waspreparedfromplantsgrownonvermiculitesuppliedwithB&Dmedium.Valuesshownaretheaverageoftwoindependentexperiments.