甜橙中多胺合成酶基因全基因组发掘和表达分析.pdf 19页

'中国科技论文在线http://www.paper.edu.cnGenome-wideidentificationandexpressionanalysisofthepolyaminebiosynthesisgeneinsweetorange(Citrus5sinensis)WuHao,LiuJihong**(CollegeofHorticultureandForestrySciences,HuazhongAgriculturalUniversity,)Abstract:Polyamines(PAs)arelowmolecularweight,aliphaticpolycationsfoundinthecellsofalllivingorganisms.Andinplants,agrowingnumberofevidencessupportthatPAsplayimportantroles10inabioticstresses.Inthisstudy,atotalof18polyaminebiosynthesisgeneswhichbelongto10kindsofdifferentpolyaminebiosynthesisenzymeswereisolatedfromtheentirecitrusgenomeandafurtheranalysisincludingthechromosomallocations,phylogeneticrelationships,functionalannotations,promoteranalysis,andgenestructureswereperformed.Tissuespecificexpressionofthesegeneswasdetectedinroot,stem,leaf,pulp,peel,andcallus.Thepolyaminebiosynthesisgenedisplayedvarious15responsestoexogenouspolyamines(putrescine,spermidine,spermine)andABAtreatments,andweredifferentiallyalteredbyabioticstresses,includingcoldandsalt.Andthechangepatternsofthreemainpolyaminesduringcoldstressinleavesandcalluswerecharacterized.Thecomprehensiveanalysisofpolyaminebiosynthesisgeneishelpfultoexploitstrategiestoimproveplanttolerancetomultipleenvironmentalstresses.20Keywords:Sweetorange(Citrussinensis);polyaminebiosynthesisgene;abioticstress;Geneexpression0IntroductionPolyamineisanorganiccompoundhavingtwoormoreprimaryaminogroups–NH2.Thereare25threemajorpolyaminesinplants,putrescine(Put),spermidine(Spd),andspermine(Spm)[1],whichplayimportantrolesinvariousprogressesofplantgrowthanddevelopment,includingcellproliferation[2],morphogenesis[3],differentiation[4],programmedcelldeath[5],andresponsestovariousenvironmentalstresses[6-8].Andtheplantstresstolerancewouldbeimprovedwhentreatedwithexogenouspolyamines.Forexample,sprayingputrescinecouldreduce30flooding-inducedoxidativedamagebyincreasingtheantioxidantproperties[9].Exogenousspermidinemaydifferentiallyaltertheactivitiesofsomescavengingsystemenzymes,hydrogenperoxideandsuperoxideradicallevelsinwater-stressedcucumberleaves[10].Applicationofsperminecouldbeexploitedtoalleviateamoderatelevelofosmoticanddehydrationstressthroughtheregulationofstress-relatedcomponentssuchasphotosyntheticpigments,plant35hormones[11]andantioxidants[12].Andalso,overexpressionsomestress-responsegenesmayimproveendogenouspolyaminecontentandobtainahigherstressresistance,likeABF[13,14],ICE1[15],MYB[16],WRKY70[17]andsoon.Throughoneareathatremainslargelyunclearishowpolyamineimprovesplantstressresistance,increasingevidencesshowedthatitmightprotecttheplantsfromstressesbyremovingfreeradicals,maintainingmembraneandcellularstructures,40keepingcation-anionbalance,regulationofionchannelsandinductionofATPsynthesis[1,18-20].Thepolyaminebiosynthesispathway(SupplementFigure1)hasbeeninvestigatedandreviewedcompletelyindetail[21,22].Briefly,putrescinecanbeproduceddirectlyfromL-ornithineBriefauthorintroduction:WuHao(1988-),male,PhDstudent,StressPhysiologyandMolecularBiologyCorrespondanceauthor:LiuJihong(1971-),male,Professor,StressPhsiologyandMolecularbiology.E-mail:liujihong@mail.hzau.edu.cn-1- 中国科技论文在线http://www.paper.edu.cnviaornithinedecarboxylase(ODC,EC4.1.1.17),orindirectlyfromL-arginineviaargininedecarboxylase(ADC,EC4.1.1.19).Spermidineissynthesizedfromputrescinebyspermidine45synthase(SPDS,EC2.5.1.16)withtheadditionofanaminopropylgroupwhichisproducedfromS-adenosylmethionine(SAM)bySAMdecarboxylase(SAMDC,EC4.1.1.50).Thenspermineandthermospermineareproducedwhentheaminopropylgroupisaddedtospermidinebysperminesynthase(SPMS,EC2.5.1.22)andthermosperminesynthase(ACL5,EC2.5.1.79).So,plantpolyaminebiosynthesispathwayincludesfivemainenzymes:ADC,SPDS,SPMS,ACL5and50SAMDC[23,24].ItisworthmentioningthatinArabidopsisthalianaODCpathwaymaybeabsent,indicatingthatputrescineispredominantlyproducedviaADC-mediatedroute.Andoverexpressionorsilenceofdifferentpolyaminebiosynthesisgeneindifferentplantspeciesmaypromoteorweakenstresstolerance[25-28].OverexpressionofPtADCgenemightreduceROScontentandinfluenceonrootgrowthconductivetodroughttolerance[29].Whenoverexpressing55SPDSgeneintomato,theenhancedpolyamineaccumulationcanaltercarotenoidmetabolismatthetranscriptionallevelinfruit[30].CaADC1-silencedpepperhasalowerlevelofpolyaminesandtheexpressionofdefenseresponsegenesweredistinctlylowerinCaADC1-silencedplantsthanthoseintheemptyvectorcontrolplants[31].Underlong-termsalinity,theSPMSandACL5mutantsaccumulatedmoreNa+andperformedworsethanwildtypeinsurvivalexperiments[32].60WhenabrogatetheupstreamopenreadingframeofSAMDC,itwillleadtopolyaminedisruptionandgrowthperturbations[33].Therefore,thereisadeepconnectionbetweenpolyaminebiosynthesisgeneandplanttoleranceofabioticstress.Citrustakesthelargestpartoftheplantareaandhighesttotalyieldintheworld.Butsevereenvironmentalfactorslimitedtheproductionofcitrus,suchascold,drought,salinityandnutrition65deficiency,especiallythecoldstress.Throughthefivedifferentpolyaminebiosynthesisenzymesmayhavedifferentmechanismsofresponsetoabioticstress,agenome-widesurveytofindtherate-limitedenzymeofpolyaminebiosynthesispathwaymayprovideanapproachtoincreasethecitruspolyaminecontentandstresstolerance.Inthisstudy,weidentifiedADC,SPDS,SPMS,ACL5,andSAMDCgenesbystudyingtheirsequencephylogeny,genomeorganization,70chromosomallocation,andcis-actingelements.Therelativeexpressionlevelsofthesegenesindifferenttissuesandvarioustreatment,includingexogenousPAstreatment,stressconditionsandABA,wereexamined.Thepolyaminecontentundercoldstresswasalsodetected.Ourresultsprovideabasicinformationthatmaybeusedforfutureinvestigationintothefunctionsofpolyaminesincitrusdevelopmentandstressresponse.751Results1.1IdentificationofpolyaminebiosynthesisgenefromsweetorangeToidentifypolyaminebiosynthesisgeneinsweetorange,theCitrussinensisAnotationprojectandphytozomewereused,andatotalof18transcriptswereisolatedfromtheentirecitrusgenome-2- 中国科技论文在线http://www.paper.edu.cn80andbelongto10differentpolyaminebiosynthesisgenes.TheywerenamedrespectivelyasADC,SPDS1,SPDS2,SPMS,SAMDC1,SAMDC2,SAMDC3,SAMDC4,ACL5-1,andACL5-2accordingtotheannotationofthetwodatabasescomparedwiththeorthologsofArabidopsisandrice(Table1).Information,suchastheCDS,theexons,thenumberofaminoacids(aa),molecularweight(MW),andtheisoelectricpointswerepredicted,theresultsofwhicharelistedinTable1.85Asshown,thecodingsequencesrangedfrom756to2262bps,deducedproteinsequencelengthsvaryfrom251to753aminoacids,andcorrespondingpIswerepredictedtobebetween4.93and5.96.Table1.Basicinformationofpolyaminebiosynthesisgenesidentifiedincitrussinensis90CDS,codingsequence;aa,aminoacid;pI,theoreticalisoelectricpoint;MW,molecularweight.1.2Phylogeneticanalysis,GenestructureandChromosomallocationTofurtherunderstandthephylogeneticrelationshipofthepolyaminesbiosynthesisgene,aphylogeneticanalysiswasperformedbasedonthefull-lengthaminoacidsequencesfromCitrussinensisandArabidopsisthalianausingClustalXandMEGAprogram(Figure1A).Accordingto95thephylogenetictree,thegenesweredividedintothreegroupsobviously,theADCgroup,theSPMSgroup(containingSPMS,SPDS,ACL5)andtheSAMDCgroup.ThedifferenttranscriptsformedbyintronssplicingwereputinthesameclassindicatedthattheywerethesamegeneandwerealreadyshowninTable1.ThenthesizeanddistributionofexonswasdeterminedinpolyaminebiosynthesisgenesinC.100sinensisbyaligningthefull-lengthcDNAtranscriptswiththecompletegenomicDNAsequences.Theexon-intronstructuresofthesegenescanprovideimportantinformationabouttheevolutionaryrelationshipsofthegenome(Figure1B).TheADCandSAMDCgroupshadfewintronsbuttheSPMSgroupcontainedatleast8intronspergenewhichwasshowninTable1andFigure1B.Thedistributionofexonsandintronsindicatedthatgeneinonegroupexhibiteda105highlyconservedstructureandSPMSgroupsmayhavemorediversefunctions.Chromosomallocationanalysisshowedthat18transcriptswhichencoded10kindsofenzymeslocatedon6chromosomesbytheMapDrawtool,whichdispersedthroughoutthegenomes-3- 中国科技论文在线http://www.paper.edu.cn(Figure1C).TheADCwasonthechromosome8,SPDS1andSPDS2werelocatedonthechromosome5and7respectively,SPMSandACL5geneswasonthechromosome9and110chromosome4.TheSAMDCgeneswerelocatedonchromosome4,7,6and9.Figure1Phylogeneticrelationship,genestructureofcitruspolyaminebiosynthesisgenes.(A)Multiplealignmentof18fulllengthPAbiosynthesisgenesfromcitrusand10fromArabidopsisthalianaweregeneratedbyClustalX2.1.Andthephylogenetictreewasconstructed115inMEGA6.0with1000bootstrapreplicatesusingtheNeighbor-Joiningmethod.(B)Exon/intronorganizationofcitruspolyaminebiosynthesisgenes.Yellowboxesrepresentexons;blacklinesrepresentintronsandpurpleboxesrepresent5’UTR(Un-translatedRegion)and3’UTR.Thesizeofexonsandintronscanbeestimatedusingthescaleatbottom.(C)Chromosomallocationsofcitruspolyaminebiosynthesisgenes.Chrstandsforchromosome.Thelengthofthechromosome120canbeestimatedusingthescaleontheleft.1.3ExpressionpatternsofpolyaminebiosynthesisgenesinresponsetoexogenouspolyaminestreatmentsincitruscallusAccordingtoFigure2B,ADC,SAMDC4,SPDS1andACL5-1weredecreasedunderPuttreatment.TranscriptlevelsofSPDS2andSPMSwereslightlyincreasedat12hand6hbutdecreased125subsequently.TheexpressionlevelofSAMDC1andACL5-2weresignificantlyincreasedat12h,24hand24h,48h,butdecreasedatothertimepoints.SAMDC2exhibitedasimilarexpressionpatternwithSAMDC3whichwasslightlyincreasedatfirstthendecreased.UpontreatmentwithexogenousSpd,theexpressionlevelofADCwasincreasedrapidlyanddecreasedalittleatlast24hours,SPDS2wasincreasedat6hand12handthendecreased130significantly.SAMDC4washighlyup-regulatedat12hand72h.SPMStranscriptlevelwasslightlyincreasedat12h,butdecreasedat48handincreasedagainat72h.TheexpressionpatternsofSAMDC1,SAMDC2,SAMDC3,SPDS2andSPDS3weresimilar.Theywerealldown-regulatedduringexogenousSpdtreatmentandstayedalowerexpressionlevel.AndtheexpressionlevelofACL5-1wasdecreasedat6h,thenreachingthemaximumvalueat12h,but135sharplydecreasedat24hagainandfinallyreachedastablelevelwhichwassamewiththeinitiallevel(Figure2B).TheexpressionpatternsofpolyaminebiosynthesisgenesunderexogenousSpmtreatmentwerequitetheoppositewithSpdtreatment.TheexpressionlevelofADC,SPDS2andSAMDC4-4- 中国科技论文在线http://www.paper.edu.cnremainedaverylowlevelatfirst24hoursandslightlyincreasedat72hafterSpdtreatment.140SPMShadaveryhighexpressionlevelat24hbutdecreasedat48hand72h.SAMDC1,SAMDC2andSAMDC3hadthesameexpressionpatternsthatincreasedsignificantlyat12hand24hthensharplydecreasedat48hand72h.SPDS1stayedaveryhightranscriptlevelexceptat12h.ACL5-2transcriptlevelwasincreasedatfirst12hoursthendecreasedat24handslightlyincreasedagainatlast48hours.TheexpressionlevelofACL5-1underwentnegligiblechanges145afterSpmtreatmentexceptalittledecreasedat24h(Figure2B).1.4ExpressionpatternsofpolyaminebiosynthesisgeneunderabioticstressesandABATofigureouttherolesofthepolyaminebiosynthesisgeneinresponsetodifferentabioticstresses,150andtoexplorewhetherthesegenesparticipateintheABAsignalingpathway,atime-courseexpressionanalysisofthesegenesinresponsetocold(4°C),salinity(NaCl)andABAtreatmentwascarriedout.Undercoldstress,thetranscriptlevelsofADC,SPDS2andACL5-1wererapidlyincreasedat12hbutdecreasedsoon.TheexpressionlevelsofSPDS1,SAMDC3,SAMDC4stayedaveryhigh155levelundercoldstressexceptalittledecreasedat24hinSAMDC4.AndSAMDC1transcriptlevelwasonlyincreasedatlast24hours.ThemRNAlevelsofSPMS,SAMDC2andACL5-2werealldecreasedandkeptaverylowlevelduringcoldstress(Figure2).TheexpressiontrendofADCfollowingsalttreatmentwassimilartothatundercoldstress,beingup-regulatedgenerallyuponexposuretothestress.SPDS1andSPDS2wereslightlydecreasedatfirst24hoursandthen160increasedtothepeakat72h.ThetranscriptlevelofSPMSwasincreasedandACL5-2wasdecreasedalongwiththesalttreatment.SAMDC2andSAMDC3expressionlevelsweredecreasedatfirstthensharplyincreasedat72hand96h.ThetranscriptlevelofSAMDC4wasincreasedtothepeakat12hthendecreasedatlast48hours.SAMDC1andACL5-1sharedasimilarexpressionpatterns,thetranscriptlevelonlyincreasedatlast48hours.165TranscriptlevelsofADCandSPDS1wereremarkablyincreasedat72hand24hseparatelysamewithSAMDC4,butSPDS2,SPMS,SAMDC2andSAMDC3werenotresponsivetoABAastheirmRNAleveldidnotchangenoticeablyfollowingABAtreatment,andevenalittledecreasedcomparedwiththecontrol(0h).ACL5-1andACL5-2sharedthesamepatternsthatthetranscriptlevelbothhadaminorincreaseat12h,24hand72hthendecreasedat72h.Thetranscriptlevel170ofSAMDC1wasnotalteredalotbutdecreasedquicklyat72hsamewithACL5-1andACL5-2.Differentwithcoldandsalttreatment,mostpolyaminebiosynthesisgenesweregenerallynotinducedinABAtreatment(Figure2C).Overall,theexpressionprofilesofthepolyaminebiosynthesisgenesvariedundercold,salinityandABAconditions.-5- 中国科技论文在线http://www.paper.edu.cn175Figure2.TheexpressionpatternsofpolyaminebiosynthesisgeneundertreatmentsofPAsandstressesincitruscallus.(A)Tissue-specificexpressionpatternsofpolyaminebiosynthesisgenes.(B)Theexpressionpatternsofpolyaminesbiosynthesisgenesundertreatmentswithputrescine(Put),spermidine(Spd)andspermine(Spm).(C)Temporal-spatialexpressionpatternsofpolyaminebiosynthesisgenesin180citruscallusundercold,saltandABAtreatment.ThetranscriptlevelsofeachgenewereperformedusingqRT-PCRapproach,andtheresultswerecalculatedusingthe2−∆∆CTmethodwithActinasinternalcontrolgene.Thesamplesthattreatedwith0hweresetascontrol.Thelog2valuesofthecalculatedresultswereusedtogenerateaheatmap.Theintensityvaluebarwasshowedontheleft,andredandgreencolorsrepresenthigherandlowerexpressionlevelsthanthe185control,respectively1.5Cis-actingelementanalysiswithinthepolyaminebiosynthesisgenepromoters.Atotalof62differentkindsofcis-actingelementswereidentifiedinthepromoterregionsofthe190examined10polyaminebiosynthesisgenes(Table2).Themostfrequentoccurrenceofthecis-actingelementisinvolvedinlightresponsiveness.AndManycis-actingelementsinvolvedinstressresponsewerealsorevealed.Forexample,TC-richrepeats(defenseandstressresponse-6- 中国科技论文在线http://www.paper.edu.cnelement)wasfoundinallofthesegenesexceptSAMDC2andSAMDC3,HSE(heatstress-responsiveelement)wasnotfoundonlyinSAMDC3.Similarly,theLTRelement(Low195temperature-responseelement)andDRE/CRTwerealsofoundinallofthesegenesexceptSPDS2.Inaddition,WUN-motif,awound-responsiveelement,wasonlydetectedinSPDS1,SAMDC1andSAMDC3.Meanwhile,severalcis-actingelementsinvolvedinhormoneresponseweredetected.TheABREelementswhosecoresequenceisCGTCinvolvedinABAresponsewerepresentinthe200promoterregionsofallthepolyaminebiosynthesisgenes.GAREmotif,TATC-boxandP-boxinvolvedingibberellinswerefoundinthepromoterregionsofADC,SPDS1,SPDS2,SPMS,SAMDC2,SAMDC4andACL5-1exceptSAMDC1,SAMDC3andACL5-2.TCAelementrelatedtosalicylicacidresponseonlydidnotexistinADC,SAMDC3andSAMDC4promoterregions.CGTCA-motifandTGACG-motif,associatedwithMeJAresponse,existedinSPDS2,SPMS,205SAMDC2,SAMDC4,ACL5-1andACL5-2promoterregions.Incontrast,theauxinresponseelement,AuRR-coreandTGA-elementwereonlyfoundinSPMSandSAMDC4.TheethyleneresponsiveelementwasfoundinSPDS1,SAMDC2,ACL5-1andACL5-2(Table2).Also,somecis-actingelementsrecognizedbytranscriptionfactorswereexistedinthepromoterregions.CACG-motif,recognizedbyNACfamily[20,48],existedinallofthepolyamine210biosynthesisgenes.MBS,recognizedbyMYBfamily[49],alsoexistedinallofthesegenesexceptSAMDC3.W-box,recognizedbyWRKYfamily[50],werefoundinSPDS1,SAMDC2,SAMDC4,ACL5-1,ACL5-2promoterregions.And,ashasbeenmentionedbefore,theDRE/CRT,ABREandEREelementswhichwererecognizedbyCBF,ABFandERFtranscriptionfactorsalsoexistedinthepromoterregionsofpolyaminebiosynthesisgene.What’smore,thereweresomeother215cis-actingelementsexisted.Forexample,AREelement,essentialfortheanaerobicinduction,as-2-box,involvedinshoot-specificexpression,CCGTCC-boxandCAT-box,relatedtomeristemexpression,GCN4-motifandSkn-1,cis-regulatoryelementsinvolvedinendospermexpressionwereallexistedinoneormoregenespromoterregions(Table2).220225230-7- 中国科技论文在线http://www.paper.edu.cnTable2.Cis-actingelementsexistedinthe2kbupstreamregionofpolyaminebiosynthesisgenes.Organismmeansthespeciesthatthecis-actingelementwasfirstidentified.“+”,thesense235strand;“-”,theanti-sensestrand.Andthepositiveornegativesignbehindthenumberrepresentthatthenumberofcis-actingelementsexistedinthesensestrandoranti-sensestrandinthegene’spromoterregion.1.6PolyaminescontentundercodstressincitrusleavesandcallusAccordingtotheFigure3A,thecontentofputrescine(Put)wasmuchhigherthanspermidine(Spd)240andspermine(Spm)incitrusleavesundernormalconditions.AndSpmisalittlebitmorethanSpd.Alongwiththetreatment,thecontentofPutwasincreasedsignificantly.ThePutlevelon36hwasalmost2.5timesthanthecontrol(0h).ButthecontentofSpdshowedanegligibleincrease.AndSpmcontentwasdecreasedlittleatfirst,thenraisedsignificantlybuttheabsolutecontentwasstillmuchlowerthanPut.However,thechangepatternofcallusundercoldstresswasquite245differentwithleaves(Figure3B).Incallus,SpmandSpdcontentweremuchhigherthanPutwhichwascompletelyoppositewiththecontentinleavesundernormalconditions.AndSpmcontentwassignificantlyincreasedat12hbutPutcontentwasdecreasedremarkablyatthesametimepoint.ThentheSpmandPutcontentsreturnedtothenormallevel.AndthecontentofSpdwasslightlyincreasedatfirst12hoursanddecreasedatlast36hours.250-8- 中国科技论文在线http://www.paper.edu.cnFigureChangesofPAlevelsundercoldstressincitrusleafandcallus.BenzoyledfreePAsfrom4-week-oldunboltedplantsthathadbeentreatedwithcoldstressweredetectedbyhigh-pressureliquidchromatography(HPLC).Meanvalues±SDofthreeindependentexperiments255areshown.Asterisksindicatesignificantdifferences(*P<0.05,**P<0.01).2Disscussion2.1CharacterizationofpolyaminebiosynthesisgeneinsweetorangeInthisstudy,atotalof10polyaminebiosynthesisgeneswereidentified.AsshowinTable1,wegotthetranscriptnamesfromphytozomedatabase,thelocusnamefromtheCitrussinensis260Annotationprojectandmadethemone-to-onecorrespondence.Thenumberoftranscriptnameswereabitmorethanthenumberoflocusnamesmeantthatseveraltranscriptssharedasimilarcodingsequences(CDS),theonlydifferentbetweenthemwasthepositionoftheintronswhichwasconfirmedbythegenestructures(Figure1A).InArabidopsis,therearetwogenesencodesargininedecarboxylase,AtADC1andAtADC2[51].Butincitrus,thereisonlyonegenelocatedon265the8chromosomeandnamedCsADC(Cs8g07560)obviously.TherearethreespermidinesynthasesinArabidopsis:AtSPDS1,AtSPDS2andAtSPDS3(alsonamedAtSPMS).ButtheAtSPDS3encodesanovelsperminesynthaseanditisaparalogofpreviouslycharacterizedspermidinesynthases:AtSPDS1andAtSPDS2.Asfarasthespermidinesynthaseandsperminesynthaseincitrus,threegeneswereidentified:Cs5g05220,Cs7g08430andCs9g18030.The270orthologsoffirsttwogenesinArabidopsiswereAT1G23820(AtSPDS1)andAT1G70310(AtSPDS2).So,Cs5g05220andCs7g08430werenamedCsSPDS1andCsSPDS2.Cs9g18030wassupposedtobenamedCsSPDS3,becauseAtSPDS3encodesasperminesynthaseinArabidopsis,soincitrus,itwasnamedtoCsSPMSaccordingtoitspredictedfunction.TheconditionofS-adenosylmethioninedecarboxylasewascomplicated.TherearefourSAMDCgenesin275Arabidopsis,AT3G02470andAT3G25570werebothannotatedtoAtSAMDC,theAT5G15950andAT5G18930werenamedtoAtSAMDC2andAtSAMDC4,andtheAT3G17715wasannotatedtoAtSAMDC3butitwasapseudogene.Soincitrus,wedirectlyusedtheannotationofUniProtKnowledgebase[52].TheCs4g02260,Cs7g12410werenamedtoCsSAMDC1andCsSAMDC2respectively,Cs6g19210wasnamedtoCsSAMDC3sequentially.TheCs9g05430wasnamedto280CsSAMDC4becauseofitsArabidopsisorthologAtSAMDC4.AndCs4g06030andCs9g06680-9- 中国科技论文在线http://www.paper.edu.cnbothwereorthologswithAtACL5(AT5G19530)inArabidopsis.Forconveniencesake,wenamedtheCs4g06030andCs9g06680toCsACL5-1(ThermosperminesynthaseACAULIS5)andCsACL5-2accordingtoitspositiononchromosomes.Inspiteofthis,thisnamingmethodjustprovidesawaytounderstandthefunctionsandmakeaconvenientwaytoresearchthesegenes.285Furtherexperimentsandresearchedshouldbeperformedtoannotatethesegenesaccuratelyandproperly.2.2Thecis-actingelementsandthestressresponsesThepolyaminebiosynthesisgeneshavebeenobservedinresponsetodifferentenvironmentalstress.Forexample,inArabidopsis,ADC1ismainlyinducedbycold[53],whileADC2expression290isstronglyinducedbyseveralabioticstresseslikedehydration,highsalinityandK+deficiency[54,55].AtSAMDC2mRNAwasinducedbysalttreatmenttransientlyat10handinresponsetocoldafter5h.AtSPMSmRNAlevelshowedarapidresponsetosaltstress.Inresponsetocoldtreatment,theAtACL5mRNAappearedafter5h,andincreasedupto24h[56].Generally,stress-inducedgeneshaveacommoncharacteristicthatvariouscis-actingelementsexistedinthe295genepromoterregion.Actually,thecis-actingelementsthatfunctioninstress-responsivegeneexpressionhavebeenpreciselyanalyzedtoelucidatethemolecularmechanismsofgeneexpressioninresponsetostress.Forexample,ABRE[57]isinvolvedindroughtstressandABAsignaltransduction.HSE[58]isinvolvedinheatstress.Thedehydration-responsiveelement(DRE)withthecoresequenceCGACwasidentifiedasacis-actingpromoterelementthatregulatesgene300expressioninresponsetodrought,salt,andcoldstressesinArabidopsis[59].AsimilarmotifwasidentifiedastheC-repeat(CRT)andlow-temperature-responsiveelement(LTR)incoldinduciblegenes[60].Inthisstudy,theqRT-PCRanalysiswasperformedtodetectthegeneexpressionsofpolyaminebiosynthesisgeneunderabioticstresses(Figure2C).Someofthesegenesexpressionlevelswereup-regulatedordown-regulatedundercoldandsalttreatments,butsomewithoutany305change.Forexample,theexpressionlevelofADC,SPDS1andSAMDC4weresignificantlyincreasedandmaintainedahighlevelincitrusleavesduringcoldstress(SupplementFigure3),especiallySAMDC4,whichindicatedthatthesegenesactedaskeymodulatorsinpreventingdamageinsweetorangeasaresultofcold-inducedstresscondition.Andofcourse,theCRTelementwasexistedonthepromoterregionsofthesegenes.Butthereareexceptionsthatthe310transcriptlevelsofSPDS2,SPMS,SAMDC2,SAMDC3andSAMDC4didnotinducedandstayaverylowleveluponABAtreatmentthoughtheABREelementexistedinthesegenespromoterregion(Figure2C,Table2).AndthesamesituationhappenedinSAMDC2andACL5-2undercoldstress.DespitetheCRTelementwasfoundinthepromoterregion,theSAMDC1andACL5-2expressionleveldidnotchangealongwiththecoldtreatment(Figure2C,Table2).315Inaddition,accordingtotheTable2,thepromoterregionsofpolyaminebiosynthesisgenecontainedvariouscis-actingelementsasmentionedabovethatrecognizedbydifferenttranscriptionfactorswhichalsoinvolvedinstressresponse.ButfewstudiesfocusedonthismechanismandmostofthemwererelatedtoADC,therate-limitedenzymeinputrescine-10- 中国科技论文在线http://www.paper.edu.cnbiosynthesis.Forexample,aMYBtranscriptionfactor,PtsrMYB,caninteracttworegionsofthe320ADCpromoterandthetransgenictobaccolinesdisplayedhighermRNAlevelsoftheADCgene[16].AnothertranscriptionfactorWRKY70fromFortunellacrassifoliaalsointeractwiththepromoterofFcADCwhichcontainedtwoW-boxelementsandcanpromoteproductionofputrescineviaregulatingADCexpression.Andrecently,anewNACtranscriptionfactor,PtrNAC72,wasprovedtobindtothepromoterofPtADCandfunctionasarepressorofputrescine325biosynthesis[20].2.3ThepolyaminecontentandpolyaminebiosynthesisgenesRecently,alargenumberofstudiesshowsthatplantpolyaminelevelsareincreasedinresponsetosuchstressesashighandlowtemperatures,salinity,drought,hyperosmosisandhypoxia[8,25].Andpreviousstudyalsorevealedthattheexpressionlevelofpolyaminebiosynthesisgeneswere330inducedindifferentstressconditions.Overexpressionpolyaminebiosynthesisgenesmayelevatetheendogenouspolyaminecontentandimprovethestresstolerance[23,61].InArabidopsis,AtADC2isresponsiblefortheincreaseofADCactivity[62].AndtheenhancedSAMDCexpressionmayleadtothesperminecatabolism[63].Asmentionedabove,inthisstudy,thePutcontentinleaveshadthehighestcontentinsteadofSpmincallusofthethreepolyaminesduringthecold335stress(Figure3).Andcomparedwiththeexpressionpatternsinleavesandcallusduringcoldstress,theputrescinebiosynthesisgeneADCexpressionlevelinleaveswashigherthanthatincalluswhichmayexplainwhythePutcontentwasthehighestinleaves.Butonthecontrary,thesperminebiosynthesisgeneSPMSexpressionlevelincalluswasnotinducedbycoldstresswhichwasoppositewiththehighestcontentofspermineincallus.(Figure2,SupplementFigure3).So340theeffectofpolyaminedegradationmustbetakenintoconsiderationinordertoexplainthisresult.PlantPAOs(polyamineoxidases)catalyzetheoxidationofthepolyaminesubstratesspermidineandspermine.SpdandSpmmaybestronglyoxidizedbyPAOunderthesestressconditions.AndsomeofthePAOswerealsoinducedbyabioticstressesinsweetorange.WangLiu(2015)[64]revealedthatCsPAO4,functionasterminalcatabolismofSpdandSpm,wassignificantlyinduced345attheearlystageofcoldtreatment.Sothegeneexpressionprofileswerenotpreciselycorrelatedwithpolyaminecontents,whichmaybecauseofvariouslevelsofregulationofpolyaminemetabolismasdiscussedabove.3ConclusionPreviousstudieshaveinvestigatedandanalyzedthefeaturesandfunctionsofsomeofthe350polyaminebiosynthesisgeneinvariousspeciessuchasArabidopsis,rice,pepper,cucumber,potatoandLotusjaponicus[27,31,65-68].Tobemoreexact,thisisthefirsttimetoanalysispolyaminebiosynthesisgeneincitrussinensiscomprehensively.Inthisstudy,thetotalof18genesbelongsto10kindsofpolyaminebiosynthesisenzymesbygenome-wideanalysiswereidentified.Thephylogenetictreeandgenestructureweregeneratedwiththefulllengthof355Arabidopsispolyaminebiosynthesisgenesequencestounderstandthepotentialbiological-11- 中国科技论文在线http://www.paper.edu.cnfunctionsbetterduringevolutionaryprocesses.Chromosomallocationanalysesshowedthat18geneswerelocatedon6chromosomesofall9chromosomesincitrus.Theanalysisofpromoterregionsofeachpolyaminebiosynthesisgeneindicatedthatthesegenesmaybeinvolvedinabioticstressesandplanthormonesresponses.Expressionpatternsofthepolyaminebiosynthesisgene360underexogenoustreatmentswithPut,SpdorSpmshowedthatthesegenewereindeedinvolvedinpolyaminebiosynthesisandmetabolism.AndthecontentofthePut,SpdandSpmundernormalgrowthconditionsandcoldstresswasdetectedtofurtherfigureouttheroleofpolyamines.Accordingtotheresultsandpreviousresearches,WeconsideredthattheADCwasoneofthemainrate-limitingenzymesduringcoldstress,wearecurrentlyinvestigatingtheroleofADCin365polyaminebiosynthesisundercoldstressconditionsbyusingtheinteractionsofgenesorproteins.4Materialsandmethods4.1Plantmaterials,growthconditionsandtreatmentsCitruscalluswasculturedonMTmediumandsubculturedeverythreeweeks.Callusgrowthof10daysonthemediumwasusedfortreatment.ThecalluswassubjectedtotreatmentswithPut,Spd,370Spm,abscisicacid(ABA),sodiumchloride(NaCl)andcold(4°C).ForPAstreatment,calluswasculturedat25°CindarkontheMTmediumaddedwith2.0mMPut,1.0mMSpdor2.0mMSpm,followedbysamplingat0,6,12,24,48and72haftertreatment.Forstresstreatments,thecalluswasculturedat25°CinthedarkonMTmediumsupplementedwith100μMABAor100mMNaClrespectively,or4°CindarkonMTmediuminalowtemperatureincubator.Calluswas375collectedat0,12,24,48,72and96hafterthecorrespondingtreatments.Inaddition,samplesatthe0htimepointwereusedascontrolsforallthestressconditions.Toinvestigatetissue-specificexpressionprofiles,roots,stemsandleavesfromsix-week-oldseedlingsofsweetorangewerecollected.Andpulpandpeelswereseparatedfromcitrusfruitspickedinorchards.Allthesampleswerefrozeninliquidnitrogenimmediatelyaftercollected,andstoredat-80℃forfurtheranalysis.380Atleastthreebiologicalreplicateswereperformedforeachsample.4.2IdentificationandSequenceanalysisThepolyaminebiosynthesisgenesequences(includingcodingsequence,transcriptsequenceandgenomesequence)ofArobidopsisthalianaandOryzasativawereobtainedfromtheTAIR[34]and385RiceGenomeAnnotationProject[35].Toidentifythepolyaminebiosynthesisgeneincitrus,thepublishedArabidopsisrelatedgenesasquerysequencesweresubjectedtoatblastnsearchagainsttheCitrussinensisgenomedatabase[36]andthephytozome[37].Tofurthercharacterizethecitruspolyaminebiosynthesisgenes,anonlineExPasyprogram[38]wasusedtocalculatethelengthofaminoacid(aa),molecularweight(Wt),andisoelectricpoint(PI)ofeachprotein.390-12- 中国科技论文在线http://www.paper.edu.cn4.3Phylogeneticanalysis,Genestructure,ChromosomallocationandPromoteranalysisGenestructurepredictionwasgeneratedusingtheonlinewebserverGeneStructureDisplayServer2.0[39].TheresultsweremanuallyadjustedaccordingtotheGT-AGrule.AndtheClustal395Xprogram(version2.1)andGeneDocsoftware(version2.7)wereusedtogeneratemultiplealignmentsoftheproteinsseparatelyaccordingtotheirfunctionsinpolyaminebiosynthesispathway.AndthephylogenetictreesweregeneratedwithMEGAprogram(version6.06)usingtheNeighbor-Joining(NJ)algorithm[40].Bootstrapanalysiswasperformedusing1,000replicatesinMEGAtoevaluatethereliabilityofdifferentphylogeneticgroups[41].MapDraw400tool[42]wasusedtodesignthemapsofphysicallocationofthesegenesandthestartingpositiononeachchromosomewassearchedandconfirmedbyBlastNagainsttheCitrussinensisAnnotationproject[36].Theduplicationeventsofpolyaminebiosynthesisgeneincitrusweresearchedbasedonpreviousparameters:e-value<1e−10andidentity>90%[43].The2000bpupstreamofthestartcodonwastakenastheputativepromoterregionintheCitrussinensis405Annotationproject[36]andsearchedforcis-actingelementsinPlantCAREdatabases[44].4.4RNAisolationandexpressionanalysisTotalRNAswereextractedaccordingtotheinstructionsofTrizolreagent(TaKaRaBioGroup,Japan).ThefirststrandcDNAsweresynthesizedusingPrimeScriptTMPTreagentKitwithgDNA410Eraser(TOYOBOCO.,LTD,Japan).Thegene-specificprimers(supplementalTableS1)weredesignedusingPrimer5.0withmeltingtemperaturesof56-58℃,primerlengthsof19-20bpsandampliconlengthswith100-200bp.Quantitativereal-timePCR(qRT-PCR)wasperformedinAppliedBiosystemsQuantStudio™7FlexReal-TimePCRSystem(ThermoFisherScientific,America)usingtheQuantiNovaTMSYBR®GreenPCRKit(TRANSGENBIOTECH,China).415Eachreactionsystemcontained5μL2×SYBRGreenPCRMasterMix,1μLQNROXReferenceDye,0.35μLofeachprimer,2.3μLwater,andappropriatelydilutedcDNA1μL.Thethermalcyclingconditionswere95°Cfor2minfollowedby40cyclesof95°Cfor5sec,60°Cfor10sec.ThecitrusACTINwasusedastheinternalreferencegene.The2−∆∆CTmethodwasusedtocalculatetheexpressionlevelofdifferentsamples[45].Therepresentativedataareexpressedasthe420meanvalues±standarddeviation(±SD).Eachtreatmentwasrepeatedthriceindependently.TheclusteranalysisandheatmapsweregeneratedusingCluster3.0andTreeviewsoftware.4.5QuantitativeanalysisoffreepolyaminesFrozentissuesweregroundinliquidnitrogenusingamortarandpestle.About0.3gpowderswere425extractedin5%precoolingHCLO4withDTT(0.05g/100ml)basedonthemethoddescribedbyFloresGalston(1982)[46]withmodifications.Sampleswerecentrifugedat12000rpmfor20minafter1hincubationinanicebath.Thesupernatantphase,containingthefreepolyaminefraction,wasstoredfrozenat-20°Cinplasticvials.Derivationandbenzoylationofthepolyamineswere-13- 中国科技论文在线http://www.paper.edu.cnperformedasdescribedbyFu.Chen.Wang,etal.(2011)[47].HPLCanalysiswasperformedwith430aprogrammableAgilentTechnologies1200seriesliquidchromatograph(AgilentTechnologies,US).ThesolventsystemconsistedofHPLC-grademethanol:water,55%:45%(v/v,A:B)to95%:5%in10minataflowrateof0.7ml/min.PAslevels,expressedasnmol/gfreshweight(FW),weretheaverageofthreereplicatesforeachsample.4.6StatisticalAnalysis435Allexperimentaldataareaveragesofatleastthreeindependentreplicates.ThedatawerestatisticallyevaluatedbyapplyingFisher’sLSDtest,intheANOVAprogramofSPSS(IBMSPSSsoftware,version22),takingP<0.05(*)andP<0.01(**)assignificantlydifferent.References[1]YangB.,WuJ.,GaoF.,etal.Polyamine-inducednitricoxidegenerationanditspotential440requirementforperoxideinsuspensioncellsofsoybeancotyledonnodecallus[J].Plantphysiologyandbiochemistry:PPB/Societefrancaisedephysiologievegetale,2014,79:41-47.[2]KimD.W.,WatanabeK.,MurayamaC.,etal.PolyamineOxidase5RegulatesArabidopsisGrowththroughThermospermineOxidaseActivity[J].Plantphysiology,2014,165(4):1575-1590.445[3]FortesA.M.,CostaJ.,SantosF.,etal.ArginineDecarboxylaseexpression,polyaminesbiosynthesisandreactiveoxygenspeciesduringorganogenicnoduleformationinhop[J].Plantsignaling&behavior,2014,6(2):258-269.[4]ConaA.,CenciF.,CervelliM.,etal.Polyamineoxidase,ahydrogenperoxide-producingenzyme,isup-regulatedbylightanddown-regulatedbyauxinintheoutertissuesofthemaize450mesocotyl[J].Plantphysiology,2003,131(2):803-813.[5]YodaH.,YamaguchiY.,SanoH.Inductionofhypersensitivecelldeathbyhydrogenperoxideproducedthroughpolyaminedegradationintobaccoplants[J].Plantphysiology,2003,132(4):1973-1981.[6]SharmaH.S.S.,FlemingC.,SelbyC.,etal.Plantbiostimulants:areviewontheprocessingof455macroalgaeanduseofextractsforcropmanagementtoreduceabioticandbioticstresses[J].Journalofappliedphycology,2013,26(1):465-490.[7]LiuJ.H.,NadaK.,HondaC.,etal.Polyaminebiosynthesisofapplecallusundersaltstress:importanceoftheargininedecarboxylasepathwayinstressresponse[J].Journalofexperimentalbotany,2006,57(11):2589-2599.460[8]LiuJ.H.,KitashibaH.,WangJ.,etal.Polyaminesandtheirabilitytoprovideenvironmentalstresstolerancetoplants[J].PlantBiotechnology,2007,24(1):117-126.[9]YiuJ.C.,JuangL.D.,FangD.Y.T.,etal.Exogenousputrescinereducesflooding-inducedoxidativedamagebyincreasingtheantioxidantpropertiesofWelshonion[J].SciHortic,2009,120(3):306-314.-14- 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