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Metabolism of Reactive Oxygen Species in the Cytoplasmic Male-Sterile Cotton Anther

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Metabolism of Reactive Oxygen Species in the Cytoplasmic Male-Sterile Cotton Anther

    Metabolism of Reactive Oxygen Species in the Cytoplasmic Male-Sterile Cotton Anther AgriculturalSciencesinChina

    2007,6(3):275280

    Availableonlineatwwwsciencedirectcom

    一?

    ScienceDirectMarch2007

    MetabolismofReactiveOxygenSpeciesintheCytoplasmicMaleSterile

    CottonAnther

    JIANGPeidong,ZHUYunguo,,WANGXiaoling,ZHUWei,ZHANGXiao

    quan,XIEHaiyanand

    ?NGXuede1

    

    DepartmentofAgronomy,CollegeofAgriculture&Biotechnology,ZhejiangUniversity,Hangzhou310029,P.R.China

    2CollegeofLifeScience&Technology,TongjiUniversity,Shanghai200092.P.R.China

    Abstract

    Reactiveoxygenspecies(ROS)inplantcell,includingsuperoxide(o/),hydrogenperoxide(H2O2),andmalondialdehyde

    (MDA),arethoughttobeimportantinduciblefactorsofcellapoptosisifexcessivelyaccumulatedincells.Toelucidate

    themetabolicmechanismofROSproductionandscavenginginanthersofthecytoplasmicmalesterile(CMS)cotton,

    CMSline,maintainer,andhybridF1anthers,wereemployedforstudyingtherelationshipbetweenCMSandmetabolism

    ofROS,bycomparingROSchangesinthesterileandfertileanthersatdifferentdevelopmenta

lstages.Theresults

    showedthatduringtheabortionpreliminarystage(sporogenouscelldivisionstage),anthersofCMSlinehadhigher

    contentsof0,H2O2,andMDAthanthoseofmaintainerorhybridF1.Simultaneously,thehigheractivitiesofsuperoxide

    dismutase(SOD),catalase(CAT),andperoxidase(POD)jnscavengingROSweremeasuredintheanthersoftheCMS1ine.

    indicatingthatanincreaseofROSinanthersofabortionpreliminarystagehadaninducibleeffectontheantioxidant

    enzymes.ButduringtheabortionpeakofCMSanther(pollenmothercellmeiosisstage),ontheonehand,contentsof0,

    H2O2,andMDAwereextraordinarilyhighinCMSanthers,ontheotherhand,theactivitiesofSOD,CAT,andPODwere

    excessively1ow,whichdisruptedthebalancebetweentheproductionandeliminationofROSand1edtopollenmother

    cellsapoptosisatthisstage.Inthefollowingtwostages(uninucleatemicrosporestageandmaturepollenstage),the

    contentsof0andH2O2intheabortedantherswereapproximatedtocontentsinthefertileanthersofthemaintainerand

    hybridF1.However,MDAcontentswerecontinuouslyraisedandenzymicactivitiesofSOD,CAT,andPODwere

    consistentlydecreasedinsterileanthers,whichindicatedthatROSstillhadharmfuleffectsontheanthersafterthe

    apoptosisofthemalecells.ExcessiveaccumulationofO,H202,andMDAandsignificantreductionofROSscavenging

    enzymeactivitieswerecoinstantaneouswithmalecellsapoptosisintheanthersofthecottonCMSline.Butwhenthe

    restorergenewastransferredintotheCMSline,excessiveproductionofROScouldbeeliminatedintheanthersofhybridF.

    Keywords:cotton,cytoplasmicmalesterility,reactiveoxygenspecies

INTRODUCTION

    Cytoplasmicmalesterility(CMS)isamaternallyinher- itedtraitthatisunabletoproduceorreleasefunctional pollens,whichplayanimportantroleintheutilization ofhybridvigor.CMSisassociatedwithabnormalre

    combinationOfmitOchOndrialgenome.however.its mechanismisnotveryplain(LinkeandB6rner2005). Mitochondrionprovidesenergyforallkindsofactivi

    tiesincellsthroughrespiration.Meanwhile.mitochon

    driongeneratesreactionoxygenspecies(ROS),which ThispaperistranslatedfromitsChineseversioninScientiaAgriculturaSinica

    J1ANGPei_dong,E.mail:jiangpd@126com;Co~espondenceWANGXue-de,E-mail:xd

    wang@zju.educn

    ~2007,CAASAll^ghtsresentedPublishedbyElsevierLtd

276JIANGPeidongeta1.

    maybedamagingtotheorganismsbythereactionof respirationchain.ROSincludesuperoxide(O),hy

    drogenperoxide(H,O.),hydroxylradical(?OH),and thesingletoxygen(.O2),inwhichO,andH2O2arethe mainsourceofROS.IfROSproductioninorganisms cannotberemovedfromcellsrapidlyandeffectively, ROSwillbeaccumulatedincellsandcanpossiblylead toapoptosisrMaxwelleta1.2002).Excessiveaccu

    mulationofROSwasreportedintheCMSanthersof rice(ChenandLiang1991;Lia1.2004),wheat(Zhao fa1.1996).andmaizefDuaneta1.1996).Nevertheless. themetabolismofROSgenerationandscavenging,and itsrelationshiptoCMSaswel1.isstillnotclear.Couon CMSwiththecytoplasmofG.harknessiiBrandegeeis

    agenetictraitthatiscontrolledbycytoplasmicgenes andrepressedbynuclearrestorergenes.TheCMS cottonanthersbegintodegenerateinthesporogenous celldivisionstagesandreachabortionpeakinthepol

    lenmothercellmeiosisstage.sofhatitcannotform tetrade.whichisnamedasnonpollenmalesterility

    (WangandPan1997),andisappropriateforstudying theCMSmechanism.Sointhisarticle.thedynamic changesontheROScontentsandROSscavenging enzymicactivityatpreliminaryabortion,peakabortion, andlaterabortionstagesofmalesterileantherswere studied.toinvestigatetherelationshipbetweencotton CMSandROSmetabolism.

    MATERIALSANDMETHODS

    Plantmaterialsandenzymesolutionpreparation ACMSlinewiththecytoplasmofG.harknessif Brandegee,JiA,itsmaintainerline,JiB,andhybridFl, JiAxZhedastrongrestorer,weregrownintheexperi

    mentalfieldsatthefarmofZhejiangUniversity,China duringthesummerseasonsfrom2003to2005.An

    thersatparticulardevelopmentalstageswerepicked outfromthecorrespondingyoungbudsandcollected forexperiments.Thewholeantherdevelopmentwas dividedintofourstagesaccordingtothemicrosporo

    genesisprogressbycheckingunderthemicroscope, thatis,sporogenouscelldivisionstage,pollenmother cell(PMC)meiosisstage,uninucleatemicrosporestage, andmaturepollenstage.Thefirstfullyexpandedleaf wasusedforcontrointhedeterminationOfROScon

tentsandantioxidantenzymicactivities.Freshlycol

    lectedanthersandleaves(1g)weregroundinliquid nitrogen.andthenmixedin10mLprecooledextraction buffer[0.1MsHCl,pH7.8,0.5mMEDTA,0.1%

    (w/v)polyVinylpyrrolidone(PVP).Afterhomoge

    nization,celldebriswaspelletedbycentrifugationat 2000xgfor10min.Thesupematantwasrecentrifugated atl2000xgfor10minandcollectedforthemeasure

    mentofenzymicactivity.

    Determinationof02=generationrate,H2O2,and MDAcontents

    O2generationratewasdeterminedasdescribedby ElstnerandHeupel(1976),bymonitoringthenitrite formationfromhydroxylamineinthepresenceofO,', whichwasnotedasnmolmg.protein20min~.H,O,

    contentswasmeasuredbymonitoringtheA4l5ofthe titaniumperoxidecomplexfollowingthemethodof Pattersoneta1.(1984),whichwasnotedasnmolg. F.MDAcontentwasmeasuredasdescribedbyHeath andPacker(1968),whichwasnotedasnmolmg. protein.

    DeterminationofROSscavengingenzyme

    activities

    SODactivitywasassayedbymeasuringtheinhibition ofphotochemicalreductionofnitrobluetetrazolium (NBT)accordingtothemethodofWangeta1.(1983). OneunitofSODactivitywasdefinedastheamountof enzymerequiredtocause50%inhibitionofthereduc

    tionofNBT.PODactivitywasdeterminedaccording toKochbaeta1.f1977).OneunitofPODactivitywas

    definedastheincreaseof0.0lODperminute.CAT activitywasmeasuredbythemethodofJiangeta1. rl982).0neunitofCATwasdefinedasthedecrease Of0.1ODperminute.

    RESULTS

    02=generationrate,H2O2andMDAcontentsin sterileandfertileanthem

    SubtlecontentsofROSareessentialtocellmetabolism. @2007.GAAS.AllnghtsroservedPublishedbyElsevierLtcl

MetabolismofReactiveOxygenSpeciesintheCytoplasmicMale

    SterileCottonAnther277

    However.ifROSareexcessivelyaccumulated,they wouldcauseaseriesofdamagetocells(Forsmark- Andreeeta1.1997).Therewerenoobviousdiffer. encesinO.'productionrate,H,O,,andMDAcontents intheleavesamongthethreematerials(JiA,JiB,and F)(Table1),whereas,significantdifferenceswere foundintheanthers(Fig.1).O,'productionrateand H,O,contentshavethesamechangetendency.During thesporogenouscelldivisionstage(stage1)andPMC meiosisstage(stage2),thegenerationrateofO,and contentsofHOinthesterileline(JiA)increasedwith alargescope.whichwas416.1.878.1%and233.0. 438.2%morethanthoseinmaintainer(JiB), respectively.Butintheuninucleatemicrosporestage (stage3)andmaturepollenstage(stage4),O,pro- ductionrateandHOcontentsobviouslyreducedin thesterileanthers,andtherewerenomoresignificant differencesbetweenJiAandJiB.Itwasworthyof

    noticethatwhenthesterilelinewashybridizedwiththe restorer,excessiveproductionofO,andH,O,inhy- bridF,couldbescavengedandkeptconsistentwith themaintainerintheantherdevelopmentalprocessbe. causeoftheintroductionofrestorergene.Itwaschar. acterizedinthisstudythattheanthersofCMScotton begandegradationinthesporogenouscelldivisionstage andreachedabortionpeakinPMCmeiosisstage.and resultedintheantherinthefollowingdevelopmental stagesnotcontainingmalecellsanylonger(Wanget f.1998).Sointheabortionstage.increasein0 generationrateandH.O.contentswithalargescale wasparalleltosubstantivemalecelldeath. SeenfromFig.1.changeoftheMDAcontentshada similarpatternbetweenJiBandF,.However,sterile lineJiAhadhighercontentsthanthatofJiBandF,in eachdevelopmentalstage.MDAcontentsinsterilean. therswereonasustainedincreaseduringthewholean. therdevelopment,whichwas210.9,411.5,5l3.3, and509.2%higherthanthatinmaintaineranthersat thefourstages.respectively.Itwasindicatedthatthe toxicityofMDAtotheantherspossiblysustainedlonger thanthatofOandH.O.,orhadahysteresis.

    ActivitiesofROSscavengingenzymes(SOD,

    CATandPOD)inthesterileandfertileanthers SOD,CAT,andPODarerecognizedastheimportant antioxidantenzymes,whicharebeneficialtomaintain- ingcellsinthenormalphysiologicalredoxstateand preventingthemfromdamage.SODcancatalyzethe conversionofO2toH2O2andO2.CATandPODcan

Table102prodcutinrate,H2O2andMDAcontentsinthecottonleafofCMSline(JiA),maintai

    ner(JiB)andhybrid(F1)

    '.'

    ?''JiB1-JiA

    Thesameasbelow.

    234234

    AntherdeveIopmentalstages

    234

    Fig.10igenerationrateandH2O2andMDAcontentsintheanthersofCMSline(JiA),maintai

    ner(JiB)andhybrid(F1)duringdifferent developmentalstages.1,sporogenouscelldivisionstage;2,pollenmothercellmeiosisstage;

    3,uninucleatemicrosporestage;4,mature pollenstage.

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    decomposeH2O2toO2andH2O.Nosignificantdiffer

    encesintheSOD,CAT,andPODactivitieswasfound amongtheleavesofJiA,JiB,andF(Table2),but distinctdifferencesexistedintheanthers(Fig.2,.As showninFig.2,duringtheprocessofanther

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    Antherdevelopmentalstages 234

    Fig.2TheactivitiesofSOD,CATandPODintheanthersofCMSline(JiA),maintainer(JiB)an

    dhybrid(F1).1,sporogenouscelldivision stage;2,pollenmothercellmeiosisstage;3,uninucleatemicrosporestage;4,maturepollensta

    ge.

    4B

    Fig.3Pollenmothercells(PMCs)andsacinthesterileanthers.A,dyingPMCsinthesterileant

    hers(x264);B,nonpollensacinthe

    sterileanthers(xl32).

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