Study
AgriculturalSciencesinChina
;2005,4(2):94—100
;://www.ChinaAgriSci.com
;StudyoftheProtectiveEffectsinPEPCTransgenicRice
;ZHANGQian1?2,JIAODe—mao1,LINGLi—li?,ZHANGYun—hua2andHUANGXue—qing
;JJiangsuAcademyofAgriculturalSciences,Nanjing210014,P.R.China
;2AnhuiAgriculturalUniversity,nefei230036,P.R.China
;JCollegeofLifeScience,SichuanUniversity,Chengdu610064,P.R.China
;ThediurnalcourseofchlorophyllfluorescenceparameterandactiveoxygenmetabolismofflagleavesinPEPCtransgenic
;anduntransformedriceKitaakewerestudied.Theresultsshowedthatthephotosyntheticrateunderhighlightintensity
;hasbeenincreasedby50%andphotoinhibitionofphotosynthesisinPEPCtransgenicwasalleviatedaftertheintroduction
;ofPEPCgenefrommaizeintorice.ItwasdemonstratedthattheincrementofphotosynthesisinPEPCtransgenicwas
;relatedtotheintroductionofPEPCgeneusingspecificinhibitorofPEPC.Photoinhibitionofphotosynthesisindifferent
;genotypesexistsatnoonundernaturalcondition.PEPCtransgenicriceexhibitedalessdecreaseinF/Fm,aless
;photoinhibitionandahigherefficiencyoflightenergyconversiontochemicalenergyandlowerthermalenergydissipation.
;Theseresultsprovidedthephysiologicalbasisonthemechanismoftolerancetophotoinhibitionandricebreedingwith
;highphOt0syntheticefficiency.
;Keywords:PEPCtransgenicrice,Photoinhibition,Chlorophyllfluorescence,Activeoxygenmetabolism,Diurnalchange
;Withrapiddevelopmentoftransgenictechnologyin
;recentyears,usinganAgrobacteriumtumefaciens-me-
;diatedtransformationsystem,themaizegeneencoding
;PEPC,oneofthekeyenzymeinCdphOtOsynthetic
;pathway,hasbeenintroducedintoCcropriceand
;high-levelexpressionofthePEPCgenewasobserved
;inthetransgenicriceplant【l】.Itexhibitedhigherlight—
;saturatedphotosyntheticrate(55%),carboxylationef-
;ficiency(50%)thanthoseoftheuntransformedWT.
;Atthesametime,theCO2compensationpointdecreased
;by27%f?.】andthegrainyieldperplantincreasedby
;10—30%【4】.indicatingthebroadprospectsforenhanc—
;ingtheproductivityofricecropsthroughgenetic ;engineering.
;Photoinhibitioniscausedwhenexcitationenergypro—
;ducedbyphotoreactionismorethanwhatcanbeused ;forphotosynthesis,resultinginthedecreaseofphoto—
;syntheticefficiencyandthedecadenceofquantumef- ;ficiencyandPSIIphotochemicalemciency.Inbotany. ;photoinhIbitionwasprovedtobeawidelyexisted ;phenomenon.Therewerenumerousreportsaboutthe ;mechanismofphotoinhibition.Nowithasbeenknown ;thatphotoinhibitionmechanismincludetwoaspects, ;namely.inactivationandbreakageofPSIIreactioncen—
;terandprotectivemechanismofphotosyntheticappa—
;ratus[5-7].
;OurpreviousresearchdemonstratedthatPS ;IIphotochemicalefficiency(F/F),photochemical ;quenching(qP)inPEPCtransgenicricedecreasedless ;thanthoseinuntransformedwidetypeKitaakeafter ;thetreatmentsofhighlightintensity(3h)and ;photooxidizerMV.indicatingthestrenghtheningcapa- ;bilityoftolerancetophotooxidationofPEPCtransgenic ;rice【2】.Inthispaper.withPEPCtransgenicriceas ;CorrespondenceJIAODe—mao,TeL/Fax:86—25—84391939,E—mail:photosyn@public1.ptt.js.ca
;
;StudyoftheProtectiveEffectsinPEPCTransgenicRice95 ;material,thediurnalchangesofthechlorophyllfluo—
;rescenceparametersandrelativeindexesofactiveoxy—
;genmetabolismandthexanthophyllcycleunderstrong ;lightwithhightemperatureinsummerwerestudied ;andphotoprotectivestrategyaboutPEPCtransgenic ;riceunderphoto—inhibitionconditionwereprimarily ;discussed.Theresearchprovidedthenewevidencefor ;elucidatingonthetraitsoftolerancetophotoinhibition ;inCdphotosyntheticenzymetransgenicriceandex—
;ploredthenewpathwayfortheresearchaboutthere—
;lationshipbetweenbiologytechniqueandphysiological ;breeding.
;MATERIALSANDMETHODS
;Plantmaterials
;ThethirdgenerationofPEPCtransgenlcriceand ;untransformedwidetype(WT,Kitaake)wereprovided ;byProf.KuinWashingtonStateUniversity.By ;propagating,detectingandidentifyinginevery
;generation,wehavegottenthe7thgenerationofstable ;materials,inwhichPEPCactivityequaled1400pmol ;m’s...asmuchas20foldcomparedtothatinwide—
;typerice.Allriceplantsweregrowninpotsinnet—
;houseofInstituteofGeneticsandPhysiology,Jiangsu ;AcademyofAgriculturalScienceofChina.Different ;genotypesofriceweresownindifferenttimesand ;sampledfrommatureflagleavesatheadstage.There ;were15potsperplant,fivehillsperpotandoneseed—
;ingforeachhil1.Theplantswerecultivatedusingstan—
;dardmanagementpracticesforricecrop. ;Diurnalchangesofthephotofluxdensityand ;temperatureinthesiteofmeasurement
;Fig.1showedthediurnalchangesofthephotoflux ;density(PPFD)andtemperature(T)attimeofoneday ;atthesiteofmeasurement.
;Measurementof02evolutionrateandtreatment ;OfDCDP
;02evolutionrateassaysRiceflagleafdiscswitha ;diameterof1cmwerecutintosmallpiecesandelimi—
;natedgasbyreducingpressureinthesolutioncontain—
;l800
;1600
;目1400
;呈l200
;金1000
;800
;600
;宝
;葛
;量
;8:00l0:0012:0014:00l6:00l8:00
;8:00l0:00l2:0014:00l6:00l8:00
;Thetimeoftheday
;Fig.1Diurnalchangesofthephotofluxdensity(PPFD)and ;temperature(T)atthetimeofoneday(July10)in ;thesiteofmeasurement
;ing100mmolL..Hepes—KOH(pH7.8)and20mmolL
;NaHCO,.TheOevolutionrateofchloroplastswas ;measuredwithaSP一2typeoxygenelectrode(madein ;InstituteofShanghaiPlantPhysiology,ChineseAcade—
;myofSciences1underPPFDof800pmolm一s’at
;25.C.
;TreatmentoftheinhibitorDCDPTheuppersur—
;faceofintactflagleavesweresmearedbythesolution ;containing200mmolL’.peculiarinhibitor3,3一dichloro一
;2一(dihydroxyphosphinoylmethy1)propenoate(DCDP) ;andl%(v/v1Tween一80.Thecontrolleavesweretreated ;withdistilledwaterwith1%fv/v1Tween一80.Plants
;wereplacedindarkorilluminatedbyweaklightunder ;PPFDOf20—30pmolm’s’fortwohourscausing
;distilledwaterandtheinhibitorfilteringintotheleaves. ;Measurementofchlorophyllfluorescencepara- ;meters
;Thechlorophyllfluorescenceparametersweremea—
;suredbyFMS一2fluorescencemeter(Hansatech,UK) ;asdescribedbyGentyt引.Withfunctionallightf1200
;pmolms..,30s),saturatingradiationpulse(4000 ;lamolms，,30s)andflash(0.9lamolms..,30s), ;eachmaterialwasmeasuredthreetimes.Thechloro—
;phyllfluorescenceparameterswerecalculatedaccord—
;
;ZHANGQianetal
;ingtothefollowingequations
;F/F
;m(F一F0)/Fm
;qP=(F’F)/(F’一F0’)
;qN=1一(F’-F0’)/(Fm-F0)
;Measurementoftheindexesofactiveoxygen ;metabolism
;Riceflagleaveswereharvestedindifferenttimesatthe ;samedayandweresentimmediatelyinliquidnitrogen ;toreserve.Using50mmolLpH7.8phosphatebuffer ;asexactionbufferaddingalittlePVP,0.5gleaveswere ;groundinice—bath.Thehomogenatewascentrifugedat ;4.C.10000gfor1,5min.Thesupernatantwasused ;forthemeasurementoftheindexesofactiveoxygen ;metabolism.Thecontentofproteinwasdetermined ;accordingtoBradfordt10】usingAlbuminbovineas
;standard.Peroxidase(POD)activitywasassayedby ;themethodofKochbaetalt”.OneunitofPODactivity
;isdefinedastheincreaseof0.1oDperminute.Supero—
;xidedismutase(SOD)activitywasassayedbythe ;methodofGiannopolitisandRiest.OneunitofSOD ;activityisdefinedastheamountofenzymethatcaused ;50%inhibitionoftheinitialrateofNBTreduction.OT ;evolutionratewasmeasuredaccordingtothemethod ;0fgetal【31.MeasurementofMDAwasaccording
;t0themethodofLingetalt141.
;Measurementofdiurnalvariationofabsorption ;at505nm
;AccordingtothemethodofBilgereta1.【51.duetothe
;absorptionofthexanthophyllswasat505nm,wecan ;testthevariationofthecontentofxanthophyllsbythe ;changesofabsorbanceatthisphase.DifferentDart0f ;thesameleafwassampledindifferenttimesofoneday ;andwassentimmediatelyintoliquidnitrogen.Leaf ;bladeswerecutintopiecesinthedarkandhomog—
;enizedusing80%acetone.Thehomogenatewascen—
;trifugedat200gfor15min.Thenusing80%acetone ;asacontrol,theopticalabsorptionofsupernatantwas ;assayedat505nmand652nm(theabsorptionof ;chlorophyl1).Becausediurnalchangesofthecontent ;ofxanthophyllsarenotobvious,wecancomparethe ;diurnalchangesofthecontentofxanthophylls(shown ;asratioofA505/A652)usingchlorophyUasacontro1. ;RESULTS
;Effe~ofDCDPtreatmentson02evolutionrate ;indifferentgeno~pesofriceundernaturaland ;photoinhibitionconditions
;ShownasFig.2,OevolutionrateinPEPCtransgenic ;ricewasincreaseedby50%comparedwiththatin ;untransformedwidetype.Toprovewhethertheincre—
;mentofOevolutionrateisrelativetotheintroduction ;ofmaizePEPCgene.differentgenotypesofricewere ;treatedbyPEPCpeculiarinhibitor(DCDP1underthe ;conditionsofnatureandDh0t0inhibiti0n.Aftertreat—
;mentitcanbeseenthatOevolutionrateinPEPC ;—Nature
;PEPCtransgenicriceKitaake
;Datashownmean~s.e.(n=3).Natare—Naturecondition;Ph0t0jnhibjti0n—
;Photoinhibitioncondition;Nature+DCDP—Undernatureconditionricewas ;treatedbyPEPCpeculiarjnhibitorDCDP:Ph0t0inhIbjtion+DCDP——Under
;photojnhibitionconditionricewastreatedbyPEPCpeculiarinhibitorDCDP.
;Naturecondition:Sampleat10:00lnthemorningataclearday;Photolnhibition
;condition:AttachedleaveswereplacedwithCO,一freeaircontaining1%O,.under ;PPFDof10oolamolill一21from8:00to10:00for2h.
;Fig.2EffectofDCDPtreatmenton02evolutionrateinPEPC ;transgenicriceandwildt3,peKitaakeundernatural ;andphOt0inhibiti0nconditions
;transgenicricedecreaseddrasticallyandreachedto ;untransformedwidetypelevelindicatingthattheen—
;hancementofph0t0syntheticcapabilityinPEPC ;transgenicriceisactuallyresultedfromtheintroduc—
;tionofmaizePEPCgene.
;Diurnalchangesofthechlorophyllfluorescence ;parametersindifferentgenotypesofrice ;Theapplianceofthetechniqueofpulsemodulatedfluo—
;rescencecanassesschloroplastPSIIprimaryphoto—
;chemicalefficiency
;m
;undernon—damagecondition
;andcandistinguishthechlorophyllfluorescencepa一
;加mO
;一..砷巨log31Bc0【】三.>.0
;
;StudyoftheProtectiveEffectsinPEPCTransgenic ;...
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;rametersphotochemicalquenching(qP)andnon—pho—
;tochemicalquenching(qN)inleavestoprovidethe ;importantbasisonunderstandingtherelationshipbe—
;tweencapabilityofphotosyntheticapparatusindiffer—
;entgenotypesofriceandenvironmentconditions. ;Fig，3AshowedthatF,,
;/】1mofleavesindifferentgeno—
;typesofricereachedpeakinthemorning.about0.84, ;presentingdescendenttendencywiththeincreaseof ;lightintensityandrecoveredgraduallywiththedecline ;oflightintensityafterl4:oointheafternoon.Through ;comparisonwecanseethatF…
;/FinPEPCtransgenic
;ricedecreasedbv8.3%.1essthanthatinuntransformed ;widetypeKitaake.whichdecreasedbV16.O%under ;highlightintensityatnoon,indicatingthatPEPC ;transgenicricewasratherresistanttophoto—inhibition.
;Fig.3一Bshowedthediurnalchangesofphotochemi—
;ca1quenching(qP)presented”V”trend.Butthede—
;scendentrangeofqPinPEPCtransgenicricewaslower
;thanthatinuntransformedwidetype(WT). ;Fig.3一Cshowedthediurnalchangesofnon—photo—
;chemicalquenchingfqN).Withtheincrementoflight ;intensityinthemorning,qNinPEPCtransgenicrice ;andinuntransformedwidetypeKitaakearebothin一
;086
;0.81
;0.76
;_山{
;一0.71
;0.66
;0.61
;0.56
;Z
;0.8
;0.7
;0.6
;0.5
;04
;6:409:4012:3015:3017:10
;C——?一PEPC
;—
;?-一WT
;6:409:4012:3015:3017:10
;Thetimeoftheday
;creasedandrecoveredgraduallywiththedecadenceof ;lightintensityintheafternoon.Comparingthetwo ;genotypes,thedissipationofexcessivelightenergy ;throughthermalenergyinPEPCtransgenicricewas ;lower.
;TheratioofA505/A652inleavesofricecanberela- ;tiveindexesassessingthexanthophyllcycleofleaves ;ofrice.ItcanbeseenfromFig.3一Dthattheindexesof
;A505/A652oftwogenotypesofricewerecloseand ;therewerenosignificantdifferencefoundatanytime ;inoneday.similartothetrendofqN.Ourprevious ;studysuggestedthatthexanthophyllcycleinindica ;wasmoreactivewhilethatinjaponicawasrelatively ;weaker.DuetobothPEPCtransgenicriceanduntrans—
;formedwidetypeKitaakewerejaponicarice,thismost ;likelywasthereasonthatthechangesofA505/A652 ;werenotobvious.
;Differentgenotypesofricewerefedwithartificial ;photooxidizerMV.TheresultsirtFig.4showedthat
;MVmadeF/FandqPinleavesofuntransformed ;widetypericedroppedsharplywhilethoseinPEPC ;transgenicricereducedmoreslightlyindicatingthat ;PEPCtransgenicricewastolerancetoDhOtOOxidatiOn. ;《
;0
;《
;095
;8:0010:0013:0015:0017:00
;D
;——
;?一PEPC
;--
;11.--WF
;0010:0012:0014:0016:0018:00
;Thetimeoftheday
;PEPC—PEPCtransgenicrice;WT—WidetypeKitaake.I:means~SE(n=3).Measuredcondition:At8:
00,PPFD:854pmolm-s一,temperature28.5.C;At12:00 ;PPFD:1620pmolm-S..,temperature36.0.C;At18:00,PPFD:690ptoolm-s’.,temperature:33.0.C. ;Fig.3DiurnalchangesofchlorophyllfluorescenceparameterofPEPCtransgenicriceandwildtypeKita
ake
;裙
;0c;
;db
;如加m?帅?
;
;98ZHANGQianetal
;Diurnalchangesoftheindexesofactiveoxygen ;metabolismofdifferentgeno~pesofrice ;FromFig.5一AandB.itcanbeseenthattheactivitiesof ;PODandSODindifferentgenotypesofricecanbe ;inducedtoincreasewiththeincrementoflightinten. ;sitvinthemorning.reachedpeakatl4:00intheafter. ;noonandrecoveredgraduallywiththedecadenceof ;thelightafterwards.Withthecomparisonoftwo ;O.85
;080
;O.75
;0.70
;0.65
;O.6O
;O.55
;O50
;?PEPC
;口WT
;PEPC+MV
;Kitaake+MV
;genotypes,enzymeactivitiesinPEPCtransgenicdee
;wereinducedtoincreasecomparativelymore.demon—
;stratingthatPEPCtransgenicricehadstrongercapa—
;bilityofscavengingactiveoxygen. ;FromFig.5一CandDwecanseethatO,7-genera—
;tionrateandthecontentofmembraneperoxidationprod—
;uctMDAalsohadtrendofdiurnalchangesindifferent
;genotypesofdee.InthemorningO3mgenerationrate
;andthecontentofMDAincreasedwiththeincrement
;?PEPC
;PEPC——PEPCtransgenicrice;WT——WidetypeKitaake;PEPC+MV——PEPCtransgenicricea
fterbeingtreatedbyMV;WT+MV——WidetypeKitaakeafterbeing
;treatedbyMV.
;Fig?4ThechangesofFJFandqPofPEPCtransgenicriceandwTKitaakeunderartificialphotooxidizerM
V
;秘g土
;048
;O44
;040
;O.36
;O32
;O.28
;O24
;,._?一PEPC
;8:0010:0012:0014:0016:0018:00 ;——
;??一PEPC
;—.
;?一Kitaake
;8:0010:0012:0014:0016:0018:00 ;Thetimeoftheday
;35
;30
;8
;{
;l量:
;—
;_.一PEPC
;—.
;?—.Kitaake
;8:0010:0012:0014:0016:0018:00
;8:0010:0012:0014:0016:0018:00
;Thetimeoftheday
;Measuredc0nditiOn:At8:00,PPFD:854lamolms’.,temperature28.
;5.C;At12:00,PPFD:1620lamolm.s..,temperature36.OoC;At18:00.PPFD:690pmolm.s
;temperature33.0.C.hmeans_-eSE(n=3).
;Fig?5Diurnalchangesofmetabolisminkat14:00intheafter—
;noonandrestoredgraduallywiththedecadenceofthe ;lightintensityafterwards.Intwogenotypes,Ogen—
;erationrateandthecontentofMDAinPEPCtransgenic ;ricewerelower,whichdemonstratedthatleavesof ;PEPCtransgenicricehadlessgeneratedandaccumu—
;latedactiveoxygenandmembraneperoxidationproduct. ;DlSCUSSlON
;Thelightenergycapturedbyplantphotosyntheticap—
;paratuscouldbeconversedbythreecompetitive ;pathways:photochemicalelectrontransport,chlorophyll ;fluorescenceemissionandthedissipationofnon--radia-- ;tiveenergy.Amongthem,chlorophyllfluorescence ;emissiononlyeliminatepartialabsorbedlightenergy ;(onlyaccountingfor3—5%oflifebody).Whenthepho—
;tochemicalelectrontransportwasblocked,thedissi—
;pationofnon—radiativeenergybecameanimportant
;pathwaytoeliminateexcessivelightenergy.Thede—
;greeofthedissipationofnon—radiativeenergycanbe
;detectedbyfluorescenceparameternon--photochemi-- ;calquenching(qN)?.Inaddition,excessiveenergy
;couldtransferelectronto02whichbecame0in ;PSIorPSII.Plentyofactiveoxygenproducedmem—
;braneperoxidation,resultedintheaccumulationofMDA, ;andhencedestroyedthephotosyntheticapparatusI1一.
;Intheresearchofthevariationofactivitiesofanti—
;oxidationenzyme.Mishrafo?