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Study_1 (2)

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Study_1 (2)

    Study

AgriculturalSciencesinChina

    ;2005,4(2):94100

    ;://www.ChinaAgriSci.com

    ;StudyoftheProtectiveEffectsinPEPCTransgenicRice

    ;ZHANGQian1?2,JIAODemao1,LINGLili?,ZHANGYunhua2andHUANGXueqing

    ;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

    ;inthetransgenicriceplantl.Itexhibitedhigherlight

    ;saturatedphotosyntheticrate(55%),carboxylationef-

    ;ficiency(50%)thanthoseoftheuntransformedWT.

    ;Atthesametime,theCO2compensationpointdecreased

    ;by27%f?.andthegrainyieldperplantincreasedby

    ;1030%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 ;rice2.Inthispaper.withPEPCtransgenicriceas ;CorrespondenceJIAODemao,TeL/Fax:862584391939,Email:photosyn@public1.ptt.js.ca

    ;

    ;StudyoftheProtectiveEffectsinPEPCTransgenicRice95 ;material,thediurnalchangesofthechlorophyllfluo

    ;rescenceparametersandrelativeindexesofactiveoxy

    ;genmetabolismandthexanthophyllcycleunderstrong ;lightwithhightemperatureinsummerwerestudied ;andphotoprotectivestrategyaboutPEPCtransgenic ;riceunderphotoinhibitionconditionwereprimarily ;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

    ;1400

    ;l200

    ;1000

    ;800

    ;600

    ;

    ;

    ;

    ;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..HepesKOH(pH7.8)and20mmolL

    ;NaHCO,.TheOevolutionrateofchloroplastswas ;measuredwithaSP2typeoxygenelectrode(madein ;InstituteofShanghaiPlantPhysiology,ChineseAcade

    ;myofSciences1underPPFDof800pmolms’at

    ;25.C.

    ;TreatmentoftheinhibitorDCDPTheuppersur

    ;faceofintactflagleavesweresmearedbythesolution ;containing200mmolL’.peculiarinhibitor3,3dichloro

    ;2(dihydroxyphosphinoylmethy1)propenoate(DCDP) ;andl%(v/v1Tween80.Thecontrolleavesweretreated ;withdistilledwaterwith1%fv/v1Tween80.Plants

    ;wereplacedindarkorilluminatedbyweaklightunder ;PPFDOf20—30pmolm’s’fortwohourscausing

    ;distilledwaterandtheinhibitorfilteringintotheleaves. ;Measurementofchlorophyllfluorescencepara- ;meters

    ;Thechlorophyllfluorescenceparametersweremea

    ;suredbyFMS2fluorescencemeter(Hansatech,UK) ;asdescribedbyGentyt.Withfunctionallightf1200

    ;pmolms..,30s),saturatingradiationpulse(4000 ;lamolms,30s)andflash(0.9lamolms..,30s), ;eachmaterialwasmeasuredthreetimes.Thechloro

    ;phyllfluorescenceparameterswerecalculatedaccord

    ;

    ;ZHANGQianetal

    ;ingtothefollowingequations

    ;F/F

    ;m(FF0)/Fm

    ;qP=(F’F)/(F’F0’)

    ;qN=1(F’-F0’)/(Fm-F0)

    ;Measurementoftheindexesofactiveoxygen ;metabolism

    ;Riceflagleaveswereharvestedindifferenttimesatthe ;samedayandweresentimmediatelyinliquidnitrogen ;toreserve.Using50mmolLpH7.8phosphatebuffer ;asexactionbufferaddingalittlePVP,0.5gleaveswere ;groundinicebath.Thehomogenatewascentrifugedat ;4.C.10000gfor1,5min.Thesupernatantwasused ;forthemeasurementoftheindexesofactiveoxygen ;metabolism.Thecontentofproteinwasdetermined ;accordingtoBradfordt10usingAlbuminbovineas

    ;standard.Peroxidase(POD)activitywasassayedby ;themethodofKochbaetalt”.OneunitofPODactivity

    ;isdefinedastheincreaseof0.1oDperminute.Supero

    ;xidedismutase(SOD)activitywasassayedbythe ;methodofGiannopolitisandRiest.OneunitofSOD ;activityisdefinedastheamountofenzymethatcaused ;50%inhibitionoftheinitialrateofNBTreduction.OT ;evolutionratewasmeasuredaccordingtothemethod ;0fgetal31.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).NatareNaturecondition;Ph0t0jnhibjti0n

    ;Photoinhibitioncondition;Nature+DCDPUndernatureconditionricewas ;treatedbyPEPCpeculiarjnhibitorDCDP:Ph0t0inhIbjtion+DCDP——Under

    ;photojnhibitionconditionricewastreatedbyPEPCpeculiarinhibitorDCDP.

    ;Naturecondition:Sampleat10:00lnthemorningataclearday;Photolnhibition

    ;condition:AttachedleaveswereplacedwithCO,freeaircontaining1%O,.under ;PPFDof10oolamolill21from8:00to10:00for2h.

    ;Fig.2EffectofDCDPtreatmenton02evolutionrateinPEPC ;transgenicriceandwildt3,peKitaakeundernatural ;andphOt0inhibiti0nconditions

    ;transgenicricedecreaseddrasticallyandreachedto ;untransformedwidetypelevelindicatingthattheen

    ;hancementofph0t0syntheticcapabilityinPEPC ;transgenicriceisactuallyresultedfromtheintroduc

    ;tionofmaizePEPCgene.

    ;Diurnalchangesofthechlorophyllfluorescence ;parametersindifferentgenotypesofrice ;Theapplianceofthetechniqueofpulsemodulatedfluo

    ;rescencecanassesschloroplastPSIIprimaryphoto

    ;chemicalefficiency

    ;m

    ;undernondamagecondition

    ;andcandistinguishthechlorophyllfluorescencepa

    ;mO

    ;..砷巨log31Bc0【】三.>.0

    ;

    ;StudyoftheProtectiveEffectsinPEPCTransgenic ;...

    ;R

    ;......

    ;i

    ;..

    ;c

    ;

    ;e

    ;97

    ;rametersphotochemicalquenching(qP)andnonpho

    ;tochemicalquenching(qN)inleavestoprovidethe ;importantbasisonunderstandingtherelationshipbe

    ;tweencapabilityofphotosyntheticapparatusindiffer

    ;entgenotypesofriceandenvironmentconditions. ;Fig3AshowedthatF,,

    ;/1mofleavesindifferentgeno

    ;typesofricereachedpeakinthemorning.about0.84, ;presentingdescendenttendencywiththeincreaseof ;lightintensityandrecoveredgraduallywiththedecline ;oflightintensityafterl4:oointheafternoon.Through ;comparisonwecanseethatF…

    ;/FinPEPCtransgenic

    ;ricedecreasedbv8.3%.1essthanthatinuntransformed ;widetypeKitaake.whichdecreasedbV16.O%under ;highlightintensityatnoon,indicatingthatPEPC ;transgenicricewasratherresistanttophotoinhibition.

    ;Fig.3Bshowedthediurnalchangesofphotochemi

    ;ca1quenching(qP)presented”V”trend.Butthede—

    ;scendentrangeofqPinPEPCtransgenicricewaslower

    ;thanthatinuntransformedwidetype(WT). ;Fig.3Cshowedthediurnalchangesofnonphoto

    ;chemicalquenchingfqN).Withtheincrementoflight ;intensityinthemorning,qNinPEPCtransgenicrice ;andinuntransformedwidetypeKitaakearebothin

    ;086

    ;0.81

    ;0.76

    ;_{

    ;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——?PEPC

    ;

    ;?-WT

    ;6:409:4012:3015:3017:10

    ;Thetimeoftheday

    ;creasedandrecoveredgraduallywiththedecadenceof ;lightintensityintheafternoon.Comparingthetwo ;genotypes,thedissipationofexcessivelightenergy ;throughthermalenergyinPEPCtransgenicricewas ;lower.

    ;TheratioofA505/A652inleavesofricecanberela- ;tiveindexesassessingthexanthophyllcycleofleaves ;ofrice.ItcanbeseenfromFig.3Dthattheindexesof

    ;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

    ;——

    ;?PEPC

    ;--

    ;11.--WF

    ;0010:0012:0014:0016:0018:00

    ;Thetimeoftheday

    ;PEPCPEPCtransgenicrice;WTWidetypeKitaake.I:means~SE(n=3).Measuredcondition:At8:

    00,PPFD:854pmolm-s,temperature28.5.C;At12:00 ;PPFD:1620pmolm-S..,temperature36.0.C;At18:00,PPFD:690ptoolm-s’.,temperature:33.0.C. ;Fig.3DiurnalchangesofchlorophyllfluorescenceparameterofPEPCtransgenicriceandwildtypeKita

    ake

    ;

    ;0c;

    ;db

    ;如加m??

    ;

    ;98ZHANGQianetal

    ;Diurnalchangesoftheindexesofactiveoxygen ;metabolismofdifferentgeno~pesofrice ;FromFig.5AandB.itcanbeseenthattheactivitiesof ;PODandSODindifferentgenotypesofricecanbe ;inducedtoincreasewiththeincrementoflightinten. ;sitvinthemorning.reachedpeakatl4:00intheafter. ;noonandrecoveredgraduallywiththedecadenceof ;thelightafterwards.Withthecomparisonoftwo ;O.85

    ;080

    ;O.75

    ;0.70

    ;0.65

    ;O.6O

    ;O.55

    ;O50

    ;?PEPC

;WT

    ;PEPC+MV

    ;Kitaake+MV

    ;genotypes,enzymeactivitiesinPEPCtransgenicdee

    ;wereinducedtoincreasecomparativelymore.demon

    ;stratingthatPEPCtransgenicricehadstrongercapa

    ;bilityofscavengingactiveoxygen. ;FromFig.5CandDwecanseethatO,7-genera

    ;tionrateandthecontentofmembraneperoxidationprod

    ;uctMDAalsohadtrendofdiurnalchangesindifferent

    ;genotypesofdee.InthemorningO3mgenerationrate

    ;andthecontentofMDAincreasedwiththeincrement

    ;?PEPC

    ;PEPC——PEPCtransgenicrice;WT——WidetypeKitaake;PEPC+MV——PEPCtransgenicricea

    fterbeingtreatedbyMV;WT+MV——WidetypeKitaakeafterbeing

    ;treatedbyMV.

    ;Fig?4ThechangesofFJFandqPofPEPCtransgenicriceandwTKitaakeunderartificialphotooxidizerM

    V

    ;g

    ;048

    ;O44

    ;040

    ;O.36

    ;O32

    ;O.28

    ;O24

    ;,._?PEPC

    ;8:0010:0012:0014:0016:0018:00 ;——

    ;??PEPC

    ;.

    ;?Kitaake

    ;8:0010:0012:0014:0016:0018:00 ;Thetimeoftheday

    ;35

    ;30

    ;8

    ;{

    ;l:

    ;

    ;_.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 ;(onlyaccountingfor35%oflifebody).Whenthepho

    ;tochemicalelectrontransportwasblocked,thedissi

    ;pationofnonradiativeenergybecameanimportant

    ;pathwaytoeliminateexcessivelightenergy.Thede

    ;greeofthedissipationofnonradiativeenergycanbe

    ;detectedbyfluorescenceparameternon--photochemi-- ;calquenching(qN)?.Inaddition,excessiveenergy

    ;couldtransferelectronto02whichbecame0in ;PSIorPSII.Plentyofactiveoxygenproducedmem

    ;braneperoxidation,resultedintheaccumulationofMDA, ;andhencedestroyedthephotosyntheticapparatusI1.

    ;Intheresearchofthevariationofactivitiesofanti

    ;oxidationenzyme.Mishrafo?

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