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Lipids, Proteins, and Structure of Seed Oil Bodies from Diverse Species Author(s): Jason T. C. Tzen, Yi-zhi Cao, Pascal Laurent, Chandra Ratnayake, Anthony H. C. Huang Reviewed work(s): Source: Plant Physiology, Vol. 101, No. 1 (Jan., 1993), pp. 267-276 Published by: American Society of Plant Biologists Stable URL: http://www.jstor.org/stable/4274959 . Accessed: 30/11/2011 17:38 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.js
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  Lipids, Proteins, and Structure of Seed Oil Bodies from Diverse SpeciesAuthor(s): Jason T. C. Tzen, Yi-zhi Cao, Pascal Laurent, Chandra Ratnayake, Anthony H. C.HuangReviewed work(s):Source: Plant Physiology, Vol. 101, No. 1 (Jan., 1993), pp. 267-276Published by: American Society of Plant Biologists Stable URL: http://www.jstor.org/stable/4274959. Accessed: 30/11/2011 17:38 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jspJSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new formsof scholarship. For more information about JSTOR, please contact support@jstor.org.  American Society of Plant Biologists is collaborating with JSTOR to digitize, preserve and extend access to Plant Physiology. http://www.jstor.org  PlantPhysiol.1993)101:267-276 Lipids,Proteins,andStructureofSeedOil Bodies fromDiverseSpecies' JasonT.C.Tzen,Yi-zhiCao,PascalLaurent,ChandraRatnayake,andAnthonyH.C.Huang*DepartmentofBotanyand PlantSciences,UniversityofCalifornia,Riverside,California2521-0124 Oil bodiesisolated from thematureseeds ofrape (BrassicanapusL.),mustardBrassicaunceaL.),cotton(Gossypiumbirsu-tumL.),flax(Linususitatissimum),maize(ZeamaysL.),peanut(ArachishypogaeaL.),and sesame(SesamumindicumL.)hadaveragediametersthat were differentbut within anarrowrange(0.6-2.0,m),asmeasuredfromelectronmicrographsof serialsections.Theircontents oftriacylglycerolsTAG),phospholipids,andproteins(oleosins)werecorrelatedwiththeirsizes.The cor-relationfits aformula that describes asphericalparticlesur-roundedby a shellofamonolayerofphospholipids embeddedwitholeosins.Oil bodies fromthe variousspeciescontainedsub-stantialamounts oftheuncommonnegativelychargedphosphati-dylserine andphosphatidylinositol,swell as smallamounts offreefattyacids. These acidiclipidsareassumed tointeractwiththebasic aminoacid residuesofthe oleosins on thesurface ofthephospholipidayer.Isoelectrofocusingevealed thatthe oilbodiesfrom thevariousspecies had anisoelectricpointof 5.7to6.6 andthuspossessedanegativelychargedsurfaceat neutralpH.Weconclude thatseed oilbodiesfromdiversespecies areverysimilarinstructure.Inrapeseedduringmaturation,TAGandoleosinsaccumulatedconcomitantly.TAG-synthesizingcyltransferaseac-tivitiesappearedatanearlierstage andpeakedduringtheactiveperiodofTAGaccumulation.Theconcomitantaccumulation ofTAGandoleosins is similarothat reportedearlierformaize andsoybean, and thefindinghas animplicationforthe mode of oilbodysynthesisduringseedmaturation. Seeds storeTAG asfoodreserves forgerminationandpostgerminativerowthof theseedlings. TheTAG arepres-entinsmall, discretentracellularrganellescalledoilbodies(YatsuandJacks,1972;Appelquist,1975;Stymne andSto-bart,1987;Huang, 1992).Isolatedoilbodieshave asphericalshape andpossessdiametersrangingfromabout0.5to2.0/im.TheycontainmostlyTAGandsmallamountsofPLandproteinscalledoleosins.It isgenerallyagreedthat theoilbodyhas amatrix ofTAGsurroundedbyalayer of PLembeddedwith oleosins.ThePL form amonolayeruchthattheacylmoieties ofthemoleculesfaceinward tointeractwith thehydrophobicTAGnthematrix,andthehydrophilicPLheadgroups areexposedto thecytosol.Theembeddedoleosinmolecule scomposedofthreestructuralomains:anN-terminalamphipathicdomain,acentralhydrophobicdo-main,and aC-terminalmphipathic-helicaldomainVanceandHuang,1987;Qu andHuang, 1990;Murphyt al.,1991;Tzenetal.,1992).Itispredicted hatthehydrophobicportionoftheoleosin moleculepenetrateshePLlayerntothe TAGmatrix,and itsamphipathicportionresides on thePLlayerorprotrudes otheexterior.Thestructurefanoilbodyasdescribedn theprecedingparagraphmplies that therelativecontentsofTAG,PL, andproteins n apurepreparationfoilbodies shouldberelatedtothe size oftheorganelles.Recently,weused themaize oilbody as amodelsystemtocorrelate herelativecontentsofTAG,PL,andoleosinstothesize oftheorganelleTzenandHuang,1992).Aformulawasderived odescribeasphericalmass ofTAGofacertaindiameterurroundedbya shellofone PLayerof2.5 nmthickness.Thelayercontains80%PLand 20%oleosin.Of eacholeosinmolecule,about20%oftheaminoacidresidues areembedded nthe PLlayer,30%arelocated intheTAGmatrix,and50%areexposedtotheexterior.ThePL of themaizeoilbodyinclude asubstantialpercentageofthenegativelychargedPIandPS(Tzenetal.,1992),which areunusualPLcomponents inplanttissues(Mudd,1980).IthasbeenproposedthatthesenegativelychargedPL,togetherwithasmallamount ofFAA,interactwith thebasicaminoacidresidues oftheoleosinson thesurfaceofthePLlayer.Computationofthechargesonthesurface ofan oilbodyreveals anisoelectricpointof6.2,which isinagreementwiththevaluemeasuredbypH-dependentaggregationandisoelectrofocusingTzen etal.,1992).Duringseedmaturationnmaize(Quet al.,1986),aswellassoybean(Herman,1981),theembryosaccumulatedTAGandoleosinsconcomitantly.Thisobservations inagreementwith,althoughs notevidenceinsupportof, amodelofoilbodysynthesis inwhichTAGandoleosinsaresynthesizedconcomitantlyntheER,fromwhichanascent matureoilbodyisformedbybudding(Huang,1992).Itisunknownwhetherourknowledgeonthestructurefthemaizeoilbodiesandtheirdevelopmentalpatterninmaturingeedcanbeextended otheseed oilbodiesofotherspecies.Thereportedrelativecontents ofTAG,PL,andproteins nseed oilbodypreparationsromseveralspeciesvarysubstantiallyAppelquist,1975;Murphyet al.,1989).Thevariationmayindicatethatourfindingsfromthemaizeoilbodiesarenotvalidfortheseedorganellesfromotherspecies.Alternatively,tmayonlyreflectdifferences nthesizeofthe oilbodiesandthepurity ofthepreparations.Anotherdiscrepancy s inthemodeofaccumulationfTAGandoleosinsduringseedmaturation.twasreportedhat,n 'SupportedbyU.S.DepartmentofAgriculturerant91-01430(A.H.C.H.).*Correspondinguthor; ax 1-714-787-4437.Abbreviations:AA,freefattyacid(s);PC,phosphatidylcholine;PE,phosphatidylethanolamine;I,phosphatidylinositol;L,phos-pholipid(s);PS,phosphatidylserine;AG,riacylglycerol(s).267  268Tzenet al.PlantPhysiol.Vol.101, 1993 rapeseedMurphy t al., 1989),unlikemaize(Quet al., 1986)andsoybean(Herman, 1987)seeds,theaccumulationofoleosinslaggedtemporallybehindthatofTAG.The datahavebeenusedto proposethat the oilbody is synthesizedfirstwithTAG, followedtemporallybyanencasementwithsubsequently ynthesizedoleosins.Whetherornot thisalter-nativemodel of oil body formation eflectsaspecies-depend-entdifferencen theorganellebiogenesissunknown.Toclarify thesematters,weanalyzedtheseedoil bodiesisolatedfrom severalspecies.Weselectedspeciesthathaveseedoilbodiesof diverseaveragediameters.Ourfindingsshowthatthestructuralmodeloftheoilbodyestablishedwiththemaize organelleisapplicabletothosein otherspecies.Wealsofoundthat,nmaturing apeaswell as maizeseeds,theaccumulationfTAGand oleosinsoccursconcom-itantly. Here, wepresentourfindingsanddiscuss the re-porteddiscrepanciesoncerningeedoil bodiesfromdifferent species.MATERIALSAND METHODSPlantMaterials Embryosof maize(Zea maysL.,inbredline MO17),tripsacum TripsacumactyloidesL.),teosinte(Z.mays ssp.mexicana,arChalco),yucca (Yuccawhipplei), ape (BrassicanapusL.varWestar),mustard(BrassicaunceaL.),cotton(GossypiumirsutumL.), jojoba(SimmondsiahinensisL.),sunflower(HelianthusannusL.),soybean (GlycinemaxL.),flax(Linumsitatissimum),esame(Sesamumndicum L.), andpeanutArachisypogaea .),endospermandembryoofpalm(Syagrus ocoides,Martius),astorbean(Ricinusommunis.),Brazilnut(Bertholletiaxcelsa),and bran of oat(Avena ativaL.)andwheat (TriticumaestivumL.)wereobtainedfrommatureseeds.The matureseedswere eitherused directly(rape,mustard, lax,andsesame) or soakedinwater for1 h(soybean, peanut, oat,andwheatbran),6 h(cottonandsunflower),24h(maize,teosinte, tripsacum,yucca,castorbean,Brazilnut,andjojoba),or72 h(palm)before use. SamplingofMaturingSeedsofMaize andRape Maizeplantsweregrownat theuniversityield stationnsummer1990. The ear wasbagged beforethesilkstartedoappearand was checkeddaily fortheappearanceofsilk.When thesilkswereabout1.5to2cmlong,they werecutwitharazorblade,andtheearwastagged.Freshpollenwascollectedon thefollowing dayand usedtopollinatethe silk.Pollinationwasrepeatedontwo consecutivedays.The sec-onddayofpollinationwasdesignatedas 0 dafterpollination.Kernelswere harvestedeveryotherdayfrom13 to33d afterpollination.Embryosrom 25kernelsof eachmaturing tagewereused.Rape plants were grown inside agreenhousemaintainedat26.5/18.50C,about14/10hday/night cycle in fall 1990.Assoonasthe flowersopened,theywerepollinated withpollenobtainedfromseveralplants.Thepollinatedflowersweretaggedand the otheropen flowerswithout artificialpollinationwereremoved.Thepodswereharvestedeveryother day from 12 to 40dafterpollination.Twohundredseeds of eachmaturing tagewereused.IsolationofOilBodiesThe tissue washomogenizedat40Cingrindingmedium(5 g oftissue/20mL)with aPolytronBrinkmann,Westbury,NY)fitted witha PTA 10generatorathighspeedfor40s.Thegrindingmediumcontained0.6 M sucrose,1 mMEDTA,10 mMKCI,1mlMgCl2,2mmDTT,and 0.15 M TricinebufferadjustedtopH7.5 withKOH. Thehomogenatewasfilteredthrougha Nitex clothhaving 20-x20-,umpores(Tetko,Elmsford,NY).Afterfiltration,each 15-mLportionof thehomogenatewasplacedatthe bottom of a30-mLcentrifugetube,and 15mLofflotationmedium(grindingmediumcontaining0.4instead of0.6 M sucrose)waslayeredontop.The tubewascentrifugedat10,000gfor 30minin aswinging-bucketotor.Theoilbodiesontopwerecollectedandresuspendedn 15 mL ofgrindingmediumcontaininganadditional M NaCl. Theresuspensionmaterialwasplacedatthe bottomofa 30-mLcentrifugeube,15 mL offloatingmedium(grindingmediumcontaining2 M NaCland0.25 M instead of 0.6 M sucrose)waslayeredontop,andthe tubewascentrifuged.The oil bodiesontopwerecollectedandresuspendedn15mLofgrindingmedium.Theresuspensionmaterial wasplaced atthebottomofa 30-mLcentrifugetube,15mLoffloatingmediumgrindingmediumcontaining0.4 insteadof0.6 M sucrose)waslayeredontop,and thetube wascentrifuged.Thisaststepof flotationwashing wasrepeatedonce.The oil bodies ontopwerecollected andresuspendedwithgrindingmediumtogiveaconcentrationof about100mgoflipid/mL.MeasurementofOilBody DiameterInapreliminary tudy, seedoilbodies isolatedfrom18specieswereobservedunderalightmicroscopewithNomar-skioptics.Theoilbodies fromsevenspecies(rape,mustard,cotton,lax, maize,peanut,andsesame),which had adiverserangeofsizes,were chosen forfurther tudies. Theoilbodypreparationswere fixedin2.5%glutaraldehyden50mmpotassiumphosphatebuffer, pH7.5,for 3 h. Afterseveralrinses withthebuffer,theywerepostfixedin 1% OSO4 inthe same bufferfor 18h.Dehydrationwascarriedout inagradedacetoneseries.ThesampleswereembeddednSpurr'sresin.Serialsections100 nm thickwere stainedwithuranylacetate and leadcitrateandphotographedunderaPhilipsEM400electronmicroscope.Thephotoofthe middlesectionof the serialsectionswas used tomarkallthe oilbodies(150-500,dependingonthespecies)within adefinedarea.Eachof these markedoilbodies wastracedthroughoutheserialsections tomeasuretslargestdiameterR).Thevolume(V)of eachoilbodywas calculatedbytheformula,V=4/3ir(R/2)3.Theaveragevolume(V)ofthe oilbodieswasobtainedbydividingthesum ofall the oilbody volumesbythe totaloilbodynumber.TheaveragediameterR)of oilbodieswascalculated romtheiraveragevolumebythe formulaV = 4/3ir (R/2)3. IEF fOilBodiesIEFwasperformedn aBio-RadRotoforCell(Richmond,CA),whichhad thefollowingdesign.Thehorizontalcylin-drical focusingcell of55 mLwasdivided verticallynto20  OilBodies from DiverseSpecies269chambersby porous partitionso minimize diffusionduringelectrophoresisnd disturbanceof thegradientduringfrac-tionation.Duringelectrofocusing,he RotoforCell rotatedat1 rpm andthusminimizedtheflotationof oil bodies. Frac-tionationofcontentsneverychamberwas achievedsimul-taneouslyn 1sbya vacuumharvesting ystem.Inthe currenttudy,weremovedthepartitionsoallowfree movementoftheoil bodies duringelectrofocusing Tzenetal., 1992).Becausehegradientwithoutthepartitionsouldnot befractionated,hepositionofthe oilbodiesalongthegradient afterelectrofocusingwasphotographed.ThepHgradient,madefrom 1%ampholyte(0.5%Bio-Lyte5-7 and0.5%Bio-Lyte7-9,both fromBio-Rad),waspreformedbyapplyingan electricield at constantpower (12 W)at 400 to500Vfor 10min.Isolatedoil bodies(about20mgoflipid)in200 ,ALof10-mMKClsuspensionwereappliedtothegradientat thehigh pH region.Anelectricield of thesamewattagewasappliedfor10minto focustheoil bodies.Movementof the oil bodies was observedvisually.ThepHgradientinthe currentsetupwas stable before and aftersample applicationandelectrofocusing,aswellasamongdifferentruns.Thiswas revealedby repeatingheruns withthe verticalpartitionsncludedin thechamber and thenfractionating he gradient nto 20 fractionsand checkinghepHineachfractionwith apH meter.Quantitationof ConstituentsofIsolated Oil BodiesNeutralipids (mainlyTAG),proteins,and PLofoilbodieswereseparatedbythefollowing procedure.A500-,uLprep-aration ofisolatedoilbodies was extractedwith 1mLofdiethylether in a1.7-mLEppendorfube.After theprepa-rationwascentrifugedat13,600gfor 4min,theupper etherlayerwascollected. The loweraqueouslayerand theinter-facial materialswereextractedwith1mL ofdiethylethertwo additional imes. Theether fractionswerepooled (3mL),and the etherwasevaporatedundernitrogen gas.Theaqueouslayer, togetherwith theinterfacialmaterials,wasallowed toevaporateundernitrogenfor 1 hto remove theremainingther.Avolume of 750,uLfchloroform:methanol(2:1, v/v) was added. Aftergentleshaking, the tubewascentrifugedt13,600gfor4min.The lowerchloroformayerand theinterfacialmaterialswerecollectedseparately.ThechloroformractionwhichcontainedPL)was washedthreetimes,each timewith1 mLofmethanol:water1:1,v/v),followedbycentrifugation.Theinterfacialmaterialswhichweremostlyproteins)were washedthreetimesby the follow- ing procedure.They were mixed with250 ,uLof water and750,uLofchloroform:methanol2:1, v/v),andthemixturewascentrifuged.Theinterfacialmaterialswere collectedandresuspendedn0.5 mL ofwater.The resuspensionmaterialwas sonicatedwitha4-mmdiameterprobenaBraun-Sonic2000 ultrasonicgeneratorFreeport, L) with a digitalmeterreadingof200 for 15s.Theproteinsntheinterfacialmaterialswerequantifiedbythe Bradfordmethodusing BSA, fractionV,asstandard(Smithetal., 1985). Theywereresolved ntovariousconstit-uentsbySDS-PAGEwiththeseparating el and thestackinggelcomposedof 12.5 and 4.75%polyacrylamide,espectively(Tzenetal.,1990).Afterelectrophoresis,hegelwasstainedwithCoomassieblue R-150 anddestained.Theneutrallipidsinthe ether fractionwereweigheddirectly about0.1gfrom1mL of oilbodysuspension)afterexhaustiveevaporationofthe ether. The chloroformractioncontainingPL wasspottedon a TLCplate(silicagel60AfromWhatman). Theplate wasdevelopedinchloro-form:aceticacid:methanol:water70:25:5:2, v/v/v/v)andthenallowed to reactwithiodinefor color development.StandardPL(Sigma,St.Louis,MO)used for identificationwere a-PC(eggyolktypeIX-E),a-PS(bovinebrain),a-PI(bovineliver),a-PE(EscherichiaolitypeV),a-phosphati-dylglycerol(dioleoyl),anda-phosphatidicacid(dioleoyl).After the different PL wereidentified,theTLCplatewasplacedin avacuumevaporatoroallow the iodine toevap-orate.Thegelcorrespondingothe differentPLwerescraped,and theirphosphatecontentswerequantifiedbytheBartlettmethodusingKH2PO4s areference(DittmerandWells,1969).ThePLcontentnthe oilbodyfractionwas measureddirectly bythe samemethod. TheidentityofthePSfrommaizeoilbodieswasconfirmedqualitativelybyhydrolysiswithphospholipase C(ClostridiumerfringensypeXIV;Sigma)followedbydetectionof thereleasedphosphoserine(derivatizedwithphenylisothiocyanate)byHPLC,usingbovinebraina-PSas acontrol courtesyofDr. M.Madore).FFAof the oilbodyfractionweredeterminedbyacolor-metricmethod (NixonandChan,1979)usingoleicacid and palmiticacid asstandardsbothgavean identicalstandardcurve).We isolated oil bodiesfromfreshly harvestedmatureseedsofrape,mustard,cotton, andmaizeto avoidthepossibilityofdetectingFFAgeneratedduringaging.DeterminationofOilBodyConstituentsn TotalExtracts fMaturingSeedsTwohundredapeor25maizeembryoswerehomogenizedin 4 mLofgrindingmediumasdescribedarlier,except thatDTTwasdeletedfrom thegrindingmedium.Afterfiltration,thehomogenatewasadjusted o 5mLwithgrindingmedium.Thehomogenatewas eitheruseddirectlyorfrozenat-800Cuntiluse.Totalproteinsnthehomogenate werequantifiedby theBradfordmethodusing BSA,fractionV. asthestandard(Smithetal.,1985).TheproteincomponentswereresolvedbySDS-PAGE,using12.5and 4.75%polyacrylamiden theseparatinggel and thestackinggel,respectively.Two iden-ticalgelswererun.OnegelwasstainedwithCoomassieblueR-150, andthe othergelwassubjected oanimmunoblottingassay (Tzenetal.,1990).Inbothcases, therelativequantityofthe20-kDoleosinswasobtainedby thefollowingmethod.Thesamples from thestage of33 d (formaize)and 36d (forrape)afterpollinationwerechosen asstandards100%) orthecalculationofthe relativeamounts romdifferenttages.Toestimate herelativeamountsofTAG andmajoroleosinsfromdifferenttages,aserialdilution 5,10, 20.... 80,90%)ofthesamplefromthestandardstage(33 or 36 dafterpollination)plusthesamplesromotherstages wereresolvedbyTLCorSDS-PAGE.Visualcomparisonwas usedtodeter-mine the relativeamountsof the TAG andoleosins.For theanalysisof totalneutralipid in the 33-drapeand
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