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TheEpidermis-SpecicExtracellularBODYGUARDControlsCuticleDevelopmentandMorphogenesisinArabidopsisWSergeyKurdyukov,a,1AndreaFaust,aChristianeNawrath,b,2SaschaBa¨r,aDerryVoisin,aNadiaEfremova,aRochusFranke,cLukasSchreiber,cHeinzSaedler,aJean-PierreMetraux,bandAlexanderYephremova,3aMax-Planck-Institutfu¨rZu¨chtungsforschung,50829Ko¨ln,GermanybDepartmentofBiology,UnitofPlantBiology,UniversityofFribourg,Perolles,CH-1700Fribourg,SwitzerlandcInstitutfu¨rZellula¨reandMolekulareBotanik,Universita¨tBonn,D-53115Bonn,GermanyTheoutermostepidermalcellwallisspecializedtowithstandpathogensandnaturalstresses,andlipid-basedcuticularpolymersarethemajorbarrieragainstincursions.
TheArabidopsisthalianamutantbodyguard(bdg),whichexhibitsdefectscharacteristicofthelossofcuticlestructurenotattributabletoalackoftypicalcutinmonomers,unexpectedlyaccumulatessignicantlymorecellwall–boundlipidsandepicuticularwaxesthanwild-typeplants.
Pleiotropiceffectsofthebdgmutationongrowth,viability,andcelldifferentiationarealsoobserved.
BDGencodesamemberofthea/b-hydrolasefoldproteinsuperfamilyandisexpressedexclusivelyinepidermalcells.
UsingStrep-tagepitope-taggedBDGformutantcomplementationandimmunolocalization,weshowthatBDGisapolarlylocalizedproteinthataccumulatesintheoutermostcellwallintheepidermis.
Withregardtotheappearanceandstructureofthecuticle,thephenotypeconferredbybdgisreminiscentofthatoftransgenicArabidopsisplantsthatexpressanextracellularfungalcutinase,suggestingthatbdgmaybeincapableofcompletingthepolymerizationofcarboxylicestersinthecuticularlayerofthecellwallorthecuticleproper.
WeproposethatBDGcodesforanextracellularsynthaseresponsiblefortheformationofcuticle.
ThealternativehypothesisproposesthatBDGcontrolstheproliferation/differentiationstatusoftheepidermisviaanunknownmechanism.
INTRODUCTIONInplants,theoutermostepidermalcellwallisspeciallyfortiedandconsistsofhydrophilicandhydrophobiccomponents.
Cellwallcarbohydratesandglycoproteinsprovideanunderlyinghydrophilicframework,whereasextracellularlipidsconstitutetheimpermeablehydrophobicouterportionofthecellwall,calledcuticle.
Someextracellularlipids(e.
g.
,waxes)aresolubleandcanbeextractedwithorganicsolvents,whereasothers(e.
g.
,cutin)areinterconnectedbyesterbonds,andtheircompositioncanonlybestudiedafterhydrolysisofthepolyesters.
Moreover,afractionofthelipidpolymers(e.
g.
,cutan)isresistanttohydrolysis.
Structurally,theoutermostlayerofthecuticleiscomposedofwax,whereastheinsolublepolymersconstitutingthecuticleproperaredepositedbeneathitandareseeninelectronmicrographsasathinlamellatelayerofdarklystainingmaterial(Kolattukudy,2001a).
Asanexteriorprotectiveandsupportingstructure,thecellwalldeterminescellshapeandisinvolvedinadhesion,defense,differentiation,andgrowthpro-cesses.
Notsurprisingly,therefore,manymutantsassociatedwithcellwallbiogenesisandfunction(Ariolietal.
,1998;Ellisetal.
,2002;Nishimuraetal.
,2003)showpleiotropicphenotypes.
Thecuticularlayerofthecellwallisdistinguishedbythepresenceoflipidmoleculesandcomprisesthepartofthecellwallwherecarbohydrateandlipidmoleculesareintimatelyinterconnected.
Itsexactcompositionisnotknown,becauseitisnotpossibletophysicallyseparatethevariousconstituents.
However,thecompositionofthelipidpolyestersthatmakeupthestructuralcomponentofthecuticleproper,cutin,hasbeenpreciselydeterminedinmanyplantspeciesthathavearelativelythickcuticularmembrane.
Typically,itischaracterizedbythepresenceofC16andC18fattyacidsbearingv-andmid-chainhydroxylgroups(Kolattukudy,2001a).
However,a,v-dicarbox-ylicfattyacids(40%)and2-hydroxyfattyacids(14%)wereidentiedasmajordepolymerizationproductsinArabidopsisthalianacutin,althoughcutin-characteristicv-hydroxyandmid-chainhydroxylatedfattyacidsweredetectedaswell(7and8%,respectively)(Frankeetal.
,2005).
Besidestheunusualchemicalcomposition,thecuticleproperisverythininArabidopsiscomparedwithmostplantsstudied(Nawrath,2003).
BiochemicalanalysisrevealedastrikingsimilaritybetweenArabidopsiscutinandrootsuberin,whichisacellwall–linkedpolyesterfoundincellsthatprovideabarriertoapoplasticwatertransportandthathaveadefensivefunction.
Itcanbeenzymat-icallydetachedandisolatedwiththeuseofpolysaccharidehydrolases.
Contrarytocutin,thealiphaticcompositionofsuberininArabidopsisisverysimilartothatdescribedpreviously1Currentaddress:ARCCentreofExcellenceforIntegrativeLegumeResearch,SchoolofEnvironmentalandLifeSciences,UniversityofNewcastle,UniversityDrive,Callaghan,NSW2308,Australia.
2Currentaddress:DepartmentofPlantMolecularBiology,UniversityofLausanne,UNIL-Sorge,BiologyBuildingBiophore,CH-1015Lausanne,Switzerland.
3Towhomcorrespondenceshouldbeaddressed.
E-mailefremov@mpiz-koeln.
mpg.
de;fax49-221-5062-113.
TheauthorresponsiblefordistributionofmaterialsintegraltothendingspresentedinthisarticleinaccordancewiththepolicydescribedintheInstructionsforAuthors(www.
plantcell.
org)is:AlexanderYephremov(efremov@mpiz-koeln.
mpg.
de).
WOnlineversioncontainsWeb-onlydata.
Article,publicationdate,andcitationinformationcanbefoundatwww.
plantcell.
org/cgi/doi/10.
1105/tpc.
105.
036079.
ThePlantCell,Vol.
18,321–339,February2006,www.
plantcell.
org2006AmericanSocietyofPlantBiologistsforotherplants(Kolattukudy,2001b;Bernards,2002).
Itischar-acterizedbyhighlevelsofvery-long-chainv-hydroxy(43%)anda,v-dicarboxylic(24%)fattyacids.
Carboxylicfattyacids,fattyalcohols,and2-hydroxyfattyacidsareminorcomponents(Frankeetal.
,2005).
Basedonbiochemicalstudiesconductedinthe1970sand1980s,manyofthestepsinthebiosyntheticpathwayforcuti-cularpolymershavebeendeduced(Kolattukudy,2001a;Lequeuetal.
,2003).
However,thefailuretoidentifymutantsinthispathwayhasmadeitdifculttoverifytheseproposals.
ItnowseemslikelythatanumberofArabidopsismutantsthatexhibitadhesionresponses(organfusions),celldeath,andretardedcelldifferentiationandgrowth(Lolleetal.
,1998;Yephremovetal.
,1999;Pruittetal.
,2000;Tanakaetal.
,2001,2004;Wellesenetal.
,2001;Chenetal.
,2003;Kurataetal.
,2003;Schnurretal.
,2004;Kurdyukovetal.
,2006)may,infact,beassociatedwithdefectsinthecuticularlayerofthecellwallorinthecuticleproper.
Inturn,molecularcloningofseveralgenesapparentlyinvolvedintheformationofcuticleinArabidopsishasraisedfurtherquestionsconcerningtheinterrelationshipsbetweenvariouscomponentsoftheepidermalcellwallandthemechanismsbywhichthesegenesfunctioninadiversesetofsignalingpathways(YephremovandSchreiber,2005).
Lipidpolymersconstitutethemajorbarrieragainstinvasionbypathogensandrestrictcell–cellinteractions.
Toallowpollen–pistilinteractionstooccurandtoenablepathogenstoinvadetheplant,lipidcellwallpolymershavetobeweakenedordegraded.
Cutinasescapableofdoingthishavebeenisolatedfromphyto-pathogenicfungi(Solidayetal.
,1984),andthepresenceofenzymeswithasimilaractivityhasbeenreportedinplants(Hiscocketal.
,1994,2002;LavithisandBhalla,1995).
However,incontrastwiththecaseinfungi,correspondingplantgeneshavenotbeenmolecularlyidentied.
Also,thepostulatedsynthasesinvolvedinthelaststepsinthebiosynthesisofcutinandsuberin,andinattachinglipidstoothercomponentsofthecellwall,havenotyetbeenidentied.
Thesynthesisandhydro-lysisofextracellularpolymershavetobecoordinatedwithexten-sionofthecellsurfaceduringgrowth,buthowthisisachievedalsoremainstobeexplained.
Here,wereportthemolecularcloningoftheBODYGUARD(BDG)gene,whichisrequiredfortheformationofthecuticleinArabidopsis.
BDGisanepidermis-specicextracellulara/b-hydrolasefold–containingprotein.
Structuralanalysisshowedthatcellwallandcuticularmaterialsarelargelymisplacedinthemutant,andacontinuousfunctionalcuticleproperisnotformed,thusexplainingitsvariousphenotypes.
Theseresultsarecon-sistentwiththeideathatBDGmayfunctioninthecuticularlayerofthecellwallorcuticleproperbyservingasasynthase,butparadoxically,thelossofitsfunctioninthebdgmutantresultsinanoverallincreaseinthelevelofcutinandwaxconstituents.
RESULTSMorphologicalandCuticularDefectsAssociatedwiththeLossoftheBDGFunctionDuringregularmutantscreensofanArabidopsispopulationmutagenizedwiththemaize(Zeamays)transposonEnhancer/Suppressor-mutator(En/Spm),twomutantfamilies,6W61and6W32,wereselectedforstudybecausetheybothsegregateddwarsh,bushyplants.
Closerexaminationrevealedthatthesemutantsoftendisplayleaffusions,whichcanbequiteprominent,especiallyiftheplantsaregrownrstundershort-daycondi-tions.
Atearlystagesofdevelopment,both6W61and6W32(Figure1C)wereclearlydistinguishablefromwild-typeplants(Figure1B),becausetheyweresmaller,oftenproducedde-formedleaves,andshowedreducedpubescence.
Attheageofseveralweeks,mutantplantsexhibitedstronggrowthretarda-tionandabnormalleafmorphology(Figure1A);however,theseverityofthesephenotypesvaried.
Generally,mutantswerecharacterizedbyhavingcurved,moreelongatedleaveswithslightlyserratededges.
Theleafdeformationswereobservedonbothmutantsindependentlyoftheoccurrenceofleaffusions.
Someleaffusionsweresostrong,itresultedintearingoflaminaeandpetiolesduringgrowth(Figure1E),similartowhatoccurredinlcr(Wellesenetal.
,2001).
Despiteageneralgrowthdeciencyinbothmutants,theyproducednormal-lookingfertileowersandgaverisetoseeds.
Itwasnotedalsothatsprayingwithfungicides,whichwerewelltoleratedbywild-typeplants,wasdeleterioustothesemutantsandresultedinnecroticlesionsandultimatelyindeath(datanotshown).
ComplementationtestsontherecessivemutantsW32andW61showedthattheywereallelic.
Becausethephenotypesofthesemutantssuggestedthattheyweredefectiveintheformationofthecuticle,themutantlocuswascalledbdg,andthemutantallelesW32andW61weredesignatedbdg-1andbdg-2,respectively.
Scanningelectronmicroscopyofplantsgrowninthegreen-houseshowedthatepidermalcellsofbdgareshrunken.
Al-thoughbdgplantswereabletodevelopmaturetrichomes,thesefrequentlyappearedtobeattenedorotherwisemisshapen(Figures1Fand1G).
Shrinkageprobablycouldaccountformostofthesedeformations,becausetrichomeslatercollapsedandappeareddehydrated,havingaatshapeandadheringtopavementepidermalcells(Figure1G);however,theinvolvementofmechanismsspecicallycontrollingcelldeathcannotbeexcluded.
Theepidermisseemedtobetheprimarytargetofthemutation;however,parenchymatouscellsbecameaffectedascelldeathspreadfromtheleaftips(Figure1D).
Examinationofsuturezonesinbdgrevealedanetworkofbrousmaterialbetweennormallyseparatepartsoftheplant(Figure1H),similartothatfoundinsuturezonesoflcrmutants(Wellesenetal.
,2001).
Thesendingssuggestedthatthecellwallsareindirectcontactwitheachotherinsuchzones.
Areductionintheisolatingpropertiesoftheoutermostepidermalcellwallaffectedinthemutantalsoaccountsforthegerminationofwild-typepollengrainsontheleafsurfaceinbdgplants(Figure1I),theenhancementofchlorophyllleachingin80%ethanol(Lolleetal.
,1997)(Figure2A),andtheincreasedstainingwithaqueoussolutionsofthecationicdyetoluidineblue(Tanakaetal.
,2004)(Figure2B).
Intheserespects,bdgissimilartomostmutantsthatshoworganfusions(Lolleetal.
,1998).
Toprovethatthecuticleisindeedimpairedinbdg,wein-spectedtheoutermostcellwalloftheepidermisbytransmissionelectronmicroscopy.
Inelectronmicrographs,thecuticleofthewild-typeleafofArabidopsisisseenasacontinuousandregularlayeredstructure:anouterelectron-densemembraneof20to322ThePlantCell30nmcorrespondingtothecuticleproperisdepositedoveralessdenselayercorrespondingtothecellwall(Figure3A).
Thestructureofthecuticleinthebdgmutant,ontheotherhand,ischaracterizedbyaseriesofirregularlayersinwhichelectron-denseandlessdensematerialsofthecellwallareintermingledwithemptyspaces(Figures3Bto3E).
Inmanycases(Figure3C),theoutermostcellwallboundaryisprotectedbyonlyamini-malamountoftheelectron-denseoverlay,whichiscomposedmainlyofextracellularlipidpolymers(e.
g.
,cutin)(Kolattukudy,2001a).
Theformationofmultilayeredstructuresiscorrelatedwithanincreaseinthethicknessofthecuticularlayer(Figures3Dand3E).
Irregulardepositionofelectron-opaquematerialoccursnotonlyattheoutermostsurfacebutalsowithinthedeeperlayersofthecellwall.
Emptyspacesareoccasionallyfound,givingthiscomplexstructureacavernousappearance(Figures3Cto3E).
Inplaces,largeamountsofelectron-opaquematerialaredepositedwithinthecellwall,whereastheouterboundaryitselfishardlyprotectedatall(Figure3E).
Thecavernousstructureofthecuticleinbdgsuggeststhatunderlyingcellwallconstituentsmaybulgeoutcontinuouslyasaresultofthefragilityoftheoutercoat.
Interestingly,thecuticularlayerinthemutantisthickeroverall,andtheamountofextra-cellularlipidpolymersdoesnotappeartobereduced.
Asexpected,nocuticleproperisformedbetweenthewallsoftheepidermalcellsinthesuturezones(Figure3F).
Thus,theoutermostlayerofthecellwallismainlyaffectedinbdgmutants,makingitlikelythatBDGisinvolvedinthepathwayresponsiblefortheformationofeitherthecuticularlayerofthecellwallorthecuticleproper.
PhenotypeofbdgunderinVitroCultureConditionsBasedonthephenotypicperformanceofbdgmaintainedinthegreenhouse,itisconceivablethattheinabilitytoformthecuticlemakesbdgplantsvulnerabletonaturalenvironmentalconditionsthatdonotadverselyaffectthegrowthofwild-typeplants.
Totestthisnotion,bdgplantsweregrownonhormone-freeMurashigeandSkoog(MS)mediumsupplementedwithagarundercon-ditionsof100%humiditytoavoiddehydrationstress.
Indeed,Figure1.
PhenotypeofthebdgMutantofArabidopsis.
Thepanelsshowphotographs([A]to[E])andscanningelectronmicro-graphs([F]to[I]).
Bars10mmfor(A),5mmfor(B)and(C),2mmfor(D),200mmfor(F),100mmfor(G),and10mmfor(H)and(I).
(A)Whengrownnaturallyinshortdaylengths(8hoflight),6-week-oldmutants(right)havesmaller,oftendeformedleavesandmaystunttheirgrowth.
Comparewiththewild-typeplantatleft.
(B)and(C)Wild-type(B)andbdg(C)plantscanbeeasilydistinguishedattheageof3weeks.
Notethesmallsizeofbdgleafbladesandthereducednumbersoftrichomesonthem.
(D)Typicalnecroticlesionsaffectingboththeepidermisandthemesophyllatthetipsofleavesinbdg(arrows).
(E)Deformationofabdgplantcausedbygraft-likefusionsofrosetteleaves.
Notetheruptureoftwopetioles(lines1and2showupperandlowerrips)causedbyextremelystrongfusionsbetweengrowingorgans.
(F)Generalviewoftheleafsurfaceshowingdefectsintheepidermisinbdg.
Sometrichomesarefullydeveloped,butmanyaremisshapenandarrestedatearlierstages.
Inmanycases,trichomesbecomeattened,bent,andeventuallyadheretotheleafsurface(arrow).
(G)Collapseofmaturetrichomes.
(H)Asuturezone(arrow)showingnosignsofcuticleproper.
(I)Germinationofwild-typepolleninducedbycontactwithbdgleafepidermis.
Epidermis-SpecicExtracellularBDG323undertheseconditions,themutantsdidnotexhibitstronggrowthretardationduringtherst2weeks.
Onthecontrary,bdgplantshadtwotimesgreaterdryweightyieldsofleafandroottissuethanwild-typeplants(Figure4A).
Mutantplants,however,exhibitedcharacteristicmorphologicalfeaturesofbdg.
Theydevelopeddeformedleaves(Figures4Band4D)thathadthick,shortpetioles,andoccasionalleaffusions.
Degenerativechangesinbdgwererstcharacterizedbytheprogressivedeteriorationoftrichomesandtheappearanceofrupturesintheleafepidermisthatwereobservedinseverelydeformedleavesandleavesthatappearedalmostwildtype.
Incontrastwithwild-typetrichomes,whicharecharacterizedbyabulbousstalkdominatinglongitudinallyexpandedsubsidiarycells(Figure4E),bdgtrichomesoftenlackedabulbousexpansionatthebase,whichappearedtobelocatedinthedepressionarea(Figure4F),Figure2.
DefectsintheEpidermalCellSurfaceofbdgLeavesRevealedbyLeachingofChlorophyllandStainingwithaCationicDye.
(A)Intactrosetteleavesofbdgreleasechlorophyllfasterthandowild-typeleaveswhenimmersedin80%ethanol,probablybecauseboththesolventandsolutemoreeasilypenetratetheleafsurface.
Eachvalueisanaverageofsixreplicates.
BarsindicateSE.
DifferencesbetweenColumbia(Col)andbdgweresignicantatP1.
8usingawindowsizeof19correspondtoapossibleN-terminalsignalpeptide(1)andahydrophobicregioninthea/b-hydrolasedomain(2).
NTHD,N-terminalhydrophilicdomain;SP,signalpeptide.
(C)Active-siteSerregionsofvariousa/b-hydrolases.
Shadinghighlightsaminoacididentitiesfoundin>35%ofcases.
Shownatleftarefunctionallyandstructurallyrelatedgenefamilies:(1)cutinasesandcutinase-likeproteins;(2)BioHfamily;(3)BDGfamily;(4)monoglyceridelipaseandlysophospho-lipases;(5)putativelysophospholipasesofArabidopsis;and(6)variouslipasesandesterases.
Epidermis-SpecicExtracellularBDG331wasdetectedthatcouldcorrespondtothenucleus,cytoplasm,vacuoles,orothercellcompartments.
Inparticular,nouores-cencethatlocalizedtoacharacteristicreticularnetworkwasob-served,suggestingthatBDGdoesnotresideintheendoplasmicreticulumandGolgiapparatus.
However,becauseofthelimitingresolutionofthemethod,itcannotbeexcludedthatperipherallylocalizedpartsoftheseorganellescontributetothesignalinthevicinityoftheoutermostcellwall.
Theuorescencesignalwasobservedintheinnerbutnottheouterportionoftheoutermostepidermalcellwallhardenedbycuticlesecretion(Figures10Fand10G).
Cutinizationoftheouterportionofthecellwallisexpectedtocauseasterichindrancetotheaccessofantibodiestoantigenicsites.
Therefore,itremainstobedeterminedwhetherBDGwasnotdetectableintheseareasofthecellwallbecauseofantigenmaskingorlowerproteinlevels.
DISCUSSIONSequenceComparisonsSuggestaNovelFunctionforBDGItisparticularlydifculttopredictthefunctionofamemberofthea/b-hydrolasesuperfamily,suchasBDG,solelyonthebasisofitsaminoacidsequence,becausesequencesimilaritiesaregenerallylow,andbecausemembersofthesuperfamilycatalyzethehydrolysisofawidevarietyofsubstratesandincludelipases,esterases,epoxidehydrolases,acyltransferases,andSerpro-teases(NardiniandDijkstra,1999).
Synthasesbelongingtothissuperfamilywerealsodescribed(Belisleetal.
,1997;Tonge,2000).
Nevertheless,familiesofproteinscanberecognizedonthebasisofsimilaritiesinthree-dimensionalstructuresandfunc-tions.
Morethan30suchfamilieshavebeendenedinthea/b-hydrolasesuperfamily(Hotelieretal.
,2004).
However,theBDGfamily,comprisingseveralgenesinArabidopsisandrice(Figure8C),doesnotappeartobecloselyrelatedtoanyofthem.
ItfollowsthatthefunctionofBDGisnovelandlikelytobespecictoplants.
BDGGeneFunctionintheFormationoftheEpidermalCellWallandCuticleAsdescribedindetailabove,mutationsinBDGresultintheseveredeformationofepidermalcellwallsandirregularFigure9.
Tissue-SpecicExpressionofBDG.
SeriesofimagesshowingGFPuorescenceintransgenicArabidopsisplantsexpressingGFPunderthecontroloftheBDGpromoter([A]to[E])andinsitumRNAexpressionpatternsofBDG([F]to[I])inwild-typeplants.
TheGFPimageswereacquiredusingaconfocallaserscanningmicroscope.
GFPuorescenceisgreen(channel520to540nm),andautouorescenceisred(channel>590nm).
FormRNAexpressionanalysis,tissuesectionswerehybridizedwithadigoxigenin-labeledriboprobeantisensetoBDGcDNA.
Afterimmunodetection,usinganalkalinephosphatase–conjugatedanti-digoxigeninantibody,thesec-tionswereinspectedwithalightmicroscope.
Bars100mmfor(A)to(C),200mmfor(D)to(F),and50mmfor(G)to(I).
(A)Longitudinalsectionthroughavegetativeapex.
(B)Lateral(secondary)rootprimordium.
(C)Opticalsectionthroughthelateralroottip.
(D)Longitudinalsectionthroughayoungoralbudshowingthepistil(pi)andthestamen(st).
(E)CrosssectionthroughaoralbudatalaterstageshowingthatstrongGFPuorescenceisretainedattheabaxial(external)sideofthepistil.
AweakerGFPsignalisalsovisibleinovules(ov),petals(pe),andse-pals(se).
(F)Crosssectionthroughavegetativeshootapexofashort-day-grownwild-typeplant.
NotetheparticularlystrongexpressionofBDGmRNAintheepidermisontheanksofleafprimordia.
(G)Thepartof(F)insidetherectangleismagniedanddisplayed.
(H)Longitudinalsectionthroughavegetativeshootapexofalong-day-grownwild-typeplant.
BDGexpressionisdetectedintheepidermis.
(I)CrosssectionthroughaoralbudshowingBDGexpressionintheadaxialepidermisinprefusioncarpels.
Atthisstage,BDGishardlydetectableinotherowerorgans.
332ThePlantCelldepositionofelectron-opaquematerial,whichrepresentscutin.
Whereasinmanycasesonlyathinelectron-densemembranecoversthelightlystainedareasoftheoutermostcellwallboundary,depositionofextracellularlipidpolymerswithinthedeeperlayersofthecellwallresultsintheformationofmultilay-eredstructures.
Thecuticularphenotypeofbdg,whichincludesthestructurallydefectivecuticleproper,suggeststhatthemutantmaybeunabletosynthesizeorsecretesufcientquan-titiesofoneormorecutinmonomers.
However,incorrespon-dencewiththeoverallenhanceddepositionofelectron-densepolymericmaterial,theamountsofinsolublelipids,representingtypicalcutinmonomers,associatedwithcellwallsareincreasedbytwofoldtothreefoldinbdgplants.
Together,theseresultsimplythatbiosynthesisofcutinprecursorsisprobablynotadirecttargetofthebdgmutationanddistinguishbdgfromthecuticularmutantsofArabidopsisisolatedinpreviousstudies.
Forexam-ple,itwasshownthattheatt1mutantformsalessosmiophiliccuticle,butmorethantwiceasthickthanthatofthewildtype,andexhibitsasignicantdecreaseinthelevelsofallcutinmonomers,exceptforoctadecadien-1,18-dioicacid.
TheATT1geneencodesCYP86A2,acytochromeP450monooxygenasecatalyzingfattyacidv-oxidation(Xiaoetal.
,2004).
LACS2encodesalong-chainacyl-CoAsynthasethatcatalyzesthesynthesisofv-hydroxyfattyacyl-CoA.
Thethicknessofthecutinlayerontheabaxialsurfaceoflacs2leavesisreducedby30%comparedwiththatofwild-typeplants,butitscompositionwasnotdetermined(Schnurretal.
,2004).
Theace/hthmutant,decientinfattyacidv-alcoholdehydrogenaseactivity,showsinleafpolyester–specicreductionofthelevels(;70to80%ofwildtype)ofa,v-dicarboxylicfattyacids,whicharethemajorconstituentsofcuticularpolyesters(Bonaventureetal.
,2004;Yephremovetal.
,2004)andcutin(Frankeetal.
,2005)inArabidopsis.
Theace/hthmutantistheonlymutantdescribedtodateinwhichthechangesinthestructureofthecuticlearereminiscentofthoseexhibitedbybdg.
Inbothmutants,thecuticlemaybediscontinuousormultilayered;however,overallenhanceddepositionofelectron-densepolymericmaterialwasnotobservedinace/hth,andthetotalamountofcovalentlyboundlipidsisinsignicantlydecreasedinace/hth(Kurdyukovetal.
,2006).
Interestingly,ace/hthandbdgnotonlybothdisplayadisruptedcuticleproperbutalsoexhibitorganfusionpheno-types,whereaslacs2andatt1/CYP86A2donotshoworganfusions.
Thebdgmutantaccumulatesapproximatelythreetimesasmuchwaxmonomersasthewildtype,andthealkaneswithoddnumbersofcarbonscontributemosttothisincrease.
RT-PCRanalysisofbdgshowsthetranscriptionalactivationofgenesinvolvedinthedecarbonylationpathway,whichcatalyzestheformationofalkaneswithodd-numberedchainlengths.
Thus,intermsofgenetics,BDGisarepressorofwaxbiosynthesis,althoughthisdoesnotmeannecessarilythatBDGisadirectrepressor.
ItismorelikelythatthelossofcrucialstructuralcharacteristicsofcellwallpolymersandthecuticleproperinbdgtriggerswaxbiosynthesisviatheupregulationofSHN1/WIN1andCER1.
Thisresponse,however,isnotabletofullycompen-sateforthestructuraldefectsinthecuticleandthereducedepidermalbarrierfunctioninbdg.
Itwasreportedthatlacs2showsa1.
5timesincreaseinthetotalwaxloadcomparedwithFigure10.
SubcellularImmunolocalizationoftheStrep-TagEpitope-TaggedBDGProtein.
Crosssectionswerecutfromtheparafn-embeddedvegetativeshootapexesofshort-day-grownplants.
Afterincubationofthesectionswithanepitope-specicmonoclonalantibody,theuorescenttyramidesignalamplicationmethodwasusedtodetectBDG–Strep-tag.
Imagesweretakenwithauorescencemicroscope([A]to[C])oraconfocallaserscanningmicroscope([D]to[G]).
Bars500mmfor(A)and(B),250mmfor(C),40mmfor(D)and(E),and15mmfor(F)and(G).
(A)Avegetativeshootapexinbdgusedasanegativecontroltogetherwiththatofawild-typeplant(datanotshown).
(B)to(G)AvegetativeshootapexinbdgcomplementedwithaconstructcarryingtheBDGproteinC-terminallytaggedwiththeStrep-tagepitopesequenceunderthecontrolofthenativeBDGpromoter.
Notethatdeformationintheshapeoftheleafprimordiaoccursinbdg(A)butnotinthecomplementedmutant(B).
Specicuorescentsignalsintheepidermis(arrows)weredetectedonlyinbdgBDG–Strep-tagplants([B]to[G])butnotinnegativecontrols.
Amagniedviewoftheleafprimordiaboxedin(B)isshownin(C).
(D)and(F)Transversesectionscorrespondingtothelightmicroscopeimagesin(E)and(G),respectively.
TheStrep-tag–specicuorescentsignalisfoundlocalizedintheoutermostepidermalcellwalls(arrows).
Epidermis-SpecicExtracellularBDG333wild-typeplants(Schnurretal.
,2004).
Thisistwicelessthanforbdg.
Furthermore,theamountsofthemajorconstituentsofthewax,C29,C31,andC33alkanes,wereincreased9.
4,4.
6,and2.
3timesinbdg,respectively,whereasinlacs2,C29andC31alkaneswereincreased1.
7and1.
3times,respectively,buttheleveloftheC33alkanewasnotchanged(Schnurretal.
,2004).
Therefore,itseemslikelythateitherdistinctmechanismsac-countfortheincreaseofthewaxaccumulationinlacs2andbdgorthatthelattershowsamuchstrongercompensatoryre-sponse.
WithregardtothefunctionoftheBDGgene,twopossiblescenarioscouldexplaintheobservedcuticulardefects.
First,toexplainthedeformationofcellwallsseeninbdg,onecanassumethatBDGisspecicallyinvolvedinthepostulatedcross-linkingoftheoutermostlayerofepidermalcellwallsthatinvolvesa,v-dicarboxylicfattyacids(Kurdyukovetal.
,2006)and/orthepolymerizationofcutinpolyesters.
Inthisscenario,cross-linkingprovidesmechanicalstabilitytotheoutermostlayerofthecellwall,whichnormallypreventstheunderlyingmaterialfrombulgingoutward.
Theincreaseintheamountsoflipidsboundtocellwallsinbdgcouldbeaccountedforif,undertheseconditions,moresitesareavailableatwhichthepolymerizationofcutinmonomerscanbeinitiated.
Indeed,thendingthatBDGisexpressedintherootdermalcellssuggeststhatBDGmayhaveamoregeneralroleinthebiogenesisoftheoutermostcellwallsthanincutinbiosynthesisinaerialtissues.
Thisndingissupportedbyreportsshowingtheexpressionofseveralgenes,suchasLACS2(Schnurretal.
,2004),FDH(Yephremovetal.
,1999;Pruittetal.
,2000),LCR(Wellesenetal.
,2001),WAX2/YRE(Kurataetal.
,2003),andACE/HTH(Krolikowskietal.
,2003;Kurdyukovetal.
,2006),thatarerequiredforcuticledevelopmentinroots.
Subaerialtissueslackavisiblecuticle-likelayeronthesurface,andtransmissionelectronmicroscopyandhistochem-icalstainingrevealdepositionofsuberininthecellwallsintheendodermisandperidermbutnotintheepidermalcellwalls(Frankeetal.
,2005).
Nevertheless,theexpressionofACE/HTH,whichisprobablydirectlyinvolvedinthebiosynthesisofa,v-dicarboxylicfattyacids,intherootepidermissuggeststhatthesemoleculescontributetocovalentlyboundlipidsassociatedwithcellwalls.
Althoughthealiphaticcompositionoftherootepider-malcellwallsisnotknown,thepresenceofaromaticcompoundsiseasilydetectedbyuorescencemicroscopy(Frankeetal.
,2005).
Therefore,itseemslikelythatlong-chaindicarboxylatesarepresentastheiresterstomonolignols(p-coumaryl,coniferyl,andsinapylalcohols)orglycerol,similartowhathasbeenproposedforsuberin(Bernards,2002).
AlthoughadirectinvolvementofBDGinthebiosynthesisofthecuticularlayerofthecellwallorcutiniscurrentlyoneofthemostappealinghypothesesforthefunctionofthisprotein,thealternativescenarioshouldbeconsidered:thatBDGplaysnopartinthebiosynthesisofcuticlebutrathercontributestothecontrolofcellproliferationanddifferentiation.
Inthisscenario,itisprimarilytheinuenceofthecellstatusthatlimitstheabilitytoformthecuticle,whereastheaccumulationofcutinmonomersispreservedorenhanced.
Basedonthemutantphenotypeundertissuecultureconditions,BDGcouldbeconsideredanegativeregulatorofcellproliferation.
Insupportofthisview,thepheno-typesofbdgandpasticcino2/pepinomutantspartiallyoverlap,becausemutationsinPASTICCINO2/PEPINO(Bellecetal.
,2002;Habereretal.
,2002)leadnotonlytouncoordinatedcelldivisions,uncontrolledgrowth,cellproliferation,andcallusformationbutalsotoorganfusions,strikinglysimilartothoseinbdg,fdh,andlcr.
Thus,BDGmaycodeforanextracellulara/b-hydrolasefold–containingprotein(e.
g.
,lysophospholipase)thefunctionofwhichisnotdirectlyrelatedtocuticle.
CouldBDGBeaSynthaseCapableofCatalyzingReactionsintheCuticularLayeroftheCellWallDespitethefactthatBDGmaybeinvolvedindistinctmolecularmechanismsnecessaryforcuticleformation,itisreasonabletoassumethatBDGactsdirectlyasanextracellularsynthase.
Therearethreelinesofevidencethatsupportthishypothesis.
First,immunolocalizationanalysis(Figure10)revealedthatBDGispolarlylocalizedinthecellandaccumulatesintheoutermostcellwalllayeroftheepidermis,atthesidewherecuticularlipidsaredeposited.
ThisresultarguesagainsttheindirectinvolvementofBDGincuticleformation.
ComputerizedanalysisoftheBDGproteinsequencerevealedthepresenceattheNterminusofaputativesignalpeptide,followedbyahydrophilicdomainandana/b-hydrolasedomain,supportingtheimmunolocalizationdataandtheideathatBDGissecretedtotheextracellularspace.
Second,thecellwalldeformationsseeninbdg,characterizedbythestraticationofthecuticularmaterial,interruptedbyunlledpockets,areverysimilartothoseseenintransgenicArabidopsisplantsthatexpressacutinasefromthepathogenicfungusFusariumsolanipisi(Sieberetal.
,2000).
Inparticular,thezonesoftheoutermostcellwallofbdg,atwhichthecuticleproperislargelyabsent,arereminiscentoftheoutermostcellwallsofleavesofcutinase-expressingplants(Figure3C)(C.
Nawrath,unpublisheddata).
Althoughcutinaseswerenamedfortheirunparalleledabilitytohydrolyzecutin(Solidayetal.
,1984),theycanmetabolizealargevarietyofcarboxylicesters(Kolattukudy,1984;Lauwereysetal.
,1991).
Therefore,defectsinthecuticularlayerofthecellwallofthesetransgenicplantsmaybeexplainedbythebroadhydrolyticactivityofthecutinase.
Similardefectsinbdgcanbeattributedtothelossofanextracellularsynthaseinvolvedintheformationofesterbondsincutinand/orthecutinizedlayerofthecellwall(e.
g.
,betweena,v-dicarboxylicfattyacidsandmonolignolsorbetweena,v-dicarboxylicfattyacidsandv-chainandmid-chainhydroxylsoffattyacids).
Third,BDGandcutinases,whicharesmallextracellularcarboxylicesterhydrolasesofphytopathogenicfungiwithmolecularmassesof;22to25kD,bothbelongtothesuperfamilyofenzymesthatcontainthea/b-hydrolasefoldandarelikelytosharethesamecatalytictriad.
GenesencodingsecretedproteinsverysimilartocutinaseshavebeendiscoveredinthegenomesofMycobacteriumspecies,whicharethecausativeagentsoftuberculosisandleprosyinhumans.
Basedonstructuralandaminoacidsequencesimilarities,theseproteinsbelongtothecutinasefamily(Hotelieretal.
,2004),butobviouslytheyarenotcutinases.
Itwasrecentlyfoundthattheyactasmycolyltrans-ferasesduringcellwallbiosynthesis,catalyzingtheestericationoftrehalose(twoD-glucoseresiduesjoinedbytheiranomericcarbons)totwomycolicacidresidues,whicharevery-long-chaina-alkyl,b-hydroxyfattyacids(Belisleetal.
,1997;Tonge,2000).
334ThePlantCellManya/b-hydrolasesshowcatalyticexibility:whereastheseenzymescatalyzecondensationsinorganicsolventscontainingonlytracesofwater,theycatalyzehydrolyticreactionsinaque-ousmedia(BornscheuerandKazlauskas,1999).
Anonpolarmilieucontainingsomewaterispresumablycharacteristicofthecu-ticularlayerofthegrowingcellwallandseemstoprovideanadequateenvironmentforbothreactiondirectionsrequiredtomaintaincuticleintegrity.
Therefore,itisnotunlikelythatBDGmayprovetoexhibitbothsynthaseandhydrolaseactivitiesinthecellwall.
Despitethesimilarityincuticlestructurebetweenthecutinase-expressingandbdgplants,theirphenotypesarenotidentical.
Thus,thelargeamountsofcuticularmaterialthataccumulatewithinthecellwallsofthebdgmutantshaveneverbeenseeninleavesofcutinase-expressingplants,implyingthat,unlikecutinase-expressingplants,bdgispossiblymorecapableofcross-linkingcutin-likepolymers.
Theenlargedcuticularlayerobservedintheleavesofbdg,however,mayhaveacertainsimilaritytothecavernousstructureofmixedcompositionofthecuticlesofstemsincutinase-expressingplants(Sieberetal.
,2000).
Thus,BDGisanattractivecandidategeneforfurtherexploringthehypothesisthatplanta/b-hydrolasesarecapableofcatalyzingcondensationreactionsinthecuticularlayerofthecellwall.
CellWallIntegrity,CellDeath,andMorphogenesisinbdgLossoftheBDGfunctionhasadramaticeffectonthemorphol-ogyoftheoutermostepidermalcellwall.
Itappearsthattheproperstrengtheningoftheprimarycellwallensuresitsregulardepositionandshouldbeinconcertwiththeexpansionofthecellsurface.
Microscopictearsintheepidermisofbdgseemtoresultfromdisruptionsofjunctionsbetweenlateralcellwalls,indicatingthatextracellularlipidpolymersarerequiredformaintainingtissueintegrity.
Thefailureofthebdgmutanttopreservein-tercellularjunctionsbetweenepidermalcellsmayaccountforuncoordinatedmorphologicalchangesandresultinthedefor-mationoforgans.
Furthermore,itispossiblethatthecompres-sionoftheinnertissuebytheepidermisisweakenedinbdg,therebyresultingingreaterextensibilityofthecorticaltissuesandpromotingboththedeformationandgrowthoforgans.
Undertissuecultureconditionsthatreducestressinjury,thiseffectisseeninbdgduringtheinitial10-dperiod;however,bdgplantsgrowninthegreenhouseexhibitstuntedgrowthfromtherstdaysandoftendie,probablyasaresultofacombinationofdehydrationandstress.
Thedeleteriouseffectofthebdgmutationoncellviabilitymaybeillustratedinbdgbythedeathofmaturetrichomecellsthatoccursasleavesunfold.
Inthecourseofthisprocess,bdgtri-chomesundergomorphologicalchangesthatarenoticeablyreminiscentofthoseobservedincyclin-dependentkinasein-hibitorICK1/KRP1-misexpressingtrichomes(Schnittgeretal.
,2003).
Strikingly,thetrichomedeathcanonlybedelayedbutnotfullyavoidedbygrowingbdgundertissuecultureconditions.
Trichomedifferentiationisalsodelayedandreducedinbdg,facilitatingtheidenticationofbdgatthethree-tofour-leafstage,beforemostfeaturesofthephenotypearevisible.
However,whetherthisisadirecteffect(e.
g.
,relatedtocellwallintegrity)oranindirecteffect(e.
g.
,relatedtoastressresponseoftheplant)ofthemutationremainstobeinvestigated.
Thewax2/yremutationstronglyreduces(toone-sixthofthatinthewildtype)theamountofepicuticularwaxandsomewhatinuencesthemorphogenesisoftrichomes(Kurataetal.
,2003).
Theun-branchedstemtrichomeswereseverelystuntedanddeformedinthedoubleyrecer1mutantbutnotinthecorrespondingsinglemutants.
BecauseCER1isalsoknowntobeinvolvedinwaxbiosynthesis,thisindicatedtotheauthorsthatepicuticularwaxisrequiredforthenormalgrowthoftrichomes(Kurataetal.
,2003).
However,accumulationofepicuticularwaxisgreatlyenhancedinbdg;therefore,anothermechanismmustberesponsiblefortheobservedtrichomedefects.
ThedecreaseinnumbersoftrichomesandchangesintrichomebranchingpatternswereobservedintransgenicplantsoverexpressingtheSHN1/WIN1gene(Aharonietal.
,2004),whichappearedtobeactivatedaswellasCER1inbdg.
SHN1/WIN1itselfisabletoupregulateCER1andseveralotherwaxbiosynthesispathwaygenes(Brounetal.
,2004).
Therefore,itislikelythatSHN1/WIN1accountsforsimilartrichomedefectsinbdg.
SHN1/WIN1,whichencodesanethyleneresponsefactor–typetranscriptionfactor,probablyplaysaroleinthecontrolofcellularprocessesthatgodistinctlybeyondwaxbiosynthesis.
Itisworthnotingthatseveralcuticularmutants,includingfdh(Yephremovetal.
,1999),lcr(Wellesenetal.
,2001),andcer10(Zhengetal.
,2005),exhibittrichomephenotypes,indicatingthatthedeformationofcuticleinitiatesacascadeofeventsultimatelyaffectingthefateofcells.
Inthisrespect,theregulationofcuticleformationisparticularlyinterestingintheseptumofthepistil,becausethistissuepar-ticipatesinepidermalcell–cellinteractionsandrespondstothecontact.
Resemblingthatinanumberofcuticularmutants(Lolleetal.
,1998),pollengerminationcouldbeinducedontheleafepi-dermisofbdg(Figure1I).
ResultsofexpressionstudiesusingtheGFPreportergeneindicatedthatthedownregulationofBDGintheinnerovarywalloftherapidlygrowingpistil(Figure9E)maycon-tributetocell–cellinteractionsbetweenpollenandtheepidermis.
TheRoleofBDGinCellSignalingResponsesItappearsthatBDGisanepidermis-specicextracellulara/b-hydrolasefold–containingproteinrequiredforcuticleformation;however,itsexactroleinthisprocessremainsunknown.
NeitherBDGmRNAnortheBDGproteintrafcsfromtheepidermistosubepidermalcells.
Therefore,whetherBDGisanenzymethatiscapableofcatalyzingcondensationreactionsinthecuticularlipidpolyestersoranchoringthelipidpolymerstothecarbohy-drateconstituentsofthecellwalls,aninterestingquestionishowitcanmodulatemeristemproliferation,changesinmorphology,andcelldifferentiation.
Also,thereisnosimpleexplanationforthestrikingincreaseintheproductionoftheepicuticularwaxinbdg,evenifitisaresponsetothelossofcellwallandcuticleintegrity.
Theinvolvementoflipid-derivedsignalshasbeenproposedtoexplainthepleiotropicphenotypeofthelcrorgan-fusionmutant,whichincludesalteredcelldifferentiationintheepidermis,re-ducedapicaldominance,delayedsenescence,andirregularshapeofleaves(Wellesenetal.
,2001).
Furtherevidenceinsupportoftheideathatcuticlemaybeasourceofsignalsforplantsisgivenbythereportthatcutinase-expressingplants(Sieberetal.
,2000)thatareverysimilartobdgwithregardtodeformationsofthecellEpidermis-SpecicExtracellularBDG335wallandcuticleshowstrongresistancetothenecrotrophicfungusBotrytiscinerea(Chassotetal.
,2004).
Interestingly,bdgalsopossessesacertainlevelofresistancetothisfungus,whereastrichomesareseverelyaffectedincutinase-expressingplantsaswellasinbdg(C.
Nawrath,unpublisheddata).
Inanycase,thenatureofthesignalsthatinducemetabolicchangesandacelldifferentiationresponseintheepidermisanddiffusefromtheepidermisthroughoutthemeristemtotriggercellproliferationisintriguing.
METHODSGrowthConditionsArabidopsisthalianaplantsweregrowninagreenhouseat22to238Cundereitheran8-hphotoperiod(shortday)ora16-hphotoperiod(longday)at50to60%RH.
Forinvitroexperiments,seedsweresurface-sterilizedbyapplyinga70%ethanolsolutionfor10minanda10%bleachsolutionfor15min.
Seedswerethenwashedundersterileconditions,keptforsynchroniza-tionin0.
1%agarforafewdaysinthedarkat48C,andplatedonagarcontaininghalf-strengthMSmediumand3%sucrose.
Finally,plantsweretransferredaftergerminationontoagar-solidhalf-strengthMSmediuminglasspotsorplatesandgrownunderlong-dayconditionsinaclimatechamber(16hoflight/8hofdark,21to228C).
MolecularIsolationofthebdg-1TransposonInsertionAlleleandReverseGeneticsLeafmaterialandseedswerecollectedfrom30mutantandwild-typeplantssegregatingfortherevertingW32mutant,andtheseedsweresownforprogenytesting.
Then,threeheterozygousandthreehomozy-gousW32mutantplantswereusedfortransposoninsertiondisplaywiththe39endtransposonprimersasdescribedpreviously(YephremovandSaedler,2000;Steiner-Langeetal.
,2001).
Asingle800-bpfragmentcommontoallsixplantswasdetected,butinitialattemptstoreamplifyDNAsequencescutfromasilver-staineddenaturingpolyacrylamidegelfailed.
Tocircumventthisdifculty,separationoftransposoninsertiondisplayproductswasconductedwithTransgenomic'sWAVEsystem(equippedwithafractioncollector)runinnondenaturingmodeat508C.
ThereversegeneticsscreenforEn/Spminsertionswasperformedessentiallyasdescribedpreviously(Steiner-Langeetal.
,2001)usinga2-kbgeneprobepreparedfromgenomicDNAbyPCR.
Characterizationofthe59and39TerminiofBDGmRNAThe59and39endsoftheRNAweredeterminedbyRACEusinga59/39RACEkit(RocheAppliedScience).
ModicationsoftheprotocolincludedreplacingdATPbydGTPfortailing.
The59and39RACEproductswereanalyzedandpuriedbyagarosegelelectrophoresisandclonedintothepGEM-Tvector.
Twentyclonesweresize-selected,andthevelongestweresequenced.
TransgeneConstructsandGenerationofTransgenicPlantsFortransgeniccomplementationofthebdg-2mutant,theBDGgene(withitsnativepromoter)wasampliedfromgenomicDNAofecotypeColwiththeprimersKS3F(59-CGAAGCTTATGTATGTCAGTCGTCGTTGACC-TCTGC-39)andKS4R(59-CTGAGCTCCGAAATTGGACGCACATTGCA-AACCACT-39),whichintroducedHindIIIandSacIcloningsites(underlined),respectively,atthe59and39ends.
ThePCRfragments,digestedwithHindIIIandSacI,wereligatedintothecorrespondingcloningsitesofthebinaryvectorpBHS(Wellesenetal.
,2001).
Acolony-poolingstrategy,whichallowedustoavoidsequencingbeforethetransformationofplants,wasappliedasfollows.
IndependentcoloniesofEscherichiacoliDH10B(eightclones)containinginsertswerecom-binedforplasmidDNAisolation.
AgrobacteriumtumefaciensGV3101wastransformedwiththeplasmidDNA,andpositivecolonies(16clones)werecombinedagainforinplantatransformationofthemutantasdescribedpreviously(Wellesenetal.
,2001).
Complementationwasindicatedbytheappearanceofplantswithwild-typemorphologyinseveral(seveninthiscase)independenttransgenicfamilies.
Forexpressionstudies,theputativepromoteroftheBDGgenewasampliedasan886-bpfragmentusingPbdgHindIII(59-GAGTCGGAAGCTTGATGCCACGCACACGTCCTTG-39)andPbdgXba(59-CGG-TCTAGACAAAATGCGTGAGAGAGAGAA-39)asprimers,subclonedintopGEM-T(Promega),andveriedbysequencing.
ThepromoterwastheninsertedintopBHS-basedreporterbinaryvectorscontainingaGUSorGFPmgfp5-ER(HaseloffandSiemering,1998)geneasareporter.
TogenerateacomplementationconstructinwhichtheBDGproteinwasfusedtotheStrep-tagIIepitopetagunderthecontrolofthenativeBDGpromoter,the3.
5-kbgenomiccopyofBDGwasampliedusingPbdgHindIIIandPbdgXma(59-GCTCTGGGCCCATTTATTGAATGAAGT-TGAGGAG-39)asprimers,digestedwithHindIIIandXmaI,andclonedintothepBHS-basedbinaryvectorpB-ctSTcontainingonecopyoftheStrep-tagIIsequence(IBA).
Intheresultingconstruct,theBDGproteinsequencewasfollowedbythesequencePGSAWSHPQFEK,whichcontainedthefour–aminoacidpeptidelinkerandtheStrep-tagIIepitopetag(underlined).
Thepromoter–reporterfusionsandtheepitope-taggedproteincon-structwerethentransformedintowild-typeArabidopsis(Col)viaAgro-bacteriumasdescribedabove.
StainingwithToluidineBlueandChlorophyllLeachingPlantsweregrowninagreenhouseundershort-dayconditionsfor7to8weeksbeforeexperiments.
ThestainingmethodwasadaptedfromTanakaetal.
(2004).
RosetteleavesfromColandbdgmutantplantswerestainedatroomtemperaturefor2minwithoutshaking,usingafreshlyprepared0.
05%solutionoftolu-idineblue.
Leaveswererinsedwithtapwaterbeforebeingphotographed.
Thechlorophyll-leachingmethodwasadaptedfromLolleetal.
(1997)withminormodications.
Sixsampleswereprepared,eachcomprising15to17rosetteleaves(;0.
5g)takenfromdistinctplants.
Thesampleswereweighedbeforetheadditionofethanol(80%).
Theextractionwasconducedat228Cinarotating(18rpm)waterbath.
Aliquotsweretakenafter10,20,40,60,and80min.
Todeterminethetotalamountofchloro-phyll,theleafsampleswereplacedat908Cfor15minandcooledonicebeforethenalaliquotwastaken.
MeasurementswereperformedusingaUVIKON810/820spectrophotometer(Kontron),andcalculationsweredoneasdescribedpreviously(Lolleetal.
,1997).
MicroscopicTechniquesandinSituHybridizationCryoscanningelectronmicroscopyandtransmissionelectronmicros-copywereperformedasdescribedbyYephremovetal.
(1999)andSieberetal.
(2000),respectively.
Forconfocalmicroscopy,plantsorplantparts(transgenicplantsexpressingGFP,andColplantsasacontrol)wereembeddedin5%low-meltagarose(Biozym,Hess)containing0.
005%SilwetL-77(OSiSpecialities).
Sections(100to150mmthick)weremadewithaLeitz1512Vibratomeandviewedwithaconfocallaserscanningmicroscope(LeicaTCS4D)(Efremovaetal.
,2004).
Forinsituhybridizationprobesynthesis,a1.
6-kbfragmentwasampliedfromaBDGcDNAcloneusingprimers(BDG-SP6,59-CTC-GAGTTTAGGTGACACTATAGAACTGGAGGAAACCCTGCTACTGCTGT-CCG-39;BDG-T7,59-CTCGAGTAATACGACTCACTATAGGGAGATCA-GACCATCTTGGTGTTCTTTGC-39)thatweredesignedtoincludeT7336ThePlantCellandSP6RNApolymerasepromotersites(BDG-specicsequencesareunderlined)ontheankingends.
TheT7polymerasetranscribedtheanti-sensedigoxigenin-labeledprobeusedforthedetectionofBDGtran-scriptsinsectionedtissueasdescribedpreviously(Perbaletal.
,1996).
ThesenseprobetoserveasanegativecontrolwasobtainedusingT7RNApolymerase.
Othercontrolsincludedepidermis-specicprobes(datanotshown).
ImmunocytochemistryUsingTyramideSignalAmplicationVegetativeapexesof30-d-oldplantsgrownundershort-dayconditionswerexedinmethanol:aceticacid(3:1)at208Covernight.
Afterxation,chlorophyllwasremovedin70%ethanolat208C.
ThetissuesweredehydratedinagradedethanolseriesandembeddedinParaplastPlus(TycoHealthcare).
Sections(6mm)werecutwithamicrotome,placedontoSuperfrostPlusGOLDmicroscopeadhesiveslides(Menzel-Glaser),air-driedfor24hat428C,deparafnizedbywashinginHistoclear,andrehydratedinanethanolseries.
Heat-inducedepitoperetrievalwasperformedwith0.
01Msodiumcitrate,pH9.
0,at808Cfor30min.
ThesectionswererinsedinTris-bufferedsaline(TBS;0.
1MTris,pH7.
5,and0.
15NaCl)containing0.
1%Tween20(TBST),incubatedin2%H2O2inTBSfor1h,blockedwith5and1%BSAinTBST,andthenincubatedovernightat48CwiththemouseIgGmonoclonalantibody(naldilution,1.
2mg/mLin1%BSAinTBST)againstStrep-tagIIpeptide(IBA).
ThesectionswerewashedinTBSTandincubatedfor1hatroomtemperaturewiththetyramidesignalamplicationkithorseradishperoxidaseconju-gateofgoatanti-mouseIgGantibody(MolecularProbes)diluted1:100in1%BSAinTBST.
Todetectsecondaryantibody,slideswerewashedextensivelywithTBST(threetimesfor30minatroomtemperature),rinsedwithTBS,andincubatedwithAlexaFluor488tyramide(MolecularProbes)for10to15minaccordingtothemanufacturer'sspecications.
AfternalwashingswithTBST(threetimesfor30minatroomtemper-atureandovernightat48C),thecoverslipsweremountedonmicroscopeslidesusinga50%solutionofglycerol.
ThesampleswereexaminedwithaLeicaMZFIIImicroscopeusingaGFPlterandaLeicaTCSSP2confocallasermicroscope.
Intheconfocalsystem,uorescencewasexcitedat488nmandemissionwasreadinthe495-to540-nminterval.
AnalysisofResidualBoundLipidsThedetailsofthisprocedurehavebeendescribedelsewhere(Frankeetal.
,2005;Kurdyukovetal.
,2006).
Briey,wild-typeandmutantplantsweregrownuntilowering.
Matureleaveswereharvestedandtheirareasmeasuredbyscanning.
Solublelipidswereremovedfromsamplesbyextensivesteepinginamethanol:chloroformmixture.
Thisdelipidationtreatmentallowedtheextractionofmostofthecharacteristicmembranelipids,asnotracesofthehighlyunsaturatedfattyacids16:3and18:3weredetected(Kurdyukovetal.
,2006).
Afterdrying,theleafextractwasweighedandstoredoruseddirectlyforGC-MSanalysis.
WaxAnalysisRosetteleaves(10to20)of5-week-oldplantswerecutandimmediatelyimmersedinchloroformfor10satroomtemperature.
Theresultingsolutionofcuticularwaxeswasspikedwith10mgoftetracosane(Fluka)asaninternalstandard.
Thesolventwasevaporatedunderastreamofnitrogen,andcompoundscontainingfreehydroxylandcarboxylgroupswereconvertedintotheirtrimethylsilylethersandesters,respectively,withbis-(N,N-trimethylsilyl)-triuoroacetamide(Machery-Nagel)inpyri-dinefor40minat708CbeforeGC-MSanalysis.
Waxconstituentswereidentiedbytheirelectron-impactMSspectra(70eV,m/z50to700)aftercapillaryGC(DB-1,30m30.
32mm,0.
1mm[J&W])onanAgilent6890Ngaschromatographcombinedwithaquadrupolemass-selectivede-tector5973N(AgilentTechnologies).
Sampleswereinjectedontothecolumnat508C,heldat508Cfor2min,andthendesorbedbyincreasingthetemperatureaccordingtothefollowingprole:408C/minto2008C,2minat2008C,38C/minto3108C,and30minat3108C.
TheowrateofHecarriergaswas2mL/min.
QuantitativedeterminationofwaxcomponentswasperformedwithanidenticalGCsystemequippedwithaame-ionizationdetector.
Theextractedleafareawasdeterminedbasedonthepixelnumberofdigitizedphotocopiesoftheleaves.
QuantitativeRT-PCRTheplantstakenforRT-PCRexperimentswerethesameplantsusedfortoluidinebluestainingandchlorophyllleaching(seeabove).
Threein-dependentsampleseachrepresentingthewildtypeandbdgwerecompared.
Foreachsample,ten0.
5-to1-cmleavestakenfromdifferentplantswerecombined,andtotalRNAwasextractedwiththeRNeasyplantminikit(Qiagen).
Eachreactioncontained500ngoftotalRNA,gene-specicprimers,andcomponentsfromtheOneStepRT-PCRkit(Qiagen).
Primersforactin2(At3g18780)andSHN1/WIN1(At1g15360)weredescribedpreviously(Aharonietal.
,2004;Brounetal.
,2004);WAX2/YRE(At5g57800)wasampliedwiththeforwardprimer59-AAGCATCCTGACCTTAGAGTTCGTGTGGTTCAT-39andthereverseprimer59-TAAGACCATACTTCATGGCTGCTTCCCACA-39;CER1(At1g02205)wasampliedwiththeforwardprimer59-CAGGAACGGAGAGGTGTA-TATCCACAACCAT-39andthereverseprimer59-GTATCTATCATACCA-CACCAGCATTGATAG-39.
AllRT-PCRtargetswereampliedwithinthelinearrange.
Optimalcyclenumberwasdeterminedforeachgeneindividually:itwas36forSHN1/WIN1and24forallothergenes.
Amplicationswererepeateduptofourtimes;PCRproductswereseparatedona1.
5%agarosegelcontaining0.
25mg/mLethidiumbromideandquantiedwithaTyphoon8600uorescencescanner(AmershamBiosciences).
AccessionNumbersTheaccessionnumbersforthegenesdiscussedinthisarticleareasfollows:At1g02205(CER1),At1g15360(SHN1/WIN1),At1g64670(BDG),At1g72970(ACE/HTH),At1g49430(LACS2),At2g26250(FDH),At2g45970(LCR),At5g57800(WAX2/YRE),At4g00360(ATT1),At3g55360(CER10),andAt5g10480(PASTICCINO2/PEPINO).
ProteinsinthealignmentshowninFigure8areasfollows(GenBankaccessionnumbersareindi-catedinparentheses):Ao_CutL,Aspergillusoryzaecutinase(D38311);Mt_Cut2,Mycobacteriumtuberculosismycolyltransferase(Z77163);Cc_Cut,Colletotrichumcapsicicutinase(M18033);Fs_Cut,Fusariumsolanicutinase(AAA33334);Ec_BioH,Escherichiacolicarboxylesterase(X15587);At_BDG,ArabidopsisthalianaBDGgeneAt1g64670;At_BDG2,ArabidopsisBDG2geneAt5g41900;At_BDG3,ArabidopsisBDG3geneAt4g24140;At_BDG4,ArabidopsisBDG4geneAt5g17780;At_BDG5,ArabidopsisBDG5geneAt5g17720;Os_HYD1,Oryzasativaputativehy-drolase(AP003818);Os_HYD2,O.
sativaputativehydrolase(AC092389);Os_HYD3,O.
sativaputativehydrolase(AAO33147);Agt_pldB,Agro-bacteriumtumefacienslysophospholipase(AE009319);Rm_pldB,Rhi-zobiummelilotilysophospholipase(AL591791);Rl_pldB,RhizobiumlotilysophospholipaseL2(AL591791);Hi_pldB,HaemophilusinuenzaelysophospholipaseL2(U32747);Hs_Llpl,Homosapienslysophospholi-pase(AB017494);At_Lpl1,ArabidopsislysophospholipaseAt1g18360(AY070740);At_Lpl2,ArabidopsislysophospholipaseAt5g11650(AAM62693);At_Lpl3,ArabidopsislysophospholipaseAt1g73480(AY045929);At_Lpl4,ArabidopsislysophospholipaseAt5g16120(AY051019);Rm_lip,Rhizo-mucormieheitriacylglycerollipase(P19515);Gs_est30,Geobacillusstearothermophilusesterase(AY186197);Dm_AchE,Drosophilamela-nogasteracetylcholinesterase(P07140);Pf_estB,Pseudomonasuo-rescenscarboxylesterase(S79600);andMm_Es1Musmusculuscarboxylesterase(AAC04708).
Epidermis-SpecicExtracellularBDG337SupplementalDataThefollowingmaterialsareavailableintheonlineversionofthisarticle.
SupplementalTable1.
CompositionAnalysisofResidualBoundLipidsinbdgandWild-TypeLeaves.
SupplementalTable2.
CompositionAnalysisofEpicuticularWaxinbdgandWild-TypeLeaves.
SupplementalFigure1.
GenomicOrganizationofBDG.
SupplementalFigure2.
RNAGelBlotHybridizationof10mgofTotalRNA.
ACKNOWLEDGMENTSWethankcolleaguesfromtheZentrumzurIdentikationvonGenfunk-tionendurchInstertionsmutagenesebeiArabidopsisthalianaproject,inparticularChristianeHorst,forhelpinguswiththereversegeneticsscreen,MartineSchorderet,UlrichRyser,andElmonSchmelzerforhelpinguswithmicroscopy,AldonaRatajek-Kuhnfortakingcareofourplants,andRIKENforprovidingcDNAclonesforourstudy.
WeespeciallyappreciatethecriticismsofthemanuscriptmadebyPaulHardy.
ThisworkwassupportedbyaMax-Planck-Gesellschaftfellow-shiptoS.
K.
,MarieCurieHostFellowshipsforearly-stageresearchertrainingtoD.
V.
,aDeutscheForschungsgemeinshaftresearchgranttoL.
S,andResearchGrantNF3100A0-104224/1fromtheSwissNationalScienceFoundationtoJ.
-P.
M.
ReceivedJuly19,2005;revisedNovember11,2005;acceptedDecem-ber2,2005;publishedJanuary13,2006.
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1105/tpc.
105.
0360792006;18;321-339;originallypublishedonlineJan13,2006;PLANTCELLRochusFranke,LukasSchreiber,HeinzSaedler,Jean-PierreMétrauxandAlexanderYephremovSergeyKurdyukov,AndreaFaust,ChristianeNawrath,SaschaBr,DerryVoisin,NadiaEfremova,ArabidopsisMorphogenesisinTheEpidermis-SpecificExtracellularBODYGUARDControlsCuticleDevelopmentandSupplementalDatahttp://www.
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