Arabinogalactan Proteins Are Required for Apical Cell Extension in the Moss Physcomitrella patens

15 Pages·2005·0.56 MB·English

ThePlantCell,Vol.17,3051–3065,November2005,www.plantcell.orgª2005AmericanSocietyofPlantBiologists
Arabinogalactan Proteins Are Required for Apical Cell
Extension in the Moss Physcomitrella patens
KieranJ.D.Lee,a,1YoichiSakata,b,1Shaio-LimMau,cFilomenaPettolino,cAntonyBacic,cRalphS.Quatrano,b
CeliaD.Knight,aandJ.PaulKnoxa,2
aCentreforPlantSciences,UniversityofLeeds,LeedsLS29JT,UnitedKingdom
bDepartmentofBiology,WashingtonUniversity,St.Louis,Missouri63130-4899
cPlantCellBiologyResearchCentreandCooperativeResearchCentreforBioproducts,SchoolofBotany,
UniversityofMelbourne,3010Victoria,Australia
Cellbiological,structural,andgeneticapproacheshavedemonstratedthepresenceofarabinogalactanproteins(AGPs)in
themossPhyscomitrellapatensandprovidedevidencefortheirfunctionincellexpansionandspecificallyintheextension
ofapical tip-growingcells.Inhibitorstudies indicated thatapicalcell expansion inP.patens isblockedbysyntheticAGP
bindingb-glucosylYarivreagent(bGlcYR).Theanti-(1!5)-a-L-arabinanmonoclonalantibodyLM6bindstosomeAGPsin
P.patens,toallplasmamembranes,andtothecellwallsurfaceatthemostapicalregionofgrowingprotonemalfilaments.
Moreover,LM6labelingofcellwallsatthetipsofapicalcellsofP.patenswasabolishedinthepresenceofbGlcYR,sug-
gestingthatthelocalizedmovementofAGPsfromtheplasmamembranetothecellwallisacomponentofthemechanism
oftipgrowth.BiochemicalandbioinformaticanalyseswereusedtoidentifysevenP.patensESTsencodingputativeAGP
core proteins from homology with Arabidopsis thaliana, Brassica napus, and Oryza sativa sequences and from peptide
fragmentsisolatedfrombGlcYR-precipitatedAGPs.Geneknockoutbyhomologousrecombinationofoneofthesegenes,
P.patensAGP1,encodingaclassicalAGPcoreprotein,resultedinreducedcelllengthsinprotonemalfilaments,indicating
aroleforAGP1inapicalcellexpansioninP.patens.
INTRODUCTION forresearchonthestructureandfunctionsofprimarycellwalls
andtheircomponents.Arabinogalactanproteins(AGPs),agroup
ThemossPhyscomitrellapatensisanestablishedexperimental
ofbiochemicallycomplexproteoglycansassociatedwithplasma
bryophyte system for the study of genetics and development,
membranes and cell walls and belonging to a Hyp-rich glyco-
and its study provides insight into land plant phylogeny and
proteinsuperfamily,aredefinedonthebasisoftheircorepeptide
evolution(Rensingetal.,2002;Nishiyamaetal.,2003).P.patens
sequence motifs, the presence of abundant type II arabino-
possessesanactivesomatichomologousrecombinationsystem
galactanglycans,andtheirabilitytospecificallyinteractwitha
that is highly suited to reverse genetic approaches to probing
classofsyntheticphenylglycosidesknownasb-glycosylYariv
genefunction(SchaeferandZryd,1997;Hiwatashietal.,2001),
reagents(Nothnagel,1997;Gasparetal.,2001;Showalter,2001;
has been the subject of significant EST generation programs
Johnson et al., 2003a). Moreover, AGPs were the first known
(Rensing et al., 2002; Nishiyama et al., 2003), and is a highly
familyofabundantplantproteinsidentifiedtohaveaglycosyl-
accessible system for cell biological analysis (Reski, 1998;
phosphatidylinositol (GPI) anchor attachment to plasma mem-
Schipper et al., 2002). For these reasons, P. patens is an at-
branes(Youletal.,1998;OxleyandBacic,1999;Sherrieretal.,
tractiveorganismtousetoelucidateprocessesfundamentalto
1999;Sveteketal.,1999).FoursubclassesofGPI-anchoredAGP
plantcelldevelopment.
backbonesarecurrentlyrecognized:classicalAGPswithachar-
Bryophyte and angiosperm evolution diverged more than
acteristic Pro-rich module for the entire predicted protein
400 million years ago, but recent studies have shown that in
betweenanN-terminalsecretionsignalsequenceandahydro-
terms of cell wall composition, considerable similarities exist
phobicCterminusthatisprocessedforGPIanchorattachment,
(Ligroneetal.,2002;PopperandFry,2003;Kremeretal.,2004;
AGPs with Lys-rich domains, arabinogalactan peptides with
Matsunagaetal.,2004).P.patensisthereforeausefulspecies
shortproteinbackbones,andfasciclin-likeAGPs(FLAs),aclass
ofchimericAGPsthatcontainbothAGPmotifsandafasciclin
domain.AfurtherclassofAGPsexists,knownasnonclassicalor
1Current address: Department of Biosciences, Tokyo University of
chimericAGPsthatdonothaveaGPIanchorandAGPmotifs
Agriculture,1-1-1Sakuragaoka,Setagaya-ku,Tokyo156-8502,Japan.
that are restricted to a part of the predicted protein sequence
2To whom correspondence should be addressed. E-mail j.p.knox@
leeds.ac.uk;fax[phone]. (Schultz et al., 2000, 2002; Johnson et al., 2003a, 2003b). In
The author responsible for distribution of materials integral to the addition to the complexity of protein components, most of an
findingspresentedinthisarticleinaccordancewiththepolicydescribed AGPmoleculecomprisescomplexbranchedglycansbasedon
in the Instructions for Authors (www.plantcell.org) is: J. Paul Knox
a(1/3)(1/6)-b-galactanframeworkdecoratedwitharangeof
([email protected]).
Article, publication date, and citation information can be found at terminalsugars,including arabinose(Nothnagel,1997;Gaspar
www.plantcell.org/cgi/doi/10.1105/tpc.105.034413. et al., 2001; Tan et al., 2003, 2004). In some instances, short
3052 ThePlantCell
oligoarabinosidesarealsopresent(Goodrumetal.,2000).AGP chloronemalcellsarisefromthebranchingofcaulonemalcells.
glycan components are heterogeneous, and evidence from Both of these cell types grow by apical extension and serial
studieswithantiglycanmonoclonalantibodiesindicatesexten- divisionoftheapicalcell(Coveetal.,1997).Subapicalcellsdo
sive regulation of AGP glycan structure in relation to cell de- notextend,buttheycandividetoformsidebranchinitialsthat
velopment,althoughthefunctionalsignificanceofthisisnotyet developeitherascaulonema,secondarychloronema,orintoa
known(Knox,1997;Showalter,2001). budthatgivesrisetoaleafyshootthatisthegametophore(Cove
Although AGPs are implicated in many aspects of cell de- etal.,1997).P.patensprotonemataaregrownreadilyonboth
velopment, including cell proliferation, cell expansion, organ solid and liquid media. Using cell biological, structural, and
extension,apoptosis,germination,andsomaticembryogenesis, bioinformaticapproaches,wehavedemonstratedthepresence
precise biochemical or cell biological functions have been ofAGPsinP.patens.Usingbothinhibitorandreversegenetic
difficult to elucidate (Majewski-Sawka and Nothnagel, 2000; tools, we demonstrate a role for AGPs, specifically that of
Gaspar et al., 2001; Showalter, 2001). Approaches to dissect P.patensAGP1,whichencodesaputativeclassicalAGPcore
AGP function have included the addition of exogenous AGPs protein, in protonemal apical cell extension, the majormecha-
to developing systems, the use of b-glycosyl Yariv reagents nismofgrowthinthefilamentousformofthisorganism.
(bGlcYRs) or anti-AGP antibodies as in vivo inhibitors of AGP
action,and,recently,thegeneticmanipulationofAGPcorepro-
teins.SeverallinesofevidenceimplicateAGPsincellexpansion RESULTS
and organ elongation. The application of bGlcYR blocks the
elongation of suspension-cultured carrot (Daucus carota) and GrowthCharacteristicsofP.patensandGrowthInhibition
tobacco (Nicotiana tabacum) cells (Willats and Knox, 1996; byAGPBindingbGlcYR
Vissenbergetal.,2001),indole-3-aceticacid–inducedextension
Foreaseofexperimentalmanipulation,smallinoculaofP.patens
ofcucumber(Cucumissativus)hypocotyls(Darleyetal.,2001),
cellscanbesubculturedvegetativelyasanalternativetospore
andcellelongationinArabidopsisthalianaseedlingroots(Willats
germination (Cove et al., 1997). This involves the removal of
andKnox,1996;DingandZhu,1997;McCartneyetal.,2003).
aclusterof;50protonemalcellsandtheirdirecttransfertofresh
Treatment of lily (Lilium longiflorum) pollen tubes with bGlcYR
media where they regenerate into new colonies within a few
uncoupledexocytosisofcellwallpolymersfromextensionatthe
pollentubeapex,indicatingarolefortheseproteoglycansintip
growth of pollen tubes (Roy et al., 1999; Mollet et al., 2002).
ExogenousAGPshavebeenreportedtopromotedevelopment
ofsomaticembryos(Showalter,2001)andhavebeenproposed
tobothpromoteandguidethetipgrowthofpollentubes(Cheung
etal.,1995;Wuetal.,2000).Arabidopsismutantswithreduced
hypocotylextension(Takahashietal.,1995)androotgrowth(Ding
andZhu,1997)havereducedlevelsoralteredAGPs.Themur1
mutant has nonfucosylated AGPs, and this also results in re-
ducedrootgrowth(vanHengelandRoberts,2002).Agibberellin-
responsivegeneexpressedincucumberhypocotylsencodesan
AGP core protein that is involved in stem elongation, its over-
expressionpromotinggrowth(Parketal.,2003).Overexpression
ofthegeneencodingtomato(Lycopersiconesculentum)AGP1
resultedinchangestobothgrowthanddevelopment(Sunetal.,
2004).Todate,mutationsingenesencodingArabidopsisAGP
coreproteinsindicateextremelysubtleeffectsonplantgrowth.
MutationscausingthelossofArabidopsisAGP17,FLA4(SOS5),
and AGP30 resulted in no altered phenotype under standard
growthconditions,anddistinctphenotypesonlybecameappar-
entwhengrowninthepresenceofAgrobacteriumtumefaciens,
high salt, or abscisic acid, respectively (Nam et al., 1999; Shi
etal.,2003;vanHengelandRoberts,2003;Gasparetal.,2004).
The polar secretion of a hybrid AGP/lipid transfer protein has
Figure1. ReversibleInhibitionofGrowthofP.patensonSolidMediaby
been identified asacomponentof cell–cellinteractions during bGlcYR.
vasculardevelopmentinArabidopsis(Motoseetal.,2004).
P.patenscultureswereinoculatedoneitherBCD/ATmediumalone(no
InP.patens,boththegerminationofhaploidsporesandthe
YRcontrol)(A)ormediumwith1mMbGlcYR(B)or1mMaManYR(C)and
regeneration of protoplasts result in the development of net-
allowedtogrowfor7d.After7dofgrowth,allculturesweretransferredto
works of branched filaments known as protonemata. A pro-
control media without YR for a further 7 d ([D] to [F]). Growth in the
tonemainitiallycompriseschloronemalcellswithcharacteristic presenceofthenon-AGPbindingYR,aManYR,wasequivalenttogrowth
perpendicularcrosswalls.Subsequently,caulonemalcells(with oncontrolmedium.ColoniesinhibitedbybGlcYRresumedgrowthupon
oblique cross walls) arise from chloronemal filaments. In turn, transferoftheculturetofreshmedium(E).Bars¼1mm.
AGPsinPhyscomitrellapatens 3053
days.Theresultingprotonematadevelopinasimilarmannerto bGlcYRwasreversibleasgrowthresumedupontransfertofresh
thosearisingfromspores,regardlessoftheinitialinoculumsize. controlmediawithoutbGlcYRforafurther7d(Figure1E).
StudieswithAGPbindingbGlcYRindicatedthatthegrowthof
P. patens is sensitive to this reagent. The inclusion of 1 mM
bGlcYRReversiblyBlocksApicalCellExtensionover
bGlcYRinsolidgrowthmediumresultedinacompleteinhibition
ShortTimePeriods
ofcolonygrowthovera7-dperiod(Figure1B).Growthwasnot
disrupted by the presence of 1 mM a-mannosyl Yariv reagent The small size of P. patens and its single-cell filamentous
(aManYR),anon-AGPbindinganalogofbGlcYR.Theeffectof growth allowed observations on single extending apical cells
Figure2. TreatmentwithbGlcYRInhibitsExtensionofApicalCellsofProtonemata.
(A)P.patensculturesinoculatedineitherliquidBCDmediaalone(noYRcontrol)orwitheither1mMbGlcYRor1mMaManYR.Imagesoftheapical
caulonemalcellunderobservationweretakenat1-hintervals.Thedottedblacklinethrougheachmicrographindicatesthemostapicalregionofthecell
atthestartofobservation.Thearrowheadsindicatethetipofapicalcellsafter1-and2-hgrowthperiods.InthepresenceofaManYR,growthisthe
sameasincontrolmediawithoutYR.InthepresenceofbGlcYR,theapicalcelldidnotextend.Bars¼10mm.
(B)HistogramshowinggrowthratesforcaulonemalandchloronemalcellsinP.patensculturesinoculatedineitherliquidBCDmediaalone(noYR
control)orwith1mMbGlcYRor1mMaManYR.Datashownareforsixexperimentspertreatment,andtheerrorbarsindicatethestandarderrorofthe
mean.
3054 ThePlantCell
Figure3. Time-LapseImagesShowingtheRapidInhibitionofCellExtensionbybGlcYRandtheRecoveryofCellExtensionActivityafterItsRemoval.
CultureswereinoculatedintoliquidBCDmediainaperfusionchamber,andmicrographsweretakenofthesameapicalcellsat1-hintervalsforupto
10h.After2h,bGlcYR(1mM)wassuppliedtothechamberandremovedafterafurther1h(t¼3).Theapicalcellwasthenobservedforafurther6h.The
images shown areofachloronemal apical cell,andthetypicalgrowth rateof suchcells is5 mm/h.Extensiongrowthceased immediately upon
applicationofbGlcYR.Theapicalcellwasunabletoextendforupto3hfollowingtheremovalofbGlcYR.Afterthistime,growthrecommenced,and
anormalgrowthrateapproaching5mm/hwasachievedby5hafterremovalofbGlcYR.Blackarrowsindicatestartpointsofthetipoftheapicalcellat
t¼0andalsoatpointofapplicationofbGlcYR(t¼2).Whitearrowheadsindicatethepositionofthetipoftheapicalcellineachimage.
in response to bGlcYR. Cultures, 3 d after initiation on agar, of 1 mM aManYR had no effect upon apical cell extension
weretransferredtoperfusionchamberscontainingliquidmedia. (Figure 2).
Uponadditionof1mMbGlcYR,animmediatecessationofapical Analysisofculturestreatedfor1hwith1mMbGlcYR,which
cell extension (within 30 s, at the resolution of our time-lapse blockedgrowth,followedbyremovalofbGlcYRbyflushingthe
equipment)wasobserved(Figure2A).Instandardgrowthcon- perfusion chamber with fresh control medium, indicated that
ditions with unilateral light of constant intensity, chloronemal abGlcYR-inhibitedapicalcellwasunabletoextendforaperiod
cellsexhibitanaverageextensionrateof5mm/h,whilecaulo- of ;3 h after removal of the inhibitor, as shown in Figure 3.
nemal cells exhibit an average extension rate of ;16 mm/h However, ;4 h after removal of bGlcYR, the extension of the
(Figure 2B). After addition of 1 mM bGlcYR, no cell extension apical cell recommenced, and the apical cell reestablished a
was observed for either cell type. The equivalent application normalrateofcellextension(Figure3).
Figure4. SDS-PAGEandProteinGelBlotsofbGlcYR-PrecipitatedMaterial,aTotalProteinExtractofP.patensGrownonSolidMedia,andMaterial
SecretedintotheMediumfromP.patensinLiquidCulture.
bGlcYR-precipitatedmaterial,atotalextractofprotonemata(Cells)andapreparationofculturemediumfroma14dliquidcultureofP.patens(Med.)
weresubjectedtoSDS-PAGEandproteingelblotting.bGlcYR-reactivematerialappearedasasmearonadenaturinggelwithanapparentmolecular
massintheregionof180kD(lane2),characteristicofAGPs.Coomassieblue(CB)stainingofanequivalentgeldidnotdetectanyproteinbandsinYR-
precipitated AGPs (lane 1) or culture medium (lane 7). Immunoblotting indicated that bGlcYR-precipitated AGPs were bound by anti-(1/5)-a-L-
arabinanLM6(lane3)andanti-AGP-glycanJIM13(lane4).Loadingperlanewas10mgcarbohydrateforYR-precipitatedAGPsandmediumpreparation
(Med.;lanes7and8)and10mgproteinforthetotalextractofP.patenscells(Cells;lanes5and6).m,markers;YR-ppt,YRprecipitate.
AGPsinPhyscomitrellapatens 3055
Furthermore,thelackof1,2-linkedRhapand1,4-linkedGalApis
Table1. Monosaccharide-LinkageCompositionofbGlcYR-Precipi-
further evidence that the bGlcYR-precipitated AGP fraction is
tatedAGPsfromP.patens
essentiallyfreeofpecticpolysaccharides.
Monosaccharide DeducedLinkage Mol%a
Rhapb Terminal 18 ImmunolocalizationofP.patensAGPsinPlanta
Araf Terminal 14
2- 1 Immunolabelingofeitherwholemountorintactcellpreparationsis
3- 1 ausefultechniquetostudytheoccurrenceanddistributionofcell
5- 21 wallpolymersatthesurfaceoforgansorcells(Willatsetal.,2001).
Manp Terminal tr TheapplicationofthistechniquetoP.patensprotonematausing
4- tr
an antihomogalacturonan monoclonal antibody JIM5 (Clausen
Glcp Terminal tr
et al., 2003) demonstrated that this cell wall epitope could be
4- 1
detectedoverthe entire surface of a protonema (Figure 5A). In
GlcApc 4- 11
Galp Terminal 1
3- 7
6- 2
3,6- 23
Note:lowlevels(<0.3mol%)ofFucp(terminal),Xyl(terminal,4-linked),
Rha(2-,4-,2,4-linked),Glc(3-,4,6-linked),Man(2,4-,4,6-linked),and
Gal (2,6-, 2,3-, 3,4-, 4,6-, 2,3,6-, 4,3,6-linked) were detected but not
includedinthetable.tr,trace(<0.5mol%).
aAverageofduplicatedeterminations.
bTerminal Rha is deduced from 1,5-di-O-acetyl-6-deoxy-2,3,4-O-
methylhexitol,etc.
cDeduced from the neutral glucosyl derivative derived from carboxyl
reductionoftheglucuronosylreside.
AnalysisofbGlcYRBindingAGPsinP.patens
ToidentifythemoleculesthatarethetargetsforbGlcYRactionin
inhibiting apicalcellextension,bGlcYRwasusedtoprecipitate
material from 7-d-old protonemata. Separation of the bGlcYR-
precipitated material by SDS-PAGE is shown in Figure 4. No
proteins were detectable by Coomassie blue staining at a gel
loading of 10 mg carbohydrate. Staining of an equivalent gel
directlywithbGlcYRindicatedmaterialofhighmolecularweightin
excess of 180 kD (lane 2, Figure 4). The immunoblotting of
equivalentamountsofmaterialwithantiglycanmonoclonalanti-
Figure 5. The Distribution of Pectic Polysaccharides and AGPs in
bodies indicated that the most effective available antibody for
P.patens.
bGlcYR-precipitatedAGPsfromP.patenswasLM6,asshownin
Figure4(lane3).LM6bindsto(1/5)-a-L-arabinan(Willatsetal., (A) Indirect immunofluorescence labeling of an intact germinated
P. patens spore (12 d) with antihomogalacturonan JIM5. The JIM5
1998),whichinangiospermsismostcommonlyastructuralfeature
epitopeisabundantatthesurfaceofallcellwalls.Bar¼1mm.
ofthepecticpolysacchariderhamnogalacturonan-I(Willatsetal.,
(B)IndirectimmunofluorescencelabelingofanintactgerminatedP.patens
1998, 1999). The anti-AGP glycan monoclonal antibody JIM13
spore,equivalenttothatshownin(A)withanti-(1/5)-a-arabinanLM6.In
(Knox et al., 1991; Yates et al., 1996) also bound the bGlcYR-
contrastwiththeoccurrenceoftheJIM5epitope,theLM6epitopeismost
precipitatedAGPsbutlesseffectivelythanLM6(lanes3and4, abundantatfilamentapices(arrowheads).Bar¼1mm.
Figure4).LM6immunoblotsofatotalextractofP.patenspro- (C) Higher magnification of an apical cell from (B) displaying mild
tonemataandofmaterialsecretedintothemediumofaP.patens plasmolysis and showing a dual location of the LM6 epitope at the
liquidcultureindicatedbindingtomaterialwiththesameproper- most apicalregion ofboththeplasmamembrane(double arrowhead)
tiesasbGlcYR-precipitatedAGPs(lanes3and8,Figure4). andthecellwall(arrowhead).Bar¼20mm.
bGlcYR-precipitated AGPs were subjected to glycan mono- (D)IndirectimmunofluorescencelabelingofP.patensprotoplastswith
LM6. LM6 bound to the plasma membrane surface of all protoplasts
saccharide linkage analysis. Results (Table 1) indicated that
preparedfromprotonemata.Bar¼20mm.
1,5-linked Araf residues were a major feature of the bGlcYR-
(E)Indirectimmunofluorescencelabelingofasectionofresin-embedded
precipitated AGP glycans, confirming the observations made
protonemalcellswithLM6.TheLM6epitopeispresentatallcellsurfaces
with the monoclonal antibody LM6. Other than the 1,5-linked
andisparticularlyabundantinthecellwallatthetipofanapicalcell
Arafresidues,thelinkagecomposition,suchasthepresenceof (arrowhead).Bar¼20mm.
3,6-linked-Galp residues, terminal Rhap and GlcAp residues, (F) The same section as shown in (E) stained with Calcofluor White
is characteristic of angiosperm type II AGP glycans (Table 1). (Calc.),whichstainscellulose.
3056 ThePlantCell
contrastwithJIM5,immunolabelingofanintactprotonemawith
anti-(1-5)-a-L-arabinanLM6,indicativeofP.patensAGPs,shows
thatthisepitopewasrestrictedtothetipregionsofapicalcells
wherecellextensionistakingplace(Figure5B).Incubationcon-
ditionsthatfavorantibodybinding(i.e.,thepresenceofbuffered
saline)resultinthepartialplasmolysisofmosscells,andexam-
inationofthetipsofapicalcellsrevealedaduallocationofthe
LM6epitopeatboththecellwallandontheplasmamembrane
(Figure 5C). The presence of the LM6 epitope on all P. patens
plasma membranes was confirmed by the immunolabeling of
protoplasts prepared from protonemata (Figure 5D). The LM6
epitope was detectable atthe plasmamembrane surface of all
protoplasts immediately after protoplast isolation. As cell walls
regenerated and covered the plasma membrane, the ability of
LM6 to detectits epitope was generally lostuntil a growing tip
formed(datanotshown).Weproposethatitisonlyatthetipof
growingapicalcellsthatthecellwallissufficientlypermeableto Figure6. ImmunofluorescenceAnalysisoftheEffectoftheApplication
ofbGlcYRontheOccurrenceofLM6andJIM5EpitopesinP.patens
allowthepassageoftheprimaryandsecondaryantibodymole-
Protonemata.
culestoaccessepitopesattheplasmamembrane.Therefore,for
mostcells,the LM6 epitope cannot bedetectedat the plasma P.patenscultures(14d)weretransferredtoliquidmediawitheither1mM
membrane using the whole mount procedure. The presence of bGlcYR([A],[C],[D],and[F])or1mMaManYR([B]and[E])for3h.Oneset
the LM6 epitope at all plasma membranes was confirmed by of protonemata were fixed with 4% formaldehyde after transfer and
incubationinpresenceofYR([A],[B],[D],and[E]),andasecondset
theLM6labelingofsectionsofresin-embeddedmaterial(Figure
wereformaldehydefixedpriortoincubationwithYR([C]and[F]).After3hin
5E).Insomecases,theLM6epitopewasseentobeparticularly
thepresenceofYR,protonematawereimmunolabeledwithLM6orJIM5.
abundant in the cell wall region at the tip of an apical cell (see
IncubationoflivingcellsinthepresenceofbGlcYRresultedinthelossofthe
Figure5E),inadditiontoitspresenceattheplasmamembrane.
LM6epitopefromapicalcells(A).Theincubationoffixed,deadcellswith
bGlcYRhadnoeffectonLM6epitopeoccurrence(C).TheJIM5epitope
ApplicationofbGlcYRResultsintheLossofLM6Binding wasunaffectedbygrowthinthepresenceofbGlcYR([D]to[F]).Three
attheSurfaceofApicalCells representativefilamentsareshownforeachtreatment.Bar¼20mm.
Toinvestigatetheeventsrelatingtothecessationofgrowthby
bGlcYR, a series of experiments were performed in which approach. For the experimental approach, a sample of the
cultures were incubated in the presence of 1 mM bGlcYR or bGlcYR-precipitatedAGPswasdeglycosylatedwithanhydrous
1mMaManYRfor3h.Inonesetofexperiments,protonemata hydrogen fluoride (HF), and the resulting peptides were sepa-
were fixed with 4% formaldehyde after treatment with Yariv rated by reverse-phase HPLC and N-terminal Edman se-
reagent(YR).Inafurthersetofexperiments,protonematawere quenced. Four consensus peptide sequences were obtained.
fixedwithformaldehydebeforetreatmentwithYRfor3h.Cells Thesepeptidefragmentsshowedcharacteristicsofangiosperm
werethenimmunolabeledwiththemonoclonalantibodiesJIM5 AGPs,includingthepresenceofHypandAla.UsingtBLASTnto
andLM6usingthewholemountimmunolabelingprotocol,and searchtheNationalCenterforBiotechnologyInformationdata-
representativeresultsareshowninFigure6.Theapplicationof basewiththemajoraminoacidsandinsertingaProresiduein
bGlcYR resulted in either the loss of the LM6 epitope or its placeofblankcyclesduringsequencing(seeTable2),matches
accessibilityatthesurfaceofapicalcells(Figure6A),whereasthe to Arabidopsis AGP10 (At4g09030), AGP9 (At2g14890), and
application of aManYR did not (Figure 6B). However, bGlcYR AGP18(At4g37450)werefoundforsequencesfromP.patens
onlyresultedinthelossoftheLM6bindingwhenappliedtoliving peptides with retention times of 7.3, 9.1, and 15 min, re-
cells. When protonemata were formaldehyde fixed prior to spectively(Table2).ThesecorrespondtotwoclassicalAGPs
application of bGlcYR, there was no effect on LM6 binding (Arabidopsis AGP9 and AGP10) and a Lys-rich Arabidopsis
(Figure6C).ThebindingofJIM5wasunaffectedbyanyofthe AGP (AtAGP18). Since the P. patens AGP preparation con-
treatments(Figures6Dto6F).Weconcludethattheapplication tained multiple peptides (as expected) and not all could be
of bGlcYR to living cells results in a disruption of the normal sequenced, we are as yet unable to assign which peptide(s)
appearanceoftheLM6bindingAGPsinthecellwallatthetip. carries the LM6 epitope on the arabinogalactan chain. Al-
thoughunlikely,itisformallypossiblethatthisepitopeisalso
presentonallpeptides.
BiochemicalAnalysesofP.patensAGPsand
In a complementary bioinformatic strategy, a search of the
BioinformaticIdentificationofESTsEncoding
P.patensESTdatabasesusingtheentireangiospermAGPcore
PutativeP.patensAGPCorePeptides
peptide sequences as queries was also performed. Six ESTs
To obtain information on the protein backbones of P. patens encoding putative AGP core peptides were identified, and the
AGPs,twoparallelapproacheswereadopted:anexperimental predictedsequencesareshowninFigures7and8.AP.patens
approach with purified AGPs and a bioinformatic database sequence predicted to encode a classical AGP core protein,
AGPsinPhyscomitrellapatens 3057
Table2. SequenceAlignmentofPeptideFragmentsObtainedfromP.patensProtonematabGlcYR-PrecipitatedMaterialwithArabidopsisAGP
CoreProteinSequencesandaDeducedAminoAcidSequenceinthePHYSCObaseContigDatabase
N-TerminalSequences Alignmenta Match(AccessionNo.)
XVPPPAGSPFQPVPS 2 VPPPAGSP 8
* ******
36 VSPPAGSP 73 AtAGP10(At4g09030)
2 VPPPAGSPFQPVPS 15
*********** *
415 VPPPAGSPFQPPTS 456 PHYSCObaseContig4134
SAGPVMAVVPPVTPPVTPSV 1 SAGPVMAVVPPVT---PPVT 16
** * **** ****
19 SA-P-----PPVTTSPPPVT 32 AtAGP9(At2g14890)
SPASSAGPPMGSPPSPAPA 2 PASSAGPPMGSPPSPAP 18
* * * ** ****
29 PTASASSPVESPKSPAP 45 AtAGP18(At4g37450)
SPLTKAEVFYELKDLKGY 5 KAEVF-YEL 12
*** * ***
9 KAEIFGYEL 17 At5g39930(expressedprotein)
AGPbackbonesequencesweresearchedusingtheNCBItBLASTnprogramandPHYSCObasecontigdatabase(http://moss.nibb.ac.jp)withthe
major amino acid sequences from the deglycosylated AGP as queries. Pro was used for the X amino acid, which is deduced from incomplete
deglycosylationofHypleadingtoablank(X)cycleduringsequencing.
aAsterisksindicateidenticalaminoacids.
designated AGP1, was identified by homology to Arabidopsis fasciclin-like P.patensAGPsequences,FLA1 andFLA2,were
AGP1(At5g64310)andaBrassicanapusAGP(T07975)(Figure identifiedwithhomologytoArabidopsisFLA1(At5g55730),FLA2
7).P.patensAGP1has29%aminoacididentitywithArabidopsis (At4g12730),andarice(Oryzasativa)FLAsequence(BAD01728).
AGP1 and 22% identity with B. napus AGP. These protein ThesequencesofP.patensAGP1andAGP3-5(PHYSCObase
sequencesarehomologous throughout, exceptforaregion of accession numbers Contig11049, Contig615, Contig6896, and
16 amino acids adjacent to the predicted N-terminal signal Contig6913,respectively)wereusedtodesignprimerstoamplify
sequence. Three related short P.patens arabinogalactan pep- the full sequence of these AGP genes from a cDNA library
tides,AGP3,AGP4,andAGP5,wereidentifiedbyhomologyto (a generous gift from Mitsuyasu Hasebe). The 59 sequences
ArabidopsisAGP12(At3g13520)andAGP16(At2g46330).Two for P. patens FLA1 and FLA2 were available in PHYSCObase
Figure7. AlignmentofClassicalAGPP.patensAGP1CoreProteinSequenceswithThoseofArabidopsisAGP1andB.napusAGP.
The putative protein sequence of classical AGP P. patens (Pp) AGP1 aligned with Arabidopsis (At) AGP1 (Arabidopsis Genome Initiative locus
At5g64310)andB.napus(Bn)AGP(accessionnumberT07975).Aminoacidscommontotwosequencesareshowninbold,andwhencommontoall
threesequencestheyareshowninboldunderlined.ThelikelyN-terminalsignalsequenceofP.patensAGP1isunderlined.Theputativerecognitionsite
ofP.patensAGP1fortheadditionofaGPIanchorisshownwithboxedoutline,andtheC-terminalhydrophobicregionisshadedingray.
3058 ThePlantCell
(accession numbers Contig6808 and Contig9936). No 39 se- KnockoutofP.patensAGP1ReducestheGrowth
quencesforeitherP.patensFLA1orFLA2wereavailableinthe ofP.patens
ESTdatabases,andsothesewereobtainedby39rapidampli-
P. patens is suited to perform functional analysis by gene
fication of cDNA ends. The amplified fragments were se-
targeting; therefore, AGP1, the first full-length sequence we
quenced, and theproducts matched the predictedsequences
identifiedencodingaclassicalAGPbackbone,wasselectedfor
fromtheinitialbioinformaticsearch.
Shortlybeforethesubmissionofthismanuscript,aP.patens manipulation.Aknockout(KO)vectorwasdesignedtoreplace
deduced amino acid sequence relating to peptide fragment 1 theendogenousAGP1withaconstructcomprisingahygromycin
wasidentifiedinthePHYSCObasedatabase(Table2,Figure8). phosphotransferasegenedrivenbythecauliflowermosaicvirus
This is a further classical AGP, designated P. patens AGP2 35S promoter, flanked by LoxP recombination sites (Sauer,
(PHYSCObase accession number Contig4134) (Figure 8). This 1998).ThisconstructwouldreplacetheendogenousAGP1bya
list of putative P. patens AGP core peptides is clearly not dualrecombinationevent88bpupstreamfromtheATGcodon
complete,asitdoesnotcontainallthesequencesofallofour and93bpdownstreamfromthestopcodonofthegene.Plants
biochemicallyidentifiedP.patensAGPpeptidefragments.The were transformed by polyethylene glycol–mediated protoplast
available Physcomitrella EST collections currently include fusionwiththeAGP1:KO,andfivestabletransformantlineswere
>15,000 transcripts, but analyses have indicated that these isolated.PCRanalysisindicatedthattwooftheselines,which
librariesarenotsaturated(Nishiyamaetal.,2003). displayedsimilarphenotypes,weredisruptedattheAGP1locus.
Figure8. PutativeProteinSequencesofP.patensAGPs.
TheputativeproteinsequencesofaclassicalAGP(AGP2)andthreeshortarabinogalactanpeptidesAGP3-5,FLA1,andFLA2.AGP2wasidentified
fromatBLASTnsearchwithpeptidefragment1(Table2)withsequencerelatingtotheisolatedpeptideshowninbold.SequencesofAGP3-5were
identifiedbyhomologywithArabidopsisAGP12(At3g13520)andAGP16(At2g46330).FLA1andFLA2wereidentifiedbyhomologywithArabidopsis
FLA1(At5g55730)andFLA2(At4g12730)andwithriceFLA(BAD01728).Fasciclindomainsareshadedingray.PredictedN-terminalsignalsequences
andC-terminalhydrophobicregionspredictedtobecleavedforGPIanchorattachmentareunderlined.aa,aminoacids.
AGPsinPhyscomitrellapatens 3059
Figure9. ExpressionofAGP1andPhenotypeoftheWildTypeandaTransgenicKOLineofP.patens.
(A)RT-PCRanalysisofwild-typeandP.patensAGP1KOcultures.AmplificationofAGP1inthewildtype(lane2)butnotaKOline(lane5).Thecontrol
3060 ThePlantCell
AnalysisoftheRNAextractedfromthewildtypeandaKOlineis ThreeofthefoursubclassesofGPI-anchoredAGPcoreprotein
shown in Figure 9A, indicating that no AGP1 transcript was sequences(classicalAGP,arabinogalactanpeptide,andFLAs)
detectable. Growth analysis of the KO lines was initially per- havesofarbeenidentifiedinP.patens.Itisofinterestthatafter
formedbystudiesofcolonygrowthonsolidmedia,andthese 400 million years of evolution, P. patens contains AGP core
indicatedthattheKOofAGP1resultedindecreasedgrowthof protein sequences that are homologous to those from angio-
inocula(asmeasuredbycolonydiameter)after3weeksonsolid spermsandsuggeststhatAGPsplayimportantrolesincellular
media(Figure9B).Themeancolonydiameterswereintheregion processes that are common to bryophytes and angiosperms.
of 75%of thatofthe wildtype throughseveral culturecycles. ThepresenceofthreeclassesofAGPsequencesinP.patens
Examination of protonemata indicated that the basis of the suggeststhattheseclassesofAGPsdivergedbeforespeciation
reduction in colony size was reduced cell length (Figure 9C). events. The observation that not all the peptide sequences
Chloronemalcells,withlessvariationincelllengththancaulo- obtainedbybiochemicalisolationofbGlcYR-precipitatedAGPs
nemalcells,werechosenforthecelllengthmeasurements.The relate to sequences identified by bioinformatic searches indi-
transitionofachloronemalfilamenttocaulonemalfilamentcan catesthatmoreAGPcoreproteingenesremaintobeidentifiedin
taketwoorthreecelldivisions,andduringthisperiod,cellscan P.patens.
showcharacteristicsofbothcelltypesandvariablecelllengths, ThebehaviorofbGlcYR-precipitatedmaterialonSDS-PAGE
making caulonemal cells less suitable for analyses. Subapical gels in terms of molecular mass and heterogeneity suggests
cellsofanypositionintheprotonemawereusedforstatistical propertiessimilartoangiospermAGPs.Asignificantandunusual
analyses, with the exception of the first cell produced on side featureofthebGlcYR-precipitatedP.patensAGPs,confirmed
branchformation,whichcanoftenbeveryshort.Wild-typesub- bybothbiochemicalanalysisandimmunochemistry,isthepres-
apicalchloronemalcelllengthsrangebetween70and100mm. enceof(1/5)-a-L-arabinofuranosylresidues.Thisglycanstruc-
SubapicalchloronemalcelllengthsoftheKOlineswereconsis- tural feature is most often associated with the pectic polymer
tently shorter than the wild type in the region of 50 to 70 mm. rhamnogalacturonan-I in angiosperms (Willats et al., 2001), al-
Representative protonemata from the wild type and a KO line though it has previously been identified in an Acacia AGP
areshowninFigure9D.Theapicalcellsofwild-typeP.patens (MollardandJoseleau,1994).
showadistalnarrowing.ThiswasabsentintheKOlines.
AGPsandPlantGrowth
DISCUSSION
We present evidence not only that AGPs are present in the
mossP.patensbutalsothattheyfunctioninapicalcellextension
P.patensAGPs
that is the basis of protonemal growth. The growth inhibitor
Previous studies have demonstrated the presence of AGPs in bGlcYR, which blocks cell extension, is likely to interact with
arangeofleafyliverwortandmossbryophytespecies,including a range of AGPs, suggesting that a range of AGPs may be
P.patens,wheretheyhavebeenimplicatedinthetransitionfrom involved in the cell expansion process. This is supported by
branchingfilamentstoparenchymatousleafyshoots(Basileand theKOofoneAGPcoreproteingene,P.patensAGP1,which
Basile,1987;Basileetal.,1999;MignoneandBasile,2000).The resultedinan;25%reductioninbothcolonygrowthandcell
presence of AGPs in P. patens has been shown here by the length.bGlcYRhaspreviouslybeenusedtoinhibittheextension
biological activity of bGlcYR and the isolation of bGlcYR- of tip-growing lily pollen tubes (Roy et al., 1999), and over-
precipitablematerialwithbiochemicalcharacteristicsofAGPs. expressionofaclassicalAGPcoreproteinledtoanincreasein
These observations, along with biochemical sequencing data organgrowth(Parketal.,2003).ThisarticlereportsthattheKOof
and bioinformatic protein sequence data using Arabidopsis, a predicted classical AGP core protein gene can result in
Brassica,andOryzasequencesasqueries,indicatethepresence reducedgrowthofapicallyextendingcellsundernormalgrowth
ofatleast10putativeAGPcoreproteinsequencesinP.patens. conditions.
Figure9. (continued).
plastocyaningenewasamplifiedinboththewildtype(lane3)andtheKOline(lane6).ThemockcDNAcontrolsindicatedthatnocontaminating
genomicDNAwaspresentineithersample(lanes4and7).Lane1showsaDNA100-bpladder.
(B)Representativeplatescontainingninecoloniesforwild-typeandKOlinesafter3weeks.
(C)Histogramofmeancolonydiametersforninecoloniesfromthreewild-typeplatesandthreeplatesofaKOlinegrownfor3weeksonsolidmedia.
Thehorizontaldashedlineindicatesthemeancolonydiameterforthethreewild-typeplates.Similarcolonydiameterdatawerecollectedfromthree
separateexperiments,andthefigureshowsrepresentativeresultsfromoneexperiment.Histogramofmeanlengthsofsupapicalchloronemalcellsof
theKOlinescomparedwiththewildtype.Errorbarsindicatestandarddeviation.Meanswerecalculatedfromatleast35subapicalcellsperline.
(D)Micrographofwild-typeP.patensprotonematashowingthewild-typebranchingpatternofchloronemalfilamentsarisingfromcaulonemalcells,
distalnarrowingofapicalcells(arrowheads),andtypicalcelllengths.MicrographoftheP.patensAGP1KOshowingtypicalcelllengthsandlackof
narrowingofapicalcells.Asterisksindicatecaulonemalorintermediatecaulonemalcellsthatgiverisetochloronemalcells.Whitelinesarealignedwith
chloronemalcellsandindicatetypicalcelllengths.Bar¼100mm.