全 文 ：毛竹茎秆纤维细胞发育过程中的细胞程序性死亡*
甘小洪1,丁雨龙2
(1西华师范大学西南野生动植物资源保护教育部重点实验室,四川 南充暋637009;
2南京林业大学竹类研究所,江苏 南京暋210037)
摘要:利用TUNEL检测、细胞学及细胞化学方法,对毛竹茎秆纤维细胞发育过程中的细胞程序性死亡进行
了研究。在次生壁形成的早期,纤维细胞出现染色质凝聚、细胞器膨胀、液泡膜解体和细胞质泡状化等典型
的细胞程序性死亡形态学特征;TUNEL检测反应呈阳性,显示此时的纤维细胞核DNA发生了片段化。此
时,在纤维细胞裂解的液泡膜、降解的细胞质和凝聚的染色质上具有ATPase活性。纤维细胞质的Ca2+水平
会随着次生壁的形成而逐渐升高,随后Ca2+聚集成块状。在初生壁形成后期,纤维细胞染色质上的酸性磷
酸酶 (APase)活性增强。随着纤维次生壁的持续增厚,ATPase、酸性磷酸酶和Ca2+将在裂解的细胞质和凝
聚的染色质上持续存在多年。结果表明,毛竹茎秆纤维细胞的次生壁形成过程是一个主动自溶的细胞程序性
死亡过程。初生壁形成后期染色质上酸性磷酸酶活性增强及次生壁形成期胞质Ca2+的聚集,与纤维细胞的
程序性死亡密切相关。ATPase,Ca2+和 APase参与了纤维细胞程序性死亡过程中原生质体的降解。
关键词:纤维细胞;毛竹;细胞程序性死亡;超微结构;超微细胞化学
中图分类号:Q944,Q945暋暋 暋暋文献标识码:A暋暋暋暋暋暋文章编号:0253灢2700(2010)04灢285灢11
ProgrammedCelDeathduringFiberCelDevelopment
inPhylostachysedulis(Poaceae)Culm
GANXiao灢Hong1,DINGYu灢Long2
(1KeyLaboratoryofSouthwestChinaWildlifeResourcesConservation(MinistryofEducation),
ChinaWestNormalUniversity,Nanchong637009,China;2BambooResearchInstitute,
NanjingForestryUniversity,Nanjing210037,China)
Abstract:ProgrammedceldeathduringfiberceldevelopmentinPhylostachysedulisculmswasstudiedby
usingtheTUNELassay,andthecytologicalandcytochemicalmethods.Attheearlystageofsecondarywal
formation,themorphologicalfeaturesofPCDsuchaschromatinagglutination,organelesweling,andthe
tonoplastdisintegrationandcytoplasm vacuolation wereinvestigatedinfibercels.Positivesignalsby
TUNELassaycouldbealsodetectedwhichevidencedthenDNAfragmentation.Mg2+灢ATPaseactivitywas
examinedonthecleavagetonoplast,disintegratedcytoplasmandagglutinatedchromatinoffibercelsatthat
time.ThelevelofCa2+infibercytoplasmwouldincreasewithsecondarywalformation,andCa2+ wouldbe
congregateddensely.APasewassparselylocalizedonthenuclearchromatinduringprimarywalformation,
butitwasdenselyaggregatedatthelatestage.Withthecontinualthickeningofsecondarywal,thecleavage
cytoplasmandagglutinatedchromatinwiththeactivityofMg2+灢ATPaseandAPaseinfibercelswerepersis灢
tentformanyyears,andCa2+ wasalsodetectedinthesestructures.Theresultsshowedthatsecondarywal
formationoffiberinP灡edulisculmwasanactiveautophagicPCD,andtheaggregationofCa2+incytoplasm
duringsecondarywalformationandAPasedenselylocalizedonthenuclearchromatinduringlateprimary
云 南 植 物 研 究暋2010,32(4):285~295
ActaBotanicaYunnanica暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋DOI:10灡3724/SP灡J灡1143灡2010灡10065
* Foundationitems:TheStartupFundforScientificResearchofChinaWestNormalUniversity (No.05B035)andtheNational
NaturalScienceFundofChina(No.30271064)
Receiveddate:2010灢03灢25,Accepteddate:2010灢06灢17
作者简介:甘小洪 (1974-)男,汉族,副教授,主要从事发育植物学、植物资源保护与利用研究。
walformation,werecorrelatedtotheoccurrenceoffiberPCD.Mg2+灢ATPase,Ca2+ andAPasewerealin灢
volvedinthedisintegrationoffiberprotoplastduringPCD.
Keywords:Fibercel;Phylostachysedulis;Programmedceldeath;Ultrastructure;Ultracytochemistry
暋暋Bamboosareeconomicalyimportantplants
withinnumeroususessuchasmakingfurniture,
construction,pulpandpaper,andotherindus灢
tries.Asoneofimportantcomponentsofbam灢
booculms,thestructureandpropertiesoffiber
haveadecisiveimpactonthepropertyofbam灢
booculms,andinturn,dramaticalyinfluence
culmutilization(GanandDing,2005).Several
investigationshave dealt with the morphology,
chemicalcomponentsandtissueratiooffibers,
whichcorrelatetothestrengthoftheculmandits
pulpingproperties(Shigematsu,1940;Ghoshand
Negi,1959;Lieseand Grosser,1972;Espiloy,
1987;Abd.Latif,1995),andthefinestructure
ofbamboofibershadalsobeeninvestigatedin
detail(TonoandOno,1962;Parameswaranand
Liese,1976,1980;Fuji,1985).Thesucces灢
sivedevelopmentofbamboofibercelscouldbe
dividedintothreestages:formationoffiberini灢
tials,primary wal andsecondary wal.The
multilamelatestructureandthickeningoffiber
celwalwereobservedwiththedevelopmentof
aculmandfurtherageing(MurphyandAlvin,
1992;Lieseand Weiner,1996;GanandDing,
2005).Theplasmolysisandapoptoticbodiesin灢
vestigatedduringearlysecondarywalformation
offibercelin Phylostachysedulisculm,re灢
vealedtheoccurrenceofprogrammedceldeath
(PCD)(Heetal灡,2000).
PCDisconsideredasanintegralpartofde灢
velopmentinplants.Suchaninbuiltdeathpro灢
grammeistriggeredinresponsetointernalor
externalsignals,andplaysanimportantrolein
plantnormaldevelopment,suchasterminaldif灢
ferentiationoftrachearyelementsandlysigenous
aerenchymaformation (McCannetal灡,2000;
vanDoornandWoltering,2005;Gunawardena,
2008),senescenceofperipheralrootcapcels
(ZhuandRost,2000)andleaves(Caoetal灡,
2003;Gunawardenaetal灡,2007).PCDisoften
associatedwiththeoccurrenceofspecificbio灢
chemicaland morphologicalfeaturessuch as
condensation ofthe nucleus and cytoplasm,
fragmentationofnuclearDNA (nDNA)(Caoet
al灡,2003;Gunawardenaetal灡,2005).Where灢
in,nDNA fragmentationisthe halmark of
PCD,whichcanbedetectedbytheTUNELas灢
say(Grooveretal灡,1997).Noisknownabout
thechangesofnDNAduringfibercelsecondary
walformationorfiberPCDsofar.
Inplants,asinanimals,PCDisanenergy
dependentprocess.PlantcelundergoingPCDis
anactiveparticipantinitsowndemise(“celular
suicide暠).ATPisanessentialenergycurrency
innatureanditsroleincelularmetabolismis
welestablished,andATPaseplaysakeyrolein
theenergyreleaseduringtheseprocesses(Cui,
1997).Byusingthecytochemicallocalizationof
ATPase,thecelcomponentsactivelyparticipat灢
inginplantcel metabolismandontogenycould
bedetected (Wangetal灡,2000).Nothingis
knownaboutthedynamicchangesofATPaseac灢
tivityduringdevelopmentofbamboofibers.
APase(Acidphosphatase)isanon灢specific
hydrolyticenzymesessentialtophosphatees灢
ters,carbohydrateandphosphatemetabolism,
andimportantforphosphorusscavengingandre灢
mobilizationinplants (Darleen etal灡,1989;
Cashikaretal灡,1997).Massiveevidencesdem灢
onstratethatAPasehascrucialrolesinmetabo灢
lismofcelwal,intercelularnutrienttranspor灢
tationandphosphatetransferreaction(Tianand
Shen,1994;Wangetal灡,1999;Ibrahimetal灡,
2002;Fertenetal灡,2003).Duringsecondaryxy灢
lemdifferentiation,APasewasreportedtoplay
functionsinPCDbyWangetal灡 (1999).
As an important second messenger in
plants,calcium playedimportantrolesduring
thegrowthanddevelopmentofplant,suchas
cel division,cel wal formationandpolarity
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growth,aswelasPCD (WhiteandBroadley,
2003).Duringthedifferentiationoftrachearyel灢
ements,Ca2+ participatedinthePCDandthick灢
eningofcel wal (GrooverandJones,1999).
LittleisknownaboutthefunctionsofCa2+onfi灢
berPCD.
Theaimofthestudywastoimprovethe
understandingoffiberPCDinbambooculms.
Threespecificquestionswereansweredbyusing
differentmethodologysuchasTUNEL (termi灢
naldeoxynucleotidyltransferasedUTPnickend
labeling)assay,cytologicaland cytochemical
methods:(1)DidfiberPCDoccuraccompanying
withsecondarywalformationoffibercel? (2)
WhatwasthereasonresultinginfiberPCDduring
secondarywalformation? (3)WhatrolesdidAT灢
Pase,Ca2+andAPaseplayedinfiberPCD?
MaterialsandMethods
Plantmaterial
Youngshootsandculmsof1to8灢year灢old Phyl灢
lostachysedulisMazelexH.deLeh.wereharvestedin
May2003attheBambooGardenofNanjingForestryUni灢
versity.Youngshootswiththeheightsof60cm,80cm,
120cmand700cm werecolected,andalsamplesfrom
themiddlepartoftheculmwalweretakenasthemethod
byGanandDing(2005).
LightmicroscopyandTUNELassay
Theblockswerefixedin2% glutaraldehydein0灡02
milimole(mM)sodiumcacodylatebuffer(pH7灡0)for4
hatroomtemperature,dehydratedinagradedethanolse灢
ries,andembeddedinTechnovit7100resin.Continued
samplesections (5毺minthickness)mountedonglass
slideswerefirstexaminedandphotographedunderanO灢
lympusBH灢2polarizingmicroscope,thentreatedwiththe
insituCelDeathDetectionKitAPaccordingtotheman灢
ufacturer曚sinstructions(Roche,Germany).Forpositive
control,DNase栺 (grade栺,0灡5 mg·ml灢1in50 mM
Tris灢HCl,pH7灡5,1mg·ml灢1 BSA)wasaddedbefore
TUNELreactionmixturefor10minatroomtemperature
toinduceDNAstrandbreaks.Thenegativecontrolwas
conductedbysimilarincubationsintheabsenceofTdT.
Sampleswerecounter灢stained with0灡5% methylgreen
for30s.
Transmissionelectronmicroscopy
Sampleswereprefixedfor2hoursin0灡025mol·L灢1
phosphatebuffersolution(pH7灡0)containing4% (v/v)
glutaraldehydeinthecaseofyoungshootsand1灢year灢old
culm,or6% (v/v)glutaraldehydeinthecaseof2to8灢
year灢oldculms.Afterwashingwiththesamebuffer,the
sampleswerepost灢fixedin1% OsO4 (alsointhesame
buffer).Folowedbyafurtherwashingwiththebuffer,
thespecimensweredehydratedinagradedethanolseries
andembeddedinEpon812resin.Aftercuttingwithadia灢
mondknifeonaLKB灢Vultramicrotome,ultrathinsec灢
tionswerestainedwithsaturatedaqueousuranylacetate
for5 min,folowedbystainingwithleadcitratefor5
min.Alsectionswereexaminedandphotographedwith
anH灢600(Hitachi,Tokyo,Japan)transmissionelectron
microscope(TEM).
UltracytochemicallocalizationofMg2+灢ATPase
Folowingprefixationinamixtureof4%paraformal灢
dehydeand2灡5%glutaraldehyde,thesamplesweretrea灢
tedasthemethodbyWangetal灡 (2000).Thecontrol
specimensweredealtwithoutsubstrate,orwithsubstrate
inthepresenceof10mMsodiumfluoride(NaF).Made
withLKB灢Vultramicrotome,ultrathinsectionsofcontrol
materialwerestainedwithuranylacetate,butexperimen灢
talsamplesnostained.Alsectionswereobservedand
photographedusinganH灢600TEM.
UltracytochemicallocalizationofCa2+
Afterpre灢fixedina mixtureof2% potassium py灢
roantimonateand2灡5% glutaraldehydein0灡1mMsodi灢
umcacodylatebuffer(pH7灡1)for3hat4曟,thesam灢
pleswerewashedwiththesamebuffer,andthenpost灢
fixedinamixtureof2% potassiumpyroantimonateand
1% OsO4 inthesamebuffer.Folowedbyafurther
washingwiththeredistiledwaterfor4times,thespeci灢
menswerewashedwiththeredistiledwater(pH7灡6)for
2times.Afterwards,alspecimensweredehydratedina
gradedethanolseriesandembeddedinEpon812resin.
TheultrathinsectionsweremadewithLKB灢Vultramic灢
rotome,andstained withuranylacrtate.Al sections
wereobservedandphotographedusinganH灢600TEM.
Thetreatmentofcontrolsamples:afterexamined
withanH灢600TEM,theultrathinsectionscongregated
withcalcium pyroantimonate weretreatedin100 mM
EGTA(ethyleneglycoltetraaceticacid)(pH8灡0)for0灡5
-1hat60曟.Thenthesesectionswereobservedand
photographedwithanH灢600TEM.
UltracytochemicallocalizationofAPase
Afterpre灢fixedinamixtureof4%paraformaldehyde
and2灡5%glutaraldehydein0灡05mMsodiumcacodylate
buffer(pH7灡2)for2hat4曟,thesampleswerewashed
7823期暋 暋GANandDING:ProgrammedCelDeathduringFiberCelDevelopmentinPhylostachysedulis…暋暋暋
inthesamebufferat4曟for1灡5h,andweredissectedin灢
toslicesofabout0灡5mminthicknessinthebuffer,and
thenrinsedwith0灡05mM Tris灢maleatebuffer(pH5灡0)
for1灡5hpriortoenzymereaction.
暋暋Theenzymereactionprocedurewasperformedfol灢
lowingthemethodby Wangetal灡 (1999).Thespeci灢
menswereincubatedinacompletereactionmedium (50
mMTris灢maleatebuffer,pH5灡0,3灡6mM Pb (NO3)2
and10mMsodium毬灢glycerophosphate)at37曟for2h.
Thecontrolspecimenswererespectivelyincubatedinthe
samemediumbutwithoutsubstrate,orwithsubstratein
thepresenceof10mMsodiumfluoride(NaF).Afterin灢
cubation,alspecimenswererinsedwith0灡05mM Tris灢
maleatebuffer (pH 5灡2)for1灡5hat4曟,andthen
rinsedwith0灡05mMsodiumcacodylate(pH7灡2)buffer
for1灡5hatthesametemperature.Folowingthis,these
sampleswerefixedat4曟 with1% osmiumtetroxidein
thesamebufferovernight.Thenalspecimenswerede灢
hydratedstepwiseinanacetoneseriesat4曟.Specimens
wereembeddedinEpon812.Ultrathinsectionsofcontrol
specimenswerestainedwithuranylacetate,whileothers
werenot.Al sections wereviewedandphotographed
withanH灢600TEM.
Results
LightmicroscopyandTUNELassay
Thefibercap(vascularstrandssheath),vessel
elements(protoxylemvesselandmetaxylemvessel)
andphloem wereeasilydistinguishedwiththelight
microscopy(Fig灡1:a).Locationsofsecondarycel
walarevisibleunderthepolarizingmicroscope:sec灢
ondarycel walisstronglyanisotropicandshows
substantialdoublerefractionandappearsbrightinpo灢
larizedlight(Esau,1977).Inthemiddlepartof
shootwiththeheightof60cm,vesselelementsand
sievetubewerevisibleinthevascularstrandsdueto
theabruptincreaseinwalbrightness,whilefibersin
fibercapwereinvisible(Fig灡1:b).Atthepresent
time,thefiberswerenotlabeledbyTUNELassay
(Fig灡1:c).Inthelowerpartofthesameshoot,fiber
walwasvisibleunderpolarizedmicroscope(Fig灡1:
d),andpositivesignalsofTUNELassay(indicated
bypurplestaining)wasdetectedinthefibers(Fig灡1:
e).Innegativecontrolsections,noTUNEL灢positive
reactionweredetectedinthefibernuclei(Fig灡1:f).
Fig灡1暋a:thevascularbundlewithlignifiedfibercapinP灡edulisshootwiththeheightof700cm,TS;b灢f:themicroscopy
ofshootswiththeheightsof60cm;b:thefiberscelwalwereinvisibleinthemiddlepartofshootunderpolarizingmicro灢
scopewhichindicatedthedevelopmentalstageofprimarycelwalformation,TS;c:thefibersinthemiddlepartofshoot
werenotlabeledbyTUNELassay,LS;d:thefibercelwalinthelowerpartofyoungshootwasvisibleduetotheabrupt
increaseinwalbrightness,whichindicatedthedevelopmentalstageofsecondarycelwalformation,TS;e:TUNELassay
revealedpositivesignals(purplestaining,arrowhead)inthefibersinthelowerpartofshoot,LS;f:thenegativecontrol,
showingnoTUNELpositivesignalinfibersinthelowerpartofshoot,LS.Bar=10毺m.Abbreviations:F:fiber;FC:fiber
cap;MV:metaxylemvessel;Ph:phloem;PV:protoxylemvessel;LS:longitudinalsection;TS:transversesection
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Transmissionelectron microscopeassay during
secondarywalformationoffibers
Early,fibercel wal thickenedbilowingly,
andintactdoublekaryothecaremainedinfibernu灢
cleus,whereinchromatincommencedtoagglutina灢
tionandnucleolusdisappeared.However,someor灢
ganelessuchasmitochondrionandroughendoplas灢
micreticulumandGolgibodiesremainedinthese
cels(Fig灡2:a).Withcontinualthickeningof
secondarywal,fiberorganelesincludingmito灢
chondrion and rough endoplasmic reticulum
gradualysweledanddisintegrated(Fig灡2:b).
Afterwards,fibercytoplasm gradualyvacuo灢
lated,anddoublekaryothecaofnucleusdisap灢
peared(Fig灡2:c).Nevertheless,plasmamem灢
brane,plasmodesmataandnucleuswiththeag灢
glutinatedchromatinremainedinthefibercels
of6灢year灢oldculms(Fig灡2:f).Alongwithsec灢
ondarywalformation,fibercelwalunderwent
continualthickening,and polylaminatestruc灢
turesarrangedinregularalterationofbroadand
narrowlamelaeappeared(Fig灡2:d灢e).
ThedynamicchangesofMg+灢ATPaseduringsec灢
ondarywalformationoffibercels
Early,Mg+灢ATPaseactivitywasdetected
ontheplasmamembrane,tonoplast,nucleus,
pits and plasmodesmata and mitochondrion
(Fig灡3:a灢b).Afterwards,Mg+灢ATPasewas
examinedontheannulatelamelaandtheagglu灢
tinatedchromatin (Fig灡3:c).Withcontinual
thickeningofsecondarycelwal,theactivityof
Mg+灢ATPaseinagglutinatedchromatin would
increase (Fig灡3:e),and Mg+灢ATPase were
denselylocalizedonthedisintegratedtonoplast
(Fig灡3:d)andinthevacuolatedcytoplasm
(Fig灡3:f).Duringfibercelsecondarywalforma灢
tion,Mg+灢ATPaseremainedontheplasmamem灢
brane,transfervesiclesinpitchannelsandplas灢
modesmataandcytoplasmremnants.Theactivity
ofMg+灢ATPaseontheagglutinatedchromatin
Fig灡2暋Theultrastructuralmodificationsoffibercelsduringsecondarywalformation.a:thefibercelattheearlystageof
secondarywalformation,showingbilowinglythickeningofcelwal,chromatincommencedtoagglutinationandsomeor灢
ganelesremainedinthecels,TS;b:ultrastructureoffibercelinbambooshoot,showingtheagglutinatedchromatin,dis灢
integratedmitochondrionandtransfervesicles,TS;c:showingthevacuolatedcytoplasmandagglutinatedchromatinofafi灢
bercelinone灢year灢oldculm,TS;d:fibercelsintwo灢year灢oldculm,showingthethickeningsecondarywaloffibercels
andtheplasmodesmata(arrow)betweenfibers,TS;e:thefibersinfour灢year灢oldculm,showingpolylaminatestructuresof
fibersecondarywalandtheplasmodesmata(arrow)betweenfibercels,TS;f:showingtheagglutinatedchromatinofafi灢
bercelinsix灢year灢oldculm,TS.Bar=1毺m.Abbreviations:G:Golgibodies;M:mitochondrion;N:nucleus;PC:paren灢
chymacel;SW:secondarycelwal;TV:transfervesicle;TSseeFig灡1
9823期暋 暋GANandDING:ProgrammedCelDeathduringFiberCelDevelopmentinPhylostachysedulis…暋暋暋
Fig灡3暋DynamicchangesofMg2+灢ATPaseinthefibersduringsecondarywalformation.a灢b:fibercelsattheearlystage
ofsecondarywalformation;a:showingthedistributionofMg2+灢ATPaseontheplasmamembrane,tonoplastandthe
plasmodesmata(arrow),TS;b:showingMg2+灢ATPaseaggregatedinmitochondrionandnucleusandontheplasmamem灢
brane,TS;c:showingMg2+灢ATPaselocalizedontheannulatelamela(star)andagglutinatedchromatininthefibercels,
TS;d:showingdenseMg2+灢ATPasedistributedonthedisintegratedtonoplast(arrow)andplasmamembrane,TS;e:
showingthedistributionofMg2+灢ATPaseontheplasmodesmata(arrow)andagglutinatedchromatinoffibersin1灢year灢old
culm,TS;f:showingthedistributionofMg2+灢ATPaseonthevacuolatedcytoplasm (star)offibercelsin2灢year灢old
culm,TS;g灢h:afibercelin6灢year灢oldculm;g:showingtheplasmamembraneandagglutinatedchromatinwithMg2+灢
ATPaseactivity,TS;h:showingtheplasmodesmata(arrow)with Mg2+灢ATPaseactivitybetweenfibercels,TS;i:
showinglittleMg2+灢ATPasedetectedonthefibercelsinthecontrolspecimenwithsubstrateinthepresenceof10mMso灢
diumfluoride(NaF),TS.Bar=1毺m.Abbreviations:Ch:chromatin;M:mitochondrion;N:nucleus;Nu:nucleolus;P:
plasmamembrane;SW:secondarywal;V:vacuole;TSseeFig灡1
wouldincreasewithaging(Fig灡3:g灢h).
LittleMg+灢ATPaseactivitywasdetectedin
thecontrolspecimens(Fig灡3:i),whichdemon灢
stratedthattheresultsabovewerecredible.
ThedynamicchangesofCa2+duringfiberdevelopment
Duringfiberinitialsformation,Ca2+ mainly
distributedonthecel wal (Fig灡4:a).During
earlyprimarywalformation,Ca2+ sparselyap灢
pearedintheinnersideoftonoplastinfiber
cels,andsomewasalsoexaminedinphagosome
andvacuole(Fig灡4:b).Afterwards,thelevel
ofCa2+invacuoleincrease,butlittlewasseenin
nucleusandcel wal (Fig灡4:c).Atthelate
stage,massive Ca2+ assembledinthecyto灢
plasm,butthelevelofCa2+ invacuolede灢
creased,andCa2+ disappearedincel waland
nucleus(Fig灡4:d).Withtheformationofsec灢
ondarywal,thelevelofCa2+infibercelcyto灢
plasmgradualyincreased(Fig灡4:e),andthen
Ca2+ denselycongregatedinfibercelcytoplasm
(Fig灡4:f).Duringsecondarywalformation,
Ca2+ remainedinthedisintegratedcytoplasm,
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agglutinatedchromatin,and pitchanneland
transfervesiclesformanyyears(Fig灡4:g灢h).
ThereactiongranulesofCa2+ disappeared
inthefibercelofthecontrolspecimens(Fig灡4:
i),whichindicatedthattheresultsabovewere
credible.
ThedynamicchangesofAPaseduringtheprocess
offiberdevelopment
Duringearlyprimary wal formation,the
APaseactivity wasexamined onthe plasma
membrane,tonoplast,karyothecaandnuclear
chromatininfibercels(Fig灡5:a灢b).Atthelate
stage,APase wasaggregatedonthenuclear
chromatinoffibercels,andsomeremainedon
theplasmamembrane,tonoplastandvacuolated
cytoplasminfibercels(Fig灡5:c灢d).Withthe
formationoffibersecondarywal,APasewas
depositedontheplasmamembrane,thedisinte灢
gratedcytoplasm,mitochondrion,andtonoplast
andtheagglutinatedchromatin(Fig灡5:e灢f).A灢
longwiththecontinualthickeningofsecondary
wal,APaseremainedontheplasmamembrane,
plasmodesmata,plasmamembraneinvagination
andtransfervesiclesandagglutinatedchromatin
Fig灡4暋DynamicchangesofCa2+inthefibercelsduringdevelopment.a:fiberinitialcels,showingCa2+localizedinthe
celwal(arrow),TS;b灢e:fibercelsduringprimarywalformation,LS;b:showingthedistributionofCa2+inthedisinte灢
gratedcytoplasm (arrow)invacuoleandonthefibercelwal;c:showingmassiveCa2+ accumulatedinvacuoleandsome
appearedintheinnersideoftonoplast(arrow);d:showingthelevelofCa2+invacuoledecreasedandsomeassembledinthe
cytoplasm (arrowhead);e:showingthelevelofCa2+incytoplasmincreased(bigarrowhead)andsomedistributedinthein灢
nersideoftonoplast(smalarrowhead);f:showingCa2+ congregateddenselyinfibercelcytoplasm (arrowhead)atthe
earlystageofsecondarywalformation,TS;g灢h:fibercelsin4灢year灢oldculm,LS;g:showingCa2+remainedinthedisin灢
tegratedcytoplasm (smalarrowhead)andagglutinatedchromatin(bigarrowhead)offibercels;h:showingCa2+ detected
inthevacuolatedcytoplasm;i:showingthereactiongranuleofCa2+disappearedinthefibercelofthecontrolspecimens,
TS.Bar=1毺m.Abbreviations:Ch:chromatin;FI:fiberinitialcel;M:mitochondrion;N:nucleus;Nu:nucleolus;PC:
parenchymacel;PW:primarywal;SW:secondarywal;V:vacuole;LS,TSseeFig灡1
1923期暋 暋GANandDING:ProgrammedCelDeathduringFiberCelDevelopmentinPhylostachysedulis…暋暋暋
Fig灡5暋DynamicchangesofAPaseduringfiberdevelopment.a:fibercelsattheearlystageofprimarywalformation,
showingAPasedistributedontheplasmamembrane,tonoplast,karyothecaandnuclearchromatin,LS;b:fibercelsdur灢
ingprimarywalformation,showingAPaselocalizedontheplasmamembrane,tonoplast,chromatinanddisintegratedpro灢
toplastinvacuole(arrow),LS;c灢d,fibercelsatthelatestageofprimarywalformation;c:showingAPasecongregated
inthevacuolatedcytoplasm (star)andtransfervesicle(arrow)invacuole,LS;d:showingAPasedetectedinthenuclear
chromatin;e灢f,fibercelsattheearlystageofsecondarywalformation,LS;e:showingthedistributionofAPaseinthe
agglutinatedchromatin,plasmamembraneandthevacuolatedcytoplasm (star),LS;f:showingAPasedistributedonthe
plasmamembrane,vacuolatedcytoplasm (star),mitochondrinanddisintegratedtonoplast(arrowhead),LS;g:thefibers
in4灢year灢oldculm,showingAPaselocalizedonplasmamembrane(arrowhead)andtransfervesicle,LS;h:fibercelsin6灢
year灢oldculm,showingAPaseremainedinagglutinatedchromatinandplasmodesmatabetweenfibers(arrow),LS;i:
showinglittleAPasedetectedonthefibercelsinthecontrolspecimenwithsubstrateinthepresenceof10mMsodiumflu灢
oride(NaF),LS.Bar=1毺m.Abbreviations:M:mitochondrion;N:nucleus;Nu:nucleolus;PW:primarywal;SW:
secondarywal;TV:transfervesicle;V:vacuole;LSseeFig灡1
offiberformanyyears(Fig灡5:g灢h).
LittleAPasewasdetectedinthefibercelof
thecontrolspecimens(Fig灡5:i),whichindica灢
tedthattheresultsabovewerecredible.
Discussion
Attheearlystage,thefibercelsinP灡edulis
culmsdisplayedcytologicalchangessuchaschroma灢
tinagglutination,organelesweling,tonoplast
disintegrationandcytoplasmcleavage,whichin灢
dicatedtheinsituautolysisoffibercelproto灢
plasts,andthetypicalmorphologicalfeaturesof
PCD (Wangetal灡,1999;Fukuda,2000;An灢
dreaetal灡,2008).AlongwiththePCD,the
cel wal underwentcontinualthickening with
aging,andthepoly灢laminatestructureofthecel
wal inalternatearrangementofnarrow and
broadlamelaealsoformedgradualy.Theultra灢
structuralchangesevidencedthatsecondarywal
formationoffibercel accompanied withthe
292暋暋暋暋暋暋暋暋暋暋暋暋 暋暋暋暋暋暋暋云暋南暋植暋物暋研暋究暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋第32卷
PCD.ThedisintegratedprotoplastsduetoPCD
wasnotbeabsorbedbysurroundingcelsasre灢
portedbyJones(2001),butwasusedtobuild
itscel walliketrachearyelements (Fukuda,
2000;Cui,2000).
TUNELassaywasperformedwiththeaim
ofunderstandingspecificdegradationofnucleus
(MittlerandLam,1995).Duringprimarywal
formation,fiber cels were not labeled by
TUNELassay,butpositivesignals (indicated
bypurplestaining)couldbedetectedinthefiber
celsduring early secondary wal formation,
whichrevealedtheDNAdegradationaccompan灢
yingwithsecondarywalformation.
Duringsecondarywalformation,Mg2+灢AT灢
Paseremainedinnuclearchromatinoffibercelsin
P灡edulisculms,andwouldgradualyincreasewith
furtheragglutinatingofchromatin.Besides,dense
Mg2+灢ATPase was detected in multi灢vesicle
bodiesanddisintegratedtonoplast.Generaly,
Mg2+灢ATPaseinnucleusduringplantnormal
developmentplayedimportantrolesinthefor灢
mationandstabilizationofphysiologicalspace
betweennucleosomes,andMg2+灢ATPaseactivi灢
tywasexaminedonthenuclearchromatinin
celswithactivemetabolism.Moreover,Mg2+灢
ATPaselocalizedinnuclearchromatinduring
PCDcorrelatedtothecondensationandrupture
ofchromatin(TianandShen,1996).Thedy灢
namicchangesofMg2+灢ATPaseonnuclearchro灢
matinshowedthatnuclearchromatinoffiber
celsactivelyparticipatedinsecondarywalfor灢
mation.ThehydrolizationofMg2+灢ATPasein
nuclearchromatinduringfiberPCDcouldpro灢
videenergyforacceleratingthecondensationand
ruptureofchromatin,whichresultedinnDNA
degeneration (Andrea etal灡,2008).Inlike
manner,Mg2+灢ATPaselocalizedindisintegrated
cytoplasmandmulti灢vesiclebodiesoffibercels,
couldbeinvolvedintheautolysisofprotoplasts,
andprovidedsubstanceforsecondarywalfor灢
mation.
Insummary,alevidencesincludingcytolo灢
gy,cytochemistryandTUNELassay,validated
thatfiber cel secondary wal formation in
P灡edulisculmswasanactivePCD.Thepersist灢
enceofplasmamembraneandagglutinatednu灢
cleuswith Mg2+灢ATPaseactivityduringPCD
showedthatfiberPCD wouldkeepforalong
timeuntiltheremovaloffibercelnucleus.
Inplants,PCDduringnormaldevelopment
hasbeensubdividedintotwocategories:thatis,
apoptoticPCDandautophagicPCDinvolvingthe
earlydisruptionofthevacuole(Bursch,2001;
Brasetal灡,2005).ApoptoticPCDinplantsis
rapid,beginningwiththedegradationofthenu灢
cleusandsubsequentincompletebreakdownof
celularorganeles,andthecytoplasmicshrink灢
ageand plasmolysisareitstypicalfeatures.
PlantcelsduringautophagicPCDappearedto
bepredisposedforquickautolysis,forexample,
therewasanenzymaticapparatusreadytomedi灢
atequickspecificDNAcleavagetriggeredbyei灢
therrapidaccidentalvacuoledisintegrationor
programmedvacuolarcolapse(Hatsugaietal灡,
2004;Andreaetal灡,2008),andthetypicalcy灢
tologicalchangesofautophagicPCDischarac灢
terizedbythecytoplasmicvacuolation(Braset
al灡,2005).Basedontheoccurrenceofplasmoly灢
sisandapoptoticbodies,Heetal灡 (2000)con灢
sideredthatthePCDoffibercelin P灡edulis
culmsoccurredduringearlysecondarywalfor灢
mation.Asinthisstudy,noplasmolysisand
apoptoticbodiesweredetectedduringfiberde灢
velopment,butthenuclearchromatinagglutina灢
tionandcytoplasmicvacuolationwereexamined
duringtheprocess,whichindicatedthecharac灢
teristicsofautophagicPCD.
TheroleofCa2+duringfiberPCD
Inplantcel,calcium maystimulatethecalci灢
um灢dependentendonucleaseandinduceDNA de灢
composition(XuandHanson,2000).Increasein
calciumconcentrationischaracteristicofPCD (Qiu
etal灡,2005).Generaly,theincreaseofCa2+incy灢
toplasm canactivateaseriesofCa2+灢dependent
hydrolaseforthedisintegrationofcytoplasmand
3923期暋 暋GANandDING:ProgrammedCelDeathduringFiberCelDevelopmentinPhylostachysedulis…暋暋暋
nucleus,andthenATP灢dependentintakesystem
ofCa2+ onkaryothecaisalsoactivatedresulting
intheincreaseofthelevelofCa2+ innucleus.
TheinflowofCa2+ incytoplasmcontributesto
thedisintegrationoftonoplastandtheceaseof
cytoplasmstream,therebyinitiatescel death
(GrooverandJones,1999).Accordingtoour
investigation,theincreaseofCa2+levelincyto灢
plasmcoincidedwiththefiberPCD,whichim灢
pliedthattheincreasedCa2+incytoplasmresul灢
tedinthedisintegrationoftonoplastandthere灢
leaseofnuclease,andeventualyinitiatedfiber
PCD.DuringfiberPCDinP灡edulisculms,the
persistenceofCa2+innucleuscouldplayimpor灢
tantrolesinchromatinagglutinationandDNA
degradation.
TheroleofAPaseduringfiberPCD
Duringprimarywalformation,APaseactivity
wasdetectedonthenuclearchromatinoffibercels
inP灡edulisculms,andwouldincreaseatthelate
stagefolowedbytheoccurrenceoffiberPCD.The
developmentalprocessoffibercelisacontinuous
process,whichincludedthreestages:thatis,the
formationoffiberinitials,primarywalandsecond灢
ary wal (He etal灡,2000;Gan and Ding,
2005).Cui(1997)heldthatthelatestageof
primarywalformationmaybecriticaltofiber
differentiationanddevelopment.Dense APase
localizedonthechromatinatthecriticalstageof
fiberdifferentiationindicatedthatAPasewasev灢
identlycorrelatedtotheoccurrenceofPCD.
Duringsecondary wal formation,APasere灢
mainedontheagglutinatedchromatin,thecyto灢
plasmandtonoplastandmitochondrionoffiber
cel,whichshowedthatAPasemaybeinvolved
intheinsituautolysisofcytoplasm,andthe
disintegration oftonoplastand mitochondrion
(Wangetal灡,1999).Evidently,APaseplayeda
crucialroleinthedisintegrationofprotoplast
duringfiberPCD.
Acknowledgements:TheauthorsthanktechnicianXihua
Gan,NanjingForestryUniversity,forassistanceinsam灢
plepreparation.
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