全 文 ：鹤顶兰胚囊发育过程中Ca2+分布的超微细胞化学定位*
李冬妹1,3,王亚琴2,叶秀粦3**,梁承邺3
(1顺德职业技术学院,广东 佛山暋528300;2华南师范大学生命科学学院,广东 广州暋510631;
3中国科学院华南植物园,广东 广州暋510650)
摘要:用焦锑酸盐沉淀法对鹤顶兰 (Phaiustankerviliae)胚囊发育过程中的Ca2+状态进行超微细胞化学
定位。观察结果发现:功能大孢子时期,珠孔端的胚囊壁上开始出现小颗粒的Ca2+沉淀,但功能大孢子
细胞内未见明显的Ca2+标记;四核胚囊时期胚囊壁上的Ca2+沉淀明显增多,液泡膜上有Ca2+沉淀出现,
珠孔处的Ca2+沉淀颗粒较大;成熟胚囊时期,胚囊壁上的Ca2+沉淀进一步增多,且胚囊内Ca2+分布明显
增多,且极性明显,珠孔端助细胞、卵细胞比合点端反足细胞有更多的Ca2+沉淀。鹤顶兰成熟胚囊内
Ca2+积累的来源有:(1)在胚囊成熟前主要由珠被细胞、珠细胞通过胞间连丝向胚囊运输;(2)以沉淀有
大量Ca2+的小泡形式跨过胚囊壁进入胚囊。
关键词:鹤顶兰;胚囊;超微细胞化学定位;焦锑酸盐沉淀法;钙
中图分类号:Q944暋暋暋暋 暋暋文献标识码:A暋暋暋 暋暋暋暋文章编号:0253灢2700(2010)06灢495灢08
UltracytochemicalLocalizationofCalciumduringEmbryoSac
DevelopmentinPhaiustankerviliae(Orchidaceae)
LIDong灢Mei1,3,WANGYa灢Qin2,YEXiu灢Lin3** ,LIANGCheng灢Ye3
(1ShundePolytechnic,Foshan528300,China;2ColegeofLifeScience,SouthChina
NormalUniversity,Guangzhou510631,China;3SouthChinaBotanicalGarden,
ChineseAcademyofSciences,Guangzhou510650,China)
Abstract:UltracytochemicallocalizationofcalciuminembryosacofPhaiustankerviliae(Aiton)Bl.wascar灢
riedoutusingpotassiumpyroantimonateprecipitationmethod.ResultsofCa2+ distributionduringembryo
sacdevelopmentareasfolowing:(1)Megasporocytestage:ThereisnoCa2+ precipitationinthemegasporo灢
cyte,nucelarcelandmicropyle;(2)Functionalmegasporestage:SmalparticlesofCa2+ precipitatesappear
ontheembryosacwalatthemicropylarend,butnoapparentCa2+ canbefoundinthefunctionalmega灢
spore;(3)4灢nucleatestage:TherearesignificantincreaseofCa2+ precipitationontheembryosacwaland
largegrainsoftheprecipitatesinthemicropyle,whilealittlestartstoappearonthemembraneofvacuole;
(4)8灢nucleateembryosacstage:Ca2+ precipitationcontinuouslyincreasesontheembryosacwal.Distribu灢
tionofCa2+inembryosacshowsstrongpolarity.MoreCa2+ precipitationsareobservedinthesynergidsand
theeggcelthantheantipodalcels.AccumulationofCa2+ precipitationisachievedby(1)transferringCa2+
toembryosacthroughtheplasmodesmatabetweenthenucelarcels,whichmainlyoccurredatthechalaza
endbeforetheembryomatured;(2)forminglargeamountsofsmalbubblesofCa2+ precipitation,whichcan
crossembryosacwaltoenterembryosac.
Keywords:Phaiustankerviliae;Embryosac;Ultracytochemistry;Potassiumantimonitetechnique;Ca2+
云 南 植 物 研 究暋2010,32(6):495~502
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10139
*
**
Foundationitem:PlanProjectsponsoredbytheDepartmentofScienceandTechnologyofGuangdongProvince(2009B020405002)
Authorforcorrespondence;E灢mail:xlye@scbg灡ac灡cn
Receiveddate:2010灢07灢29,Accepteddate:2010灢10灢09
作者简介:李冬妹 (1970-)女,博士,副教授,主要从事兰科生殖生物学研究。
暋 ThedistributionandevolutionofCa2+ has
profoundinfluenceontheprocessofmetabolism
anddevelopment(Antoineetal灡,2000).The
effectofCa2+onthesexualreproductionofplant
appearstobeoneoftheimportantfactorsinthe
studyonplantreproductivebiology(Geetal灡,
2007).PreviousstudiesdemonstratedthatCa2+
signalsareinvolvedinthewholeprocessfrom
thebeginningofpolenreachingthestigmato
theendofdoublefertilization (Yang etal灡,
2002).Moststudieshavefocusedonpolende灢
velopment,polengerminationandgrowthin
vitro,thedistributioninmatureembryosac,as
welastheeffectsduringdoublefertilization.
HighcontentofCa2+ wascommonlyobservedin
thesynergidscels(TianandRussel1997;Yu
etal灡,1998,1999),andintheregionofpolen
germinationandgrowth.Inaddition,Ca2+isof灢
tenfoundintheapoplast(Yuetal灡,1998;Mao
etal灡,1992;Zhangetal灡,1995,1997),indica灢
tingthatCa2+ playsakeyroleintheprocessof
polentubesenteringembryosacandspermcels
releasing.However,howdoestheCa2+ distri灢
butionchangebeforeembryosac maturation?
Forinstance,megasporogenesisandembryosac
development,towhatextentaretheyrelatedto
thedistributionofCa2+ withinembryosac(Qiu
etal灡,2005;2008).Therehavebeenquitefew
reportsonthesequestionssofar.Tofurtherex灢
ploreeffectsofCa2+ onsexualreproduction,we
haveconductedultracytochemicallocalizationof
calciuminembryosacofPhaiustankerviliae
(Aiton)Bl.Wepresenttheresultsofasyste灢
maticinvestigationonthedistributionofCa2+
anddistributionchangesduringmegasporogene灢
sisandembryosacearlydevelopment.Andthe
waysthatCa2+ accumulatesinembryosacare
alsodiscussedinthispaper,whichmayhavefar灢
reachingsignificanceonsexualreproduction.
Materialsandmethods
Materials暋Phaiustankerviliae (Aiton)Bl.plants
weregrowninSouthChinaBotanicalGarden.Flowers
werepolinatedatanthesis.Ovulesweredissectedatdif灢
ferentstagesduringovarydevelopment.
Methods暋Sampleswerepreparedusingpotassiumpy灢
roantimonateprecipitationmethod.Ovuleswerefixedin2%
glutaraldehydein0灡1mol·L灢1 potassiumphosphatebuffer
(PBS,pH7灡6)containing1% K2H2Sb2O7·4H2Ofor4h
atroomtemperature,washedinfivechangesof1% K2H2
Sb2O7·4H2Oin0灡1mol·L灢1 PBSbuffer(20mineach)
andpostfixedin1% OsO4for16hat4曟in0灡1mol·L灢1
PBSbuffercontaining1% K2H2Sb2O7 ·4H2O.Control
waspreparedwithfolowingmodifications:1% K2H2Sb2
O7·4H2O wasomittedfromsolutionsduringprocess灢
ing,andwashedinfivechangesof0灡1mol·L灢1PBSbuff灢
erwithoutantimonite.Specimensweredehydratedingra灢
dedethanol,displacedbyepoxydimethylmethaneand
embeddedinEpon812resin.Thinsectionsatthicknessof
80-90nmwerecutusingLK灢Sultramicrotome,stained
with2% uranylacetatefor60-90 min,thenobserved
withJapaneseTEM灢1010transmissionelectron micros灢
copeat90kVandtookphotograph.
Results
TheembryosacbelongstoaPangianum
typeinitsdevelopment,whichisconsistentwith
previousreports(Yeetal灡,1996;Tungetal灡,
2000).Inthissection,thedistributionofCa2+
precipitatesanditsevolutionduringthedevelop灢
mentofembryosacaredescribed.
Megasporocytestage
Themegasporocyte,whichoriginatesfrom
archesporialcel,possesseslargevolume,thick
wal anddistinctnucleus.Beforethe meiotic
prophase,themegasporocyteishighlypolarized
andthenucleustendstolocateatthemicropylar
end.The density ofcytoplasmiccontentsis
higheratthechalazalendwhichcontainsalarge
amountoforganeles,suchas mitochondrion,
dictyosome,endoplasmicreticulum,etc.Incon灢
trast,themicropylarendhaslesscytoplasm.At
thisstage,noCa2+ precipitatescanbefoundin
the megasporocyte,nucelarcel,embryosac
walandmicropyle.(Plate栺:1)
Functionalmegasporestage
Themegasporocyteundergoesmeiosis,and
givesrisetoatetrad.Thecelatchalazalend
growsfurtherandformsfunctionalmegaspore,
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whiletheotherthreecelsclosetothemicropy灢
larenddegenerate(Plate栺:2).Thefunctional
megasporecontainsnumeroussmalvacuoles,in
whichsomeflocsbegintoappear.Thecytoplas灢
micelectrondensityislow.SmalCa2+ precipi灢
tationsstarttoaccumulateontheembryosac
walnearthemicropylarend,butnotatthein灢
terioroffunctionalmegaspore.SomeCa2+ pre灢
cipitationsareobservedatthemicropyle,andin
thenucleiofnucelarcels.(Plate栺:2,white
arrowheads).
4灢nucleateembryosacstage
Thefunctionalmegasporeundergoesmitotic
divisionandgivesrisetoa2灢nucleateembryo
sac.Rightafterthat,thesecondmitoticdivision
occurs,leadingto4灢nucleateembryosac(Plate
栺:3A,3B).IncreasesofCa2+ precipitations
areevidentontheembryosacwal,andnume灢
roussmalgrainsofCa2+ precipitationsbeginto
appearontheembryosacwalsthatarecloseto
thechalazalend.Atthechalazalend,continu灢
ousdistributionofCa2+precipitationsexitbet灢
weentheembryosacwalandthecytoplasmof
nucelarcels(Plate栻:4).ThecontentofCa2+
atouterintegumentisverymuchatthestageof
four灢nucleateembryo sac (Plate栻:5,6).
ThereisaCa2+stream whichstartsfromthe
outerintegumenttoembryosacwal,inazigzag
wayacrosstheinnerintegument(Plate栻:7).
Alargenumberofgranularprecipitationsare
observedinthevacuolesof4灢nucleateembryo
sac,degeneratednucelarcelsandinnerintegu灢
ment,especialyonthecelwalofouterintegu灢
ment.ContinuousdistributionofCa2+ canalso
beseenattheapoplastsystemofouterintegu灢
ment,innerintegument,nucelarcelsandem灢
bryowal (Plate栻:7).
8灢nucleateembryosac
Throughthethird mitoticdivision,eight灢
nucleateembryosacreplacesthefour灢nucleate
ES,someCa2+ precipitationsareobservedatthe
visualnucleusandcytoplasm (Plate栻:8).
ThereisafurtherincreaseofCa2+ precipitation
onembryosacwalandsignificantCa2+ precipi灢
tationatthemicropyle(Plate栻:9).Thema灢
tureembryosacofPhaiustankerviliae(Aiton)
Bl.containssevencels.DistributionofCa2+
precipitationsshowsstrongpolaritywithinma灢
tureembryosac,relativelowCa2+ precipitation
ispresentatthechalazalend,spottyprecipita灢
tionsinantipodalcelsandafewsmaldotson
thevacuolarmembraneofcentralcel (Plate栿:
10),largeprecipitationgrainsremainatthecel
walofinnerdegeneratedintegument(Plate栿:
11),smalbubblescontainingalargeamountof
Ca2+ precipitationcanbeseennearthewalin灢
sideembryosac(Plate栿:12).Beforefertiliza灢
tion,asynergidceldistinctivelyhasdegenera灢
ted(Plate 栿:13).Ca2+ precipitationsinegg
cel (Plate栿:14)andsynergids(Plate栿:15)
areabundant,Smalbubbles,whichexhibitag灢
gregatesofprecipitation dotsthroughoutits
membrane,areoccasionalyfound withinem灢
bryosac.
Discussion
Ca2+ playveryimportantrolesonthestruc灢
tureandphysiologicalfunctionofplantcel,es灢
pecialyindoublefertilizationofangiosperm,
butitisunfeasibleforcelstohavehighcontent
ofCa2+forlongtime,owingtothecalciumtoxi灢
city whichisassociated withthereactionbe灢
tweenCa2+ andPO43灢 (Rasmussen,1981;Ras灢
mussenandBarrett,1984).Atmegasporocyte
stage,ourobservationresultissimilartoLet灢
tucethatthereisnoCa2+ precipitatescanbe
found(Qiuetal灡,2005).Thereisapolardis灢
tributionofCa2+ precipitationinmatureembryo
sacinPhaiustankerviliae.Thepolardistribu灢
tionofCa2+ precipitationisgeneralyconsistent
withtheobservationsfromBrassicanapus,rice
andcotton (Yuetal灡,1998;1999;Zhanget
al灡,1997).Sofar,fewworksonhowtheCa2+
accumulateattheembryosac.
Inthisstudy,weemployinganidealexperi灢
mentmaterial,Phaiustankerviliae,about45
7946期暋暋LIDong灢Meietal灡:UltracytochemicalLocalizationofCalciumduringEmbryoSacDevelopment...暋 暋
daysfrom megasporocytetomatureembryosac
(Lietal灡,2006).ThedistributionofCa2+ and
itsevolutionduringmegasporogenesisandearly
embryosacdevelopmentwererevealedsystem灢
aticalywithtransmissionelectronmicroscopy.
ThetransferofCa2+fromouterintegument
tonucelarcelsandembryosacisresponsible
fortheaccumulationofCa2+inthematureem灢
bryosac.Accordingtothestudyontheforma灢
tionanddevelopmentofembryosacwalinrice
(Liuetal灡,1997),twowaysofnutritioncon灢
veyancebeforethe8灢nucleateembryosacfor灢
mats7celsareassumed.Inoneway,nutrition
passesthroughthecytoplasmofcelslinkedby
plasmodesmata (thesymplast),which mainly
occurredonembryosacatthechalazalend,in
theotherway,nutritionpenetratesintoembryo
sacthroughtheembryosacwalandthespaces
betweencels(theapoplast).Aftertheforma灢
tionof7cels,nutritionpenetratingintoembryo
sacthroughtheapoplastbecomesthe major
way.Ultracytochemicallocalizationofcalcium
wascarriedoutinthedegeneratingnucelusand
endosperm cel ofricepostflowering (Wei,
2002),suggestingthatthecalcium wastrans灢
portedtoembryosacthroughtwopatterns:one
wayisthatcalciumwastransportedbysomelit灢
tlebubblescontainingaggregatesofCa2+precipi灢
tations;theother wayisthataCa2+ stream
startsfromthedegeneratedvacuoleofnucelar
celsinazigzagwayacrossembryosacwal.We
observedthesimilarphenomenaduringembryo
sacdevelopmentofPhaiustankerviliae.Atthe
stageof4灢nucleateembryosac,remarkableCa2+
precipitationsarefoundattheplasmodesmata
betweenthenucelarcelsandembryosacinthe
chalazalend(Plate栻:4),andmoreabundant
Ca2+ precipitationswerefoundinouterintegu灢
mentthaninnerintegument(Plate栻:5,7).As
theembryosacmatured,thedegeneratedinner
integumenthasmuchCa2+ precipitation (Plate
栿:11),smal bubblescontainingalargea灢
mountofCa2+ precipitationcanbeseennearthe
walinsideembryosac(Plate栿:12).
Basedontheseobservations,wepropose
thatCa2+ accumulatesinmatureembryosacin
twoways:beforeembryosacmaturation,Ca2+
is mainlytransportedthrough plasmodesmata
fromintegumenttonucelarcels,thentoem灢
bryosac;atthestageofmatureembryosac,
Ca2+ migratesthroughembryosacwalinthe
formofsmal bubblecontainingaggregatesof
Ca2+ precipitation.
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ExplanationsofPlates
A.Antipodalcel;DS.Degeneratedsynergidcel;DM.Degen灢
eratedmegaspore;E.Eggcel;EW.Embryosacwal;FM.
Functionalmegaspore;II.Innerintegument;M.Megasporo灢
cyte;Mit. Mitochondrion;N.Nucleus;NC.nucelarcel;
OI.Outerintegument;S.Synergidcel;V.Vacuole
Plate栺:1灢3B暋 Micropyledownwardinfiguresandnotethe
Ca2+ depositswithwhitearrowheads暋1.Developmentofame灢
gasporocyte,thelargenucleusislocatedatthemicropylarend,
noCa2+precipitationshavebeenobserved.bar=7毺m;2.Func灢
tionalmegasporeshowingsomeCa2+ precipitationsinthemicro灢
pyle,nucelarcelsandthenucleioffunctionalmegaspore(white
arrowhead).bar=2毺m;3A.4灢nucleateembryosac.bar=5
毺m;3B.4灢nucleateembryosac (anothergrid),twonucleiare
locatedatthechalazalend,alargenumberofgranularCa2+ pre灢
cipitationsareshowingatthevacuole.bar=5毺m
Plate栻:4灢9暋MicropyledownwardinfiguresandnotetheCa2+
depositswith whitearrowheads暋4.Higher magnificationof
squareat3BshowingCa2+ distributionattheembryosacwal
andnucelarcelsatthechalazalend.bar=500nm;5.Abun灢
dantCa2+ atouterintegumentof4灢nucleateembryosac.bar=1
毺m;6.Integumentof8灢nucleateembryosac,Ca2+ precipitation
atouterintegumentislowerthanthatatthestageof4灢nucleate
embryosac.bar=2毺m;7.Integumentof4灢nucleateembryosac
showingCa2+ precipitationsdistributionatouterintegument,in灢
nerintegumentandembryosacwal.bar=2毺m;8.8灢nucleate
embryosac,thesynergidsandeggcelarelocatedatthemicropy灢
larend.bar=7毺m;9.Highermagnificationofsquareatthe
micropyleof8,Ca2+ precipitationsaresignificant.bar=500nm
Plate栿:10灢15暋Micropyledownwardinfiguresandnotethe
Ca2+ depositswithwhitearrowheads暋10.Matureembryosac,
threeantipodalcelsarelocatedatthechalazalend,thenucleiof
eggcelisatanothergrid,showingrelativelowcontentofCa2+
precipitationatantipodalcelsandsomesmalgrainsatthevacu灢
olemembraneofcentralcel.bar=1毺m;11.Highermagnifica灢
tionofsquareaat10,largeCa2+ precipitationsgrainsremainat
cel wal ofinnerdegeneratedintegument.bar=1毺m;12.
Highermagnificationofsquarebat10,smalbubblecontaining
significantCa2+inmatureembryosac.bar=200nm;13.Ma灢
tureembryosacbeforefertilization,asynergidceldistinctively
hasdegenerated.bar=5毺m;14.Highermagnificationofegg
celat13,showingCa2+precipitationgrainsatthecytoplasmand
nucleusofeggcel.bar=200nm;15.Highermagnificationof
squareat13,abundantCa2+precipitationatthedegeneratedsyn灢
ergidcel.bar=500nm
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暋李冬妹等:图版栺暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋LIDong灢Meietal灡:Plate栺
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暋李冬妹等:图版栻暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋LIDong灢Meietal灡:Plate栻
1056期暋暋LIDong灢Meietal灡:UltracytochemicalLocalizationofCalciumduringEmbryoSacDevelopment...暋 暋
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