Ultracytochemical Localization of Calcium during Embryo Sac Development in Phaius tankervilliae(Orchidaceae)

2010·35.3 MB·

by  Li Dong-Mei-

云 南 植 物 研 究 , ():
暋2010 326 495~502
ActaBotanicaYunnanica : /
暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋DOI10灡3724SP灡J灡1143灡2010灡10139
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 ,,王亚琴 ,叶秀粦**,梁承邺
13 2 3 3
顺德职业技术学院 广东 佛山 华南师范大学生命科学学院 广东 广州
(1 , 暋528300;2 , 暋510631;
中国科学院华南植物园 广东 广州
3 , 暋510650)
摘要:用焦锑酸盐沉淀法对鹤顶兰 胚囊发育过程中的 状态进行超微细胞化学
(Phaiustankervilliae) Ca2+
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 沉淀 但功能大孢子
。 : , Ca2+ ,
细胞内未见明显的 标记 四核胚囊时期胚囊壁上的 沉淀明显增多 液泡膜上有 沉淀出现
Ca2+ ; Ca2+ , Ca2+ ,
珠孔处的 沉淀颗粒较大 成熟胚囊时期 胚囊壁上的 沉淀进一步增多 且胚囊内 分布明显
Ca2+ ; , Ca2+ , Ca2+
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 沉淀 鹤顶兰成熟胚囊内
, , 、 Ca2+ 。
积累的来源有 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 以沉淀有
Ca2+ :(1) 、 ;(2)
大量 的小泡形式跨过胚囊壁进入胚囊
Ca2+ 。
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: 文献标识码: 文章编号:
Q944暋暋暋暋 暋暋 A暋暋暋 暋暋暋暋 0253灢2700(2010)06灢495灢08
UltracytochemicalLocalizationofCalciumduringEmbryoSac
DevelopmentinPhaiustankervilliae Orchidaceae
( )
1,3 2 3** 3
LIDong灢Mei ,WANGYa灢Qin,YEXiu灢Lin ,LIANGCheng灢Ye
(1ShundePolytechnic,Foshan528300,China;2CollegeofLifeScience,SouthChina
NormalUniversity,Guangzhou510631,China;3SouthChinaBotanicalGarden,
ChineseAcademyofSciences,Guangzhou510650,China)
Abstract
:UltracytochemicallocalizationofcalciuminembryosacofPhaiustankervilliae(Aiton)Bl.wascar灢
2+
riedoutusingpotassiumpyroantimonateprecipitationmethod.ResultsofCa distributionduringembryo
sacdevelopmentareasfollowing:(1)Megasporocytestage:ThereisnoCa2+precipitationinthemegasporo灢
cyte,nucellarcellandmicropyle;(2)Functionalmegasporestage:SmallparticlesofCa2+precipitatesappear
ontheembryosacwallatthemicropylarend,butnoapparentCa2+canbefoundinthefunctionalmega灢
spore;(3)4灢nucleatestage:TherearesignificantincreaseofCa2+precipitationontheembryosacwalland
largegrainsoftheprecipitatesinthemicropyle,whilealittlestartstoappearonthemembraneofvacuole;
(4)8灢nucleateembryosacstage:Ca2+precipitationcontinuouslyincreasesontheembryosacwall.Distribu灢
2+ 2+
tionofCa inembryosacshowsstrongpolarity.MoreCa precipitationsareobservedinthesynergidsand
theeggcellthantheantipodalcells.AccumulationofCa2+precipitationisachievedby(1)transferringCa2+
toembryosacthroughtheplasmodesmatabetweenthenucellarcells,whichmainlyoccurredatthechalaza
endbeforetheembryomatured;(2)forminglargeamountsofsmallbubblesofCa2+precipitation,whichcan
crossembryosacwalltoenterembryosac.
Keywords:Phaiustankervilliae;Embryosac;Ultracytochemistry;Potassiumantimonitetechnique;Ca2+
* 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 女 博士 副教授 主要从事兰科生殖生物学研究
: (1970-) , , , 。
云 南 植 物 研 究 第 卷
4暋96暋暋暋暋暋暋暋暋暋暋暋 暋暋暋暋暋暋暋 暋 暋 暋 暋 暋 暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋 32
2+
暋 ThedistributionandevolutionofCa has ferentstagesduringovarydevelopment.
Methods
profoundinfluenceontheprocessofmetabolism 暋Sampleswerepreparedusingpotassiumpy灢
anddevelopment(Antoineetal灡,2000).The roantimonateprecipitationmethod.Ovuleswerefixedin2%
2+ glutaraldehydein0灡1mol·L灢1potassiumphosphatebuffer
effectofCa onthesexualreproductionofplant
(PBS,pH7灡6)containing1%K2H2Sb2O7·4H2Ofor4h
appearstobeoneoftheimportantfactorsinthe
atroomtemperature,washedinfivechangesof1% K2H2
studyonplantreproductivebiology(Geetal灡, Sb2O7·4H2Oin0灡1mol·L灢1PBSbuffer(20mineach)
2+
2007).PreviousstudiesdemonstratedthatCa andpostfixedin1% OsO4for16hat4曟in0灡1mol·L灢1
signalsareinvolvedinthewholeprocessfrom PBSbuffercontaining1% K2H2Sb2O7·4H2O.Control
thebeginningofpollenreachingthestigmato waspreparedwithfollowingmodifications:1%K2H2Sb2
theendofdoublefertilization (Yangetal灡, O7·4H2Owasomittedfromsolutionsduringprocess灢
2002).Moststudieshavefocusedonpollende灢 ing,andwashedinfivechangesof0灡1mol·L灢1PBSbuff灢
velopment,pollengerminationandgrowthin erwithoutantimonite.Specimensweredehydratedingra灢
vitro,thedistributioninmatureembryosac,as dedethanol,displacedbyepoxydimethylmethaneand
wellastheeffectsduringdoublefertilization. embeddedinEpon812resin.Thinsectionsatthicknessof
2+ 80-90nmwerecutusingLK灢Sultramicrotome,stained
HighcontentofCa wascommonlyobservedin
thesynergidscells(TianandRussell1997;Yu with2% uranylacetatefor60-90min,thenobserved
withJapaneseTEM灢1010transmissionelectronmicros灢
etal灡,1998,1999),andintheregionofpollen
2+ copeat90kVandtookphotograph.
germinationandgrowth.Inaddition,Ca isof灢
tenfoundintheapoplast(Yuetal灡,1998;Mao Results
etal灡,1992;Zhangetal灡,1995,1997),indica灢
2+ TheembryosacbelongstoaPangianum
tingthatCa playsakeyroleintheprocessof
typeinitsdevelopment,whichisconsistentwith
pollentubesenteringembryosacandspermcells
2+ previousreports(Yeetal灡,1996;Tungetal灡,
releasing.However,howdoestheCa distri灢
2+
2000).Inthissection,thedistributionofCa
butionchangebeforeembryosac maturation?
precipitatesanditsevolutionduringthedevelop灢
Forinstance,megasporogenesisandembryosac
mentofembryosacaredescribed.
development,towhatextentaretheyrelatedto Megasporocytestage
2+
thedistributionofCa withinembryosac(Qiu
Themegasporocyte,whichoriginatesfrom
etal灡,2005;2008).Therehavebeenquitefew
archesporialcell,possesseslargevolume,thick
reportsonthesequestionssofar.Tofurtherex灢
2+ wallanddistinctnucleus.Beforethe meiotic
ploreeffectsofCa onsexualreproduction,we
prophase,themegasporocyteishighlypolarized
haveconductedultracytochemicallocalizationof
andthenucleustendstolocateatthemicropylar
calciuminembryosacofPhaiustankervilliae
(Aiton)Bl.Wepresenttheresultsofasyste灢 end.Thedensityofcytoplasmiccontentsis
2+ higheratthechalazalendwhichcontainsalarge
maticinvestigationonthedistributionofCa
amountoforganelles,suchasmitochondrion,
anddistributionchangesduringmegasporogene灢
dictyosome,endoplasmicreticulum,etc.Incon灢
sisandembryosacearlydevelopment.Andthe
2+ trast,themicropylarendhaslesscytoplasm.At
waysthatCa accumulatesinembryosacare
2+
alsodiscussedinthispaper,whichmayhavefar灢 thisstage,noCa precipitatescanbefoundin
themegasporocyte,nucellarcell,embryosac
reachingsignificanceonsexualreproduction.
wallandmicropyle.(Plate栺:1)
Functionalmegasporestage
Materialsandmethods
Materials暋Phaiustankervilliae(Aiton)Bl.plants Themegasporocyteundergoesmeiosis,and
weregrowninSouthChinaBotanicalGarden.Flowers givesrisetoatetrad.Thecellatchalazalend
werepollinatedatanthesis.Ovulesweredissectedatdif灢 growsfurtherandformsfunctionalmegaspore,
期
6 暋暋LIDong灢Meietal灡:UltracytochemicalLocalizationofCalciumduringEmbryoSacDevelopment...暋49暋7
2+
whiletheotherthreecellsclosetothemicropy灢 onembryosacwallandsignificantCa precipi灢
larenddegenerate(Plate栺:2).Thefunctional tationatthemicropyle(Plate栻:9).Thema灢
megasporecontainsnumeroussmallvacuoles,in tureembryosacofPhaiustankervilliae(Aiton)
2+
whichsomeflocsbegintoappear.Thecytoplas灢 Bl.containssevencells.DistributionofCa
2+
micelectrondensityislow.SmallCa precipi灢 precipitationsshowsstrongpolaritywithinma灢
2+
tationsstarttoaccumulateontheembryosac tureembryosac,relativelowCa precipitation
wallnearthemicropylarend,butnotatthein灢 ispresentatthechalazalend,spottyprecipita灢
2+
terioroffunctionalmegaspore.SomeCa pre灢 tionsinantipodalcellsandafewsmalldotson
cipitationsareobservedatthemicropyle,andin thevacuolarmembraneofcentralcell(Plate栿:
thenucleiofnucellarcells.(Plate栺:2,white 10),largeprecipitationgrainsremainatthecell
arrowheads). wallofinnerdegeneratedintegument(Plate栿:
nucleateembryosacstage
4灢 11),smallbubblescontainingalargeamountof
2+
Thefunctionalmegasporeundergoesmitotic Ca precipitationcanbeseennearthewallin灢
divisionandgivesrisetoa2灢nucleateembryo sideembryosac(Plate栿:12).Beforefertiliza灢
sac.Rightafterthat,thesecondmitoticdivision tion,asynergidcelldistinctivelyhasdegenera灢
2+
occurs,leadingto4灢nucleateembryosac(Plate ted(Plate栿:13).Ca precipitationsinegg
栺:3A,3B).IncreasesofCa2+ precipitations cell(Plate栿:14)andsynergids(Plate栿:15)
areevidentontheembryosacwall,andnume灢 areabundant,Smallbubbles,whichexhibitag灢
2+
roussmallgrainsofCa precipitationsbeginto gregatesofprecipitation dotsthroughoutits
appearontheembryosacwallsthatarecloseto membrane,areoccasionallyfoundwithinem灢
thechalazalend.Atthechalazalend,continu灢 bryosac.
2+
ousdistributionofCa precipitationsexitbet灢
Discussion
weentheembryosacwallandthecytoplasmof
nucellarcells(Plate栻:4).ThecontentofCa2+ Ca2+playveryimportantrolesonthestruc灢
atouterintegumentisverymuchatthestageof tureandphysiologicalfunctionofplantcell,es灢
four灢nucleateembryosac (Plate栻:5,6). peciallyindoublefertilizationofangiosperm,
2+
ThereisaCa stream whichstartsfromthe butitisunfeasibleforcellstohavehighcontent
2+
outerintegumenttoembryosacwall,inazigzag ofCa forlongtime,owingtothecalciumtoxi灢
wayacrosstheinnerintegument(Plate栻:7). citywhichisassociatedwiththereactionbe灢
2+ 3灢
Alargenumberofgranularprecipitationsare tweenCa andPO4 (Rasmussen,1981;Ras灢
observedinthevacuolesof4灢nucleateembryo mussenandBarrett,1984).Atmegasporocyte
sac,degeneratednucellarcellsandinnerintegu灢 stage,ourobservationresultissimilartoLet灢
ment,especiallyonthecellwallofouterintegu灢 tucethatthereisnoCa2+ precipitatescanbe
2+
ment.ContinuousdistributionofCa canalso found(Qiuetal灡,2005).Thereisapolardis灢
2+
beseenattheapoplastsystemofouterintegu灢 tributionofCa precipitationinmatureembryo
ment,innerintegument,nucellarcellsandem灢 sacinPhaiustankervilliae.Thepolardistribu灢
bryowall(Plate栻:7). tionofCa2+precipitationisgenerallyconsistent
nucleateembryosac
8灢 withtheobservationsfromBrassicanapus,rice
Throughthethirdmitoticdivision,eight灢 andcotton (Yuetal灡,1998;1999;Zhanget
2+
nucleateembryosacreplacesthefour灢nucleate al灡,1997).Sofar,fewworksonhowtheCa
2+
ES,someCa precipitationsareobservedatthe accumulateattheembryosac.
visualnucleusandcytoplasm (Plate栻:8). Inthisstudy,weemployinganidealexperi灢
2+
ThereisafurtherincreaseofCa precipitation mentmaterial,Phaiustankervilliae,about45
云 南 植 物 研 究 第 卷
4暋98暋暋暋暋暋暋暋暋暋暋暋 暋暋暋暋暋暋暋 暋 暋 暋 暋 暋 暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋 32
daysfrommegasporocytetomatureembryosac wallinsideembryosac(Plate栿:12).
(Lietal灡,2006).ThedistributionofCa2+and Basedontheseobservations,wepropose
2+
itsevolutionduringmegasporogenesisandearly thatCa accumulatesinmatureembryosacin
2+
embryosacdevelopmentwererevealedsystem灢 twoways:beforeembryosacmaturation,Ca
aticallywithtransmissionelectronmicroscopy. is mainlytransportedthroughplasmodesmata
ThetransferofCa2+fromouterintegument fromintegumenttonucellarcells,thentoem灢
tonucellarcellsandembryosacisresponsible bryosac;atthestageofmatureembryosac,
fortheaccumulationofCa2+inthematureem灢 Ca2+ migratesthroughembryosacwallinthe
bryosac.Accordingtothestudyontheforma灢 formofsmallbubblecontainingaggregatesof
2+
tionanddevelopmentofembryosacwallinrice Ca precipitation.
(Liuetal灡,1997),twowaysofnutritioncon灢
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tionofsquareaat10,largeCa2+precipitationsgrainsremainat
ExplanationsofPlates
cellwallofinnerdegeneratedintegument.bar=1毺m;12.
Highermagnificationofsquarebat10,smallbubblecontaining
A.Antipodalcell;DS.Degeneratedsynergidcell;DM.Degen灢 significantCa2+inmatureembryosac.bar=200nm;13.Ma灢
eratedmegaspore;E.Eggcell;EW.Embryosacwall;FM. tureembryosacbeforefertilization,asynergidcelldistinctively
Functionalmegaspore;II.Innerintegument;M.Megasporo灢 hasdegenerated.bar=5毺m;14.Highermagnificationofegg
cyte;Mit.Mitochondrion;N.Nucleus;NC.nucellarcell; cellat13,showingCa2+precipitationgrainsatthecytoplasmand
OI.Outerintegument;S.Synergidcell;V.Vacuole nucleusofeggcell.bar=200nm;15.Highermagnificationof
Plate栺:1灢3B暋Micropyledownwardinfiguresandnotethe squareat13,abundantCa2+precipitationatthedegeneratedsyn灢
Ca2+depositswithwhitearrowheads暋1.Developmentofame灢 ergidcell.bar=500nm
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李冬妹等 图版
暋 : 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
期
6 暋暋LIDong灢Meietal灡:UltracytochemicalLocalizationofCalciumduringEmbryoSacDevelopment...暋50暋1
李冬妹等 图版
暋 : 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
云 南 植 物 研 究 第 卷
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