全 文 ：武汉植物学研究 2003, 21( 4) : 281～287
Journal of Wuhan Botanical Research
西瓜生殖细胞向圆球形变化的超微结构研究

刘 林1, 2, 李淑兰1
( 1.临沂师范学院生命科学系, 山东临沂　276005; 2.山东师范大学生命科学学院, 济南　250014)
摘　要: 从超微结构水平上研究了西瓜生殖细胞从凸透镜形到圆球形的变化。细胞变圆过程中发生细胞器重新分
布: 刚产生的细胞中细胞器随机分布,变圆的细胞中细胞器分布呈现极性,与花粉壁相邻区域内质网堆叠而线粒体
稀少, 相反, 向着花粉中心区域线粒体大量聚集而内质网稀少; 细胞器重新分布表明生殖细胞在结构上建立了极
性。细胞变圆有助于脱离花粉壁。细胞核对着花粉壁的一面有凹陷,凹陷中有内质网和核糖体。
关键词: 西瓜; 生殖细胞; 细胞器重新排布; 生殖细胞变圆
中图分类号: Q944. 4　　　　　文献标识码: A　　　　　文章编号: 1000-470X ( 2003) 04-0281-07
Ultrastructural Study on Generative Cell During Rounding
out in Citrullus lanatus
LIU Lin1, 2 , L I Shu-Lan1
( 1. Dep artment of L if e Sci ences, L inyi T eachers Univ ersity , Linyi, Sh andong　276005, Ch ina;
2.Coll eg e of L if e S ci ences, Shand ong Normal Univ er si ty, Jin an　250014, Ch ina)
Abstract: Generative cells in po llen of Citr ullus lanatus were characterized ult rast ructur al ly as
they round out . Organelle rear rangement occur red in response to cell s rounding out . Organelles in
nascent cells w ere dist ributed r andomly . As rounding out , however, the cells show ed polarity , ex-
tensively abundant endoplasm ic ret icula and very sparse mito chondria in the region near po llen
w all, w hile much less endoplasm ic ret icula and a lot more copious mitochondr ia in the o ther side
tow ards pollen center. T he cells r ounding provides an insight into the mechanism for cel ls depar-
ture f rom po llen w all. Nucleus show ed deep invag inat ions w her e endoplasm ic r et icula and ribo-
somes w ere dist ributed.
Key words: Cit rullus lanatus; Generat ive cell; Organelle re-arr angement ; Generat iv e cell s
rounding
　　Gener at ive and vegetat ive cells in pollen in an-
giosperms dist inct ly dif fer in dimension. Vegetat ive
cells are lar ge, taking up almost the w hole inner
space of pollen, while generat ive cells quite a lo t
smaller . Nascent generat ive cells, revealing a con-
vex shape, ar e located along po llen int ine, and
separ ated fr om vegetat ive cells by curved w alls.
Such shape and location of gener at ive cells do no t
r emain long , for they round out soon af terw ards
and move into vegetat ive cel ls
[ 1 5] .
Generat ive cells rounding out is a remarkable
event w ith respect to their depar ture from pol len
w all . Mechanism for cell shaping has been studied.
In plants m icrotubule and act in cytoskeletons form
an interconnected array of f ilaments that span the
cytoplasm ic space of cells, w hich determines cells


Received date: 2003-01-15, Accep ted date: 2003-04-17.
Fou ndat ion item :T his s tudy is supported w ith Linyi Teachers University Fou ndat ion( YJB01002) .
Biog raph y: LIU L in ( 1963- ) , male, Ph. D., Prof., res earch interes t mainly in sexual plant reproduction .
shape. It has been demonst rated in some species
that the shape of g enerat iv e cell is def ined by cy-
to skeletal reor ganizat ion [ 6 9] . N ormally , there is a
clo se relat ionship between org anelle arrangement
and cytoskeleton arr ays, but almost nothing has
been known on o rganelle arrangement in generat ive
cells during their r ounding pr ocess.
Gener at ive cells in watermelon is convex-
shaped when they are born, and start rounding be-
fo re they enter v egetat ive cells
[ 1] . An ef fort w as
made in this study to character ize or ganelle rear -
rangement corresponding to r ounding-out of gener -
ative cells in this species.
1　Materials and Methods
Anthers in w atermelon, Citrullus lanatus
( T hunb. ) Mansfeld, were dissected from flow er
buds of dif ferent developmental stag es. Place the
isolated anthers in a mix ture containing 2%
paraformaldehyde, 2. 5% glutar aldehyde, and
0. 1 mo l/ L phosphate buf fer , pH 7. 2, and incubate
fo r 10 hours at r oom temper ature. Wash the t issues
w ith phosphate buffer, and then incubate them in
1% aqueous osmium tet ro xide for 10 hours at 4℃.
Dehydr ate the t issues by successive 10 m in incuba-
tions in 30% , 50%, 60% , 70%, 80%, 90% , 100%
ethanol solut ions r espect ively . Replace the f inal al-
cohol t reatment with propylene ox ide and incubate
fo r 10 m in at room temperature. Repeat the propy-
lene oxide w ash tw ice. Add an equal v olume of
mix ture of Epon-812 to the last propylene ox ide
w ash and m ix them thor oughly. After 4 hours tis-
sues w er e embedded in resin. Bake the resin at 60℃
overnight .
Sect ions 2
m thick w ere first cut to localize
the generat ive cells needed for ult rastr uctural ob-
servat ion. 2
m thick sect ions w ere heated to dry
and stained w ith a solut ion containing 1% toluidine
blue O and 1% borax . Sat isfactory samples w ere
further cut at 70 nm . The ult rathin sections w ere
double stained w ith ur anyl acetate - lead cit rate,
and observat ions w ere done using a t ransmission
elect ron microscope.
2　Results
T he nascent g enerat iv e cell is small and show s
a convex shape. Nearly half sur face of the cell is
contact w ith pollen int ine ( P late Ⅰ: 1- 4; Plate Ⅱ:
5- 7) . The cell has a cur ved wall containing f ibril-
lous materials, the edge o f the w all joining pol len
int ine ( Plate Ⅰ: 1 - 4; Plate Ⅱ: 5 - 7) . T he cell is
thus bounded w ith a f ibr illous w all and pollen in-
t ine ( Plate Ⅰ: 1- 4; Plate Ⅱ: 5- 7) . A lot of plas-
modesmata connect the tw o cells ( P late Ⅰ: 1- 4;
P late Ⅱ: 5- 7) . Soon the cell begins to round out .
As the cell becomes more spherical, the contact
area between it and pollen intine becomes smaller,
it go es deeper into the vegetative cell , and its w all
g radually disappear unt il the space betw een the
tw o cells becomes elect ron t ranslucent ( Plate Ⅰ:
1- 4; P late Ⅱ: 5 - 7 ) . Plasmodesmata can reach
over 180 nm in diameter ( Plate Ⅱ: 6) . T he genera-
t ive cell that has just entered vegetat iv e cel l takes a
spherical shape, and plasmodesmata ar e very few
( Plate Ⅱ: 8) .
It is recognizable that , at the edge of the ge-
nerat iv e cell , w all and cell membr ane ar e more se-
parated ( P late Ⅰ: 2) .
Nucleus of the nascent generat ive cell is flat
and is localized on the side in contact w ith pol len
int ine. Space betw een nucleus and cell membrane is
so nar row that it is impossible for o rganelles such
as endoplasm ic reticula and mitochondr ia to g o in-
to. The nucleus can have deep invaginations on side
adjacent to pollen w all. In invag inat ions are endo-
plasm ic ret icula, vacuoles, and r ibosomes ( P late Ⅰ:
1, 2) .
Generat ive cells display a w hole suit of or-
ganelles except plast id, the richest among them are
m ito chondria, rough endoplasmic r et icula, and ribo-
somes, and there are also some dictyo somes. Mito-
chondria in cel ls o f differ ent developmental stag es
display dif ferent shapes and st ructur es. They are
more spher ical and w ith relat ively less cristae in
nascent cells ( Plate Ⅰ: 1- 4) than in tho se that al-
most depart f rom pollen w all ( Plate Ⅱ: 5, 6) .
T he arr angement o f o rganelles in the convex-
282 武 汉 植 物 学 研 究　 　　　　　　　　　　　　　　　　第 21 卷　
shaped gener at ive cell is dist inct ly dif ferent to that
in the r ounded cell . Or ganelles in convex-shaped
cells ar e dist ributed in cytoplasm random ly ( Plate
Ⅰ: 1- 3) . As the cel l is rounding out , arrangement
of endoplasmic ret icula and mitochondria becomes
asymmetrical and makes the cell evidently po la-
rized. Rough endoplasmic ret icula are accumulated
in the pole that is contact w ith pollen int ine. Endo-
plasmic reticulum stacks tend to be o rientated par -
allel to cel l membrane. In between endoplasmic
ret icula are copious poly ribo somes. Mitochondria
are v ery few in this pole. In the pole point ing to
po llen center , how ever , m itochondria ar e str ikingly
abundant , w hile endoplasmic ret icula are relatively
few ( Plate Ⅱ: 5, 6) .
3　Discussion
3. 1　Signif icance of generative cell polarization
In concomitance w ith gener at ive cells roun-
ding out in w atermelon, there occurs rearrange-
ment of org anelles, which makes the cells po lar i-
zed. Instead of dist ribut ing r andomly as in the
nascent cell, r ough endoplasmic ret icula in roun-
ding cells accumulate in the pole in contact w ith
po llen w all , and mitochondr ia pile up in the other
po le tow ards pol len center. Microspores undergo
po larizat ion before they div ide and give rise to tw o
very differ ent cells, a generat ive cell and a vegeta-
tiv e cell
[ 10]
. Polarizat ion of m icrospo res is dif ferent
to that of gener at ive cells. Microspore polarizat ion
in watermelon is a result of vacuolizat ion, nucleus
and most org anelles in one end and relatively lit t le
cy toplasm and few o rganelles in the o ther end
[ 11]
.
Generative cell polarizat ion in w atermelon, how-
ever , is fo rmed by different o rganelles accumulated
in dif ferent amount in oppo site ends. Sperms,
w hether produced in a 3-celled po llen grain o r in a
po llen tube of 2-cel led po llen g rain, are dif ferent
ultr ast ructur al ly
[ 12, 13] . Such considerat ions lead to a
quest ion, does generat iv e cell polarizat ion con-
tr ibute to dif ferentiat ion of sperm cells? Or , is g e-
nerat ive cell polarizat ion necessary fo r fo rmat ion of
dif ferent types of sperms?
The generat iv e cell polarizat ion implies cy-
toskeleton reorg anizat ion, for it has been r epeated-
ly demonst rated that there is a close relat ionship
betw een o rganelle arr angement and cy to skeleton
arr ay s. M icrotubules and act in filaments pr ovide
the necessary int racel lular highw ay sy stem fo r the
movement o f or ganelles f rom one place to ano ther
and thus decide their spat ial locat ion
[ 14, 15]
.
3. 2　Generative cel ls rounding out provides a dy-
namic mechanism for their departure from pollen
intine
What ar e the dynam ic facto rs that cause ge-
nerat iv e cell r ounding? Or ganizat ion of micro tubule
and microf ilament cy to skeletal arrays plays an im-
portant r ole in def ining cell shape in plants
[ 6, 16]
.
Relationship betw een generat ive cells rounding
out and cytoskeletal r eo rganization w ere discovered
in some species such as N icotiana tabacum
[ 7]
,
Gagea lutea
[ 8]
, and Hyacinthus oriental is
[ 9]
.
Generat ive cel ls in most plants are born to be
in contact with pollen wall , and then g radually de-
part f rom the w all and enter vegetat ive cells f inal-
ly
[ 1 5]
. There should be an int ricate mechanism for
the depar ture. But only some supposit ions about
the mechanism have been made so far, and these
supposit ions can be summarized briefly as follow-
ing. ( a) Wall thickening w as observ ed in the con-
tact area betw een generat iv e cells and pollen in-
t ine, leading to a guess that the w all thickening
contributes to the departure
[ 2, 17 19]
. ( b) A layer of
l ipid bodies around the gener at ive cell in some
species w as demonst rated
[ 18 21] , and a speculat ion is
that interfacial tension of these lipid bodies is help-
ful for the departure
[ 2 2] . ( c) There is also a v iew
that cytosckeleton is of importance for tr ansfer of
g enerat iv e cells. It w as demonst rated that in L ilium
longif lor um large numbers of m icro tubles circle
r et icular walls that links generat ive cell to pol len
int ine, implying that micro tubules provide mot ivi-
ty[ 19] . Our opinion is that the generat ive cell s
r ounding out plays an impor tant r ole in the depar-
ture.
In w atermelon the contact surface area be-
tw een nascent generat ive cell and po llen int ine is as
w ide as nearly half of the generat iv e cells total
283　第 4 期　　　　　　　　　　 　刘 林等:西瓜生殖细胞向圆球形变化的超微结构研究(英)
surface w hen the cell is convex-shaped, and it de-
creases to almost a po int w hen the cell is rounded.
Apparent ly , the more spherical the gener at ive cells
are, the less the contact area becomes. In the edge
the cell membr ane is mor e separated f rom w al l, sig-
nifying that cell membrane moves centripetally, but
at the same t ime w all does not move, per haps for
this reason fibrillous w all in betw een the tw o cells
diminishes soon. Such move is caused, mo re or
less, by the cells rounding out . T hese facts lend
pr obability to a statement that g enerative cells
rounding out provides a dynamic mechanism for
their departure f rom pollen int ine. But the depar -
ture process should be more int ricate, for the dir ect
result of departure is the entry of the generat ive
cell into the vegetativ e cell . From the po int of vege-
tat ive cells, the entry o f generat ive cells into vege-
tat ive cells is an endocy t ic process, or phagocy to-
sis. Phagocytosis is energy-dependent , and is medi-
ated by clathrins o r proteins of this type
[ 23, 24] . Con-
tents in endocy tic v esicles are to be degenerated.
Generative cells enclosed in cytoplasm of v egetat ive
cells, however, are not harmed at all. So the puta-
tiv e phagocytosis of g enerative cells is supposed to
be more complicated.
3. 3　Function of nuclear invaginations
The nucleus of the nascent generat ive cell in
w atermelon is flat and l ies against cell membrane.
Theoret ically , the nucleus rounding is necessary
fo r a flat cell to become rounded. In pr esent study
w e found that the f lat nucleus has invaginat ions,
and the invaginations contain rough endoplasmic
ret icula and ribosomes. The substant ial incr ease in
nuclear surface ar ea result ing f rom these invagina-
tions suggests that the st ructures might function in
mRNA expor t f rom the nucleus and in pr otein im-
por t fr om the cytoplasm to the nucleus
[ 25] , and in
signaling fr om the cytoplasm to the nucleus
[ 26] .
The invag inat ions per haps mean amoebo id move-
ment , and perhaps in such w ay does the nucleus
move. It has been demonst rated that nuclei in some
types of plant cells can be dynamic, hav ing the abi-
lity to change shape and move
[ 27]
. P robably nuclear
invag inat ions in w atermelon should be considered
in association w ith generative cells rounding.
3. 4　Close communication between generative and
vegetative cells
A lot o f plasmodesmata in w atermelon inter-
connect the generat ive and vegetat ive cells w hen
the former are becoming rounded. It means highly
ef fect ive communicat ion betw een the two cells is
necessary for the generat ive cell s rounding. Gener-
ally plasmodesmata measure approx imately 40 nm
in diameter
[ 28] . However, the diameter reaches 180
nm in this species. T his needs fur ther understan-
ding .
Ref erences:
[ 1 ]　L iu L , Zhang H Y, Fan S G , et al. Further observ a-
tion on tapetum and po llen development in w aterme-
lon[ J] . J W uhan Bot Res, 2000, 18( 2) : 85 90.
[ 2 ]　Heslop-Harr ison J. Synchronous pollen mito sis and
fo rma tion of t he gener ativ e cell in massulate or chids
[ J] . J Cell Sci, 1968, 3: 457 477.
[ 3 ]　Sassen M M A, K roh M . The “cell w all”a round the
generative cell [ J] . A cta Bot N eerl, 1974, 23: 354
355.
[ 4 ]　M aruyama K , Gay H, Kaufmann B P . The natur e of
t he w all betw een gener ative and veget ativ e nuclei in
T radescantia p aludosa[ J] . Am J Bot, 1965, 52: 602
610.
[ 5 ]　Sanger J M , Jackson W T . F ine str uct ur e study of
pollen development in H aemanthus kather inae Baker .
I . F ormation o f vegetative and genera tiv e cells [ J] . J
Cell S ci, 1971, 8: 289 301.
[ 6 ]　Szymanski D B. A rabidop sis tr ichome morphgenesis:
a g enet ic approach t o study ing cyt oskeleta l function
[ J] . J Plant Grow th R egulation, 2001, 20: 131 140.
[ 7 ]　T heunis C H, Pierson E S, Cresti M . The micro tubule
cy toskelet on and t he r ounding o f isolated gener ative
cells of Nicotiana tabacum [ J ] . Sex P lant Rep rod ,
1992, 5: 64 71.
[ 8 ]　Zhang H Q , Bohdanow icz J, Pierson E S, et al. M icro-
tubular or ganization during asymmet rical division of
t he gener ative cell in Gagea lutea [ J ] . J Plant Res ,
1995, 108: 269 276.
[ 9 ]　Del Casino C, T iezzi A , Wagner V T , et al. The or g a-
nization of the cyto skeleton in the gener ative cell and
sperms of Hyacinthus or ientalis [ J ] . P rotop lasma,
1992, 168: 41 50.
[ 10]　Park S K , How den R, Tw ell D. The A rabidop sis
284 武 汉 植 物 学 研 究　 　　　　　　　　　　　　　　　　第 21 卷　
thaliana gametophy tic mutat ion Gemini po llen dis-
rupts micr ospor e polar ity , div ision asymmet ry and
po llen cell fate [ J ] . Development, 1998, 125 ( 19 ) :
3 789 3 799.
[ 11]　L iu L , Xu Q S, Zhang Z J, et al. O rg anellar changes in
micr opor es of Citrullus vulgaris Schrad[ J] . J Wuhan
Bot Res , 2000, 18( 4) : 266 270.
[ 12]　Cai X , Wei L B. U ltr astr uctur e features o f sperm cell
in Amary llis vittata [ J ] . Dev elop Rep r oduc Biol,
1996, 5( 1) : 53 59.
[ 13]　Roug ier M , Jnoud N , Said C, et al . Male gametophyte
development and formation of the male germ unit in
Dopulus d elloid es fo llow ing compat ible pollination
[ J] . Protop lasma, 1991, 162: 140 150.
[ 14]　Holzinger A , Monajembashi S, Greulich K O , et al.
Impairment o f cy toskeleton-dependent vesicle and o r-
ganelle translocation in gr een algae: combined use of
a micr ofo cused infrar ed laser as micr obeam and opti-
cal tw eezers[ J] . J M icrosc, 2002, 208( 2) : 77 83.
[ 15]　Romagnoli S, Cai G , Cresti M . I n v itr o assay s demon-
st rate that pollen tube or ganelles use kinesin-related
mot or pr ot eins t o move along micro tubules[ J] . Plant
Cell, 2003, 15( 1) : 251 269.
[ 16]　Ket elaar T , De Ruijter N C, Em ons A M . Unstable
factin specifies t he area and micro tubule dir ection of
cell expansion in A rabidop sis ro ot hairs [ J ] . Plant
Cell, 2003, 15( 1) : 285 292.
[ 17]　Cresti M , Ciampolini F , Kapil R N . Gener ativ e cells
of some ang iosperm s with part icular emphasis on
their micr otubes [ J ] . J Submicr osc Cy tol, 1984, 16:
317 326.
[ 18]　Angold R E. The fo rm ation of gener ativ e cell in t he
po llen g rain o f E ndymion non-scrip tus ( L . ) [ J ] . J
Cell Sci, 1968, 3: 573 578.
[ 19]　Nakamura S, M iki-Hir osig e H. Fine-st ructural st udy
on t he fo rmat ion of the gener ativ e cell w all and in-
tine-3 layer in a g rowing po llen g rain of L ilium longi-
f lorum[ J] . Am J Bot, 1985, 72: 365 375.
[ 20]　Brighigna L , F iordi A C, Palandri M R . U ltra st ruc-
tur al investigations on the t wo-nucleate pollen of
T illandsia caputmedusae Morr ( Brom elliaceae ) [ J] .
A m J Bot, 1981, 68: 1 033 1 041.
[ 21]　M igia M , W ilms H J, G resti M . U ltr astrctur al com-
parison of pollen g r ains fr om 2n, 3n, and 4n plants of
E up hor bia dulcis [ J ] . A m J B ot, 1987, 74: 1 665
1 671.
[ 22]　Luo Yu-Ying, Hu Shi-Y i. T he development and
str uct ur e o f t he gener ativ e cell w all in P olygonatum
simizui [ J] . A cta Botanica Sinica, 1995, 37 ( 1) : 7
13.
[ 23]　Ho lstein S E. Clathrin and plant endocyt osis [ J ] .
T raf f ic, 2002, 3( 9) : 614 620.
[ 24]　Peters J P , Gao M , Gaschet J, et al. Char act erization
o f co ated vesicles that part icipate in endocy tic recy-
cling[ J] . T raf f ic, 2001, 2( 12) : 885 895.
[ 25]　Co llings D A , Car ter C N , Rink J C, et al. Plant nuclei
can contain ex tensive g ro oves and invag inations [ J] .
P lant Cell , 2000, 12( 12) : 2 425 2 440.
[ 26]　Lui P P Y , Kong S K , Kw ok T T , et al. The nucleus
o f HeLa cell contains tubular st ructures for Ca2+ sig-
nalling [ J] . Biochem Biophy s Res Commun, 1998, 247:
88 93.
[ 27]　Chyt ilov a E, Macas J, Sliw inska E, et al. Nuclear dy-
namics in A rabidop sis thaliana [ J ] . Mol Biol Cell,
2000, 11: 2 733 2 741.
[ 28]　T ilney L G, Cooke T J, Connelly P S, et al . The
str uct ur e o f plasmodesmata as r evealed by plasmo ly-
sis, deter gent ex tr action, and protease dig estion[ J] . J
Cell B iol, 1991, 112( 4) : 739 747.
285　第 4 期　　　　　　　　　　 　刘 林等:西瓜生殖细胞向圆球形变化的超微结构研究(英)
刘 林等: 图版Ⅰ LIU Lin et al .: P late Ⅰ
d. Dict yo some; er . Endoplasm ic ret iculum; g . Groove; GN . Gener ativ e nucleus; m. M ito chondr ion; p. Plasm odesma ;
V . Vesicle ; VN . Vegeta tiv e nucleus; w . Wall
1. Show ing a nascent gener ativ e cell and a vegeta tiv e nucleus. T he gener ativ e cell convex-shaped, or ganelles distr ibut ed r an-
domly , nucleus r ev ealing deep g r ooves, w all cur ved. Scale bar= 3. 3
m; 2. Magnification o f t he gener ative cell show n in F ig .
1. Nuclear gr oo ves containing vesicle and ER , nuclear surface nearly touching cell membrane , w all containing fibr illous mat e-
rials and linking po llen intine. At the edge of the cell, cell membrane ( arr ow ) is mo re separated to w all. Scale bar= 6. 7
m;
3. Show ing a generat ive cell, nucleus still flat , o rg anelles r andom ly distr ibut ed. Scale ba r= 2. 5
m; 4. Magnification o f the
part o f the gener ativ e cell in show n in Fig . 3. Showing fibr illo us w all, ribo somes, and mit ochondr ia . Scale bar= 400 nm
286 武 汉 植 物 学 研 究　 　　　　　　　　　　　　　　　　第 21 卷　
刘 林等: 图版Ⅱ LIU Lin et al .: P late Ⅱ
5. Showing a gener ativ e cell. ERs stacked in reg ion linked t o po llen w all, m ito chondr ia accumula ting in reg ion near pollen
cent er. Scale bar= 2
m; 6. Magnificat ion o f the pa rt o f g ener ativ e cell show n in Fig . 5. M itochondria elongated, and contai-
ning mo re cr istae. There a re many plasmodesmata. Scale bar= 1
m; 7. M agnifica tion o f por tion of g ener ative cell show n in
Fig . 5. I n betw een ERs ther e ar e a lot of poly ribo somes. Only small a rea linked to po llen intine ( + ) . Scale bar = 1
m; 8.
Show ing a genera tiv e cell in cy toplasm of a veget ativ e cell, w all tr anslucent , plasmodesmata ver y few . Scale bar= 1. 2
m
287　第 4 期　　　　　　　　　　 　刘 林等:西瓜生殖细胞向圆球形变化的超微结构研究(英)