全 文 ：Received 17 Nov. 2003 Accepted 12 Mar. 2004
Supported by the National Natural Science Foundation of China (39600008), the Teaching and Research Award Program for Outstanding
Young Teachers in Higher Education Institutions of Ministry of Education of China (2002383), and Guangdong Provincial Natural Science
Foundation for group project (200023).
* Author for correspondence. E-mail: ; .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (7): 829-838
Abnormal Behavior of Nuclei and Microtubule (MT) Organizational Changes
During Embryo Sac Development in the Poly-Egg Mutant, APⅣ of Rice
LIU Xiang-Dong1, LU Yong-Gen1, ZHU Hong-Liang1, XU Xue-Bin1,
FENG Jiu-Huan1, XU Shi-Xiong (Zee S Y)2*
(1. Guangdong Provincial Key Laboratory of Plant Molecular Breeding, South China Agricultural University,
Guangzhou 510642, China;
2. Department of Botany, the University of Hong Kong, Pokfulam Road, Hong Kong)
Abstract: APⅣ is a rice mutant that develops poly-egg apparatus in its embryo sac. All the eggs that
make up the poly-egg apparatus can be fertilized respectively resulting in the development of polyembryony.
The routes taken in the development of polyembryony appear to fall mainly into three variant polygonum
pattern types, designated as “5-2-1”, “5-3-0” and “6-2-0” types. Out of the embryo sacs of APⅣ studied
about 50% exhibited variant polygonum type with associated abnormal nuclear behavior and microtubule
organizational changes. Some of the major abnormal features shown by the three variant polygonum types
were described and they included the following: For the “5-2-1” type — At the beginning of the four-
nucleate embryo sac development, one pair of nuclei became located to the micropylar end and the other
pair to the chalazal end. As embryo sac further developed, long connecting microtubule (MT) bundles that
existed between the two nuclei in the chalazal end play a role in the movement and positioning of that
nucleus. As a result of the activities of these MT, one of the nuclei in the chalazal end moved to the
micropylar end resulting in the micropylar end having three nuclei and the chalazal end only one. For the
“5-3-0” type — In the two-nucleate embryo sac of the “5-3-0” type, one nucleus remained at the
micropylar end, while the other one became located near the central region. In the four-nucleate embryo
sac, the pair of nuclei aligned in parallel to the micropylar-chalazal axis often having one of its nuclei
relocated to the micropylar end as a result of associated MT activities. For the “6-2-0” type — All the
nuclei in the megaspore, two- and four-nucleate embryo sacs became located to the micropylar end. At
the early stages of the eight-nucleate embryo sac development, the two nuclei in the central region of the
embryo sac (originally at the micropylar end) became polar nuclei. All the other nuclei remained at the
micropylar end were surrounded by reticulate MT. The relationship between abnormal behavior of nuclei
and MT organi-zation in the development of rice embryo sac was discussed.
Key words: rice (Oryza sativa); embryo sac; mutant; polyembryony
The development of embryo sac is a very important
and complex event in plant. Embryo sacs are wrapped
around by the ovary and it is technically very difficult to
observe the detail events taking place during embryo sac
development without resort to using sectioned materials
or whole cleared mounts, etc. These involve techniques
that are both very time consuming and difficult. For this
reason, so far, only a few gametophytic mutants in rice
have been studied with respect to their embryo sac
development. Among those already reported are SB-1 and
APⅣ (Liu et al., 1994; 1996a; 1996b; 1996c; 1996d; 1997).
Rice plant possesses a highly specialized floral structure
and each embryo sac has only one egg cell. Liu et al.
(1996a; 1997) found a rice mutant, APⅣ that contains more
than one egg (sometimes refer to as poly-egg apparatus)
in its embryo sac. All the eggs in the embryo sac could be
fertilized resulting in polyembryony. The number of egg
cells present within an embryo sac varies. The most com-
mon is with three eggs in one embryo sac. The poly-egg
apparatus developed in the embryo sac has been classi-
fied and placed into three groups, designated as “5-2-1”,
“5-3-0” and “6-2-0” variant polygonum types (Liu et al.,
1996a; 1996b). In the “5-2-1” type the mature embryo sac
usually contains three eggs and two synergids at the mi-
cropylar pole, two polar nuclei above the egg-apparatus
and one group of antipodal cells at the chalazal pole. In the
“5-3-0” type the mature embryo sac contains three eggs
and two synergids at the micropylar end and two polar
Acta Botanica Sinica 植物学报 Vol.46 No.7 2004830
nuclei as well as one group of antipodal cells above the
egg-apparatus. In the “6-2-0” type the mature embryo sac
contains three eggs and two synergids at the micropylar
end, two polar nuclei above the egg-apparatus and one
group of antipodal cell at the micropylar end (Liu et al.,
1996a; 1996b). Abnormal nuclei behavioral patterns have
been found in these variant polygonum types during the
development of the embryo sac (Liu et al., 1996b) and they
may appear to be affected by microtubule organizational
changes .
Microtubules (MT) are generally believed to play an
important role in determining cell shape, the position and
orientation of nuclei, etc. (Willemse and van Lammeren,
1988; Derksen et al., 1990; Brown and Lemmon, 1992; Huang
and Sheridan, 1994). During the development of the mu-
tant embryo sac, studies on microtubule organization could
be useful in understanding the roles played and the con-
trol mechanism involved during abnormal embryo sac de-
velopment at both the cytoplasmic and genetic level
(Staiger and Cande, 1990). Recently, Zhu et al. (2002) stud-
ied the pattern of changes of MT organization during me-
gasporogenesis in normal rice IR36 and APⅣ and some of
their preliminary findings indicated that the role played by
microtubules in nuclei movement is important. In this paper,
the abnormal nuclear behavior and the changes in MT
organization during megagametogenesis in APⅣ are de-
scribed in much more detail and some new features that
have not been reported before are also described.
1 Materials and Methods
A normal rice cultivar IR36 (used as control) and a poly-
egg rice mutant APⅣ were planted in the farm of South
China Agricultural University. Florets were collected from
the stage of megasporocyte formation to embryo sac ma-
turity and fixed for 1 h at room temperature with a fixative,
which contained 10% DMSO, 0.01% Triton X-100, 0.01%
MSB (3-Maleimidobenzoic acid N-hydroxysuccinimide
ester) and 4% paraformaldehyde prepared in PEMS buffer
(50 mmol/L Pipes, 2 mmol/L MgSO4, 5 mmol/L EGTA, 4%
sucrose, pH 6.8). Rinsed briefly with PEMS buffer, samples
were then dehydrated in a graded series of ethanol (V/V)
(10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and
100%), infiltrated in a polyethylene glycol (PEG) mixture
and then embedded following the protocol described by
Xu et al. (2001a; 2001b). The samples were purposely cut
into 50 mm thick sections to include all the embryo sac
nuclei in most sections using razor blades and an AO rota-
tional microtome. The sections were incubated in 100 mL of
monoclonal antibody anti-a-tubulin (Sigma T-9026) diluted
1:40 in PBS for 1 h. After being washed, the samples were
then incubated in anti-mouse IgG (Whole molecule) FITC
conjugate (Sigma F-0257) diluted 1:200 with PBS for 1 h.
The nucleus was stained with PI and the nucleoli showed
much stronger red fluorescence (Richard, 1996). Nuclei and
microtubules were observed by using a Leica SP2 laser
scanning confocal microscopy. FITC and PI at two differ-
ent excitation wave lengths (494 nm and 535 nm) were used
for fluorescence observation. The sections containing all
the nuclei of a selected embryo sac were identified (Liu et
al., 1996a; 1996b) and then serially scanned at different
levels (or layers) with each layer about 2 mm in thickness.
The optical sections obtained were then reconstructed into
a 3-D or complete embryo sac with the help of computer
graphics. The images of the MT and nuclei were printed
out using a Polaroid HR 7000 Digital Palette File Recorder.
In order to observe the developmental process of em-
bryo sacs of all the variant polygonum types of APⅣ
systematically, a large number of ovaries from the stage of
megasporocyte to embryo sac maturity were fixed in FAA
(formaldehyde, acetic acid, 50% ethanol, mixed in the fol-
lowing ratio, 5:6:89) for 24 h at room temperature for check-
ing and screening. After rinsing twice with 50% ethanol,
the samples were dehydrated in a graded series of ethanol
(50%, 30% and distilled water) and stained with 10 mg/L
eosin (dissolved in 4% sucrose) for 12 h. Samples were
washed in distilled water and dehydrated in another graded
series of ethanol (30%, 50%, 70%, 80%, 90% and 100%
ethanol). Dehydrated samples were transferred into a mix-
ture of absolute ethanol and methyl salicylate (1:1) for 1 h
and cleared for 1 h in pure methyl salicylate solution. Nu-
merous slides were prepared from the cleared samples.
Nuclei stained with eosin (which produced a red
fluorescence) in embryo sacs were examined with a Leica
SP2 Laser scanning confocal microscope. Thirty layers of
sections for each embryo sac were normally scanned. They
were then integrated and the images of the whole embryo
sac were printed out using a Kodak Professional 8660 Ther-
mal Printer (Zhang et al., 2003).
2 Results
The megasporocyte of the rice mutant, APⅣ under-
went what appeared to be quite normal meiosis producing
four megaspores. Gradually three out of four of the me-
gaspores near the micropylar end degenerated, but the
chalazal-most megaspore survived and became the func-
tional megaspore. The functional megaspore divided mi-
totically three times to ultimately develop into a mature
embryo sac. Our fluorescence observations showed that
LIU Xiang-Dong et al.: Abnormal Behavior of Nuclei and Microtubule (MT) Organizational Changes During Embryo Sac
Development in the Poly-Egg Mutant, APⅣ of Rice 831
Acta Botanica Sinica 植物学报 Vol.46 No.7 2004832
about one half of the functional megaspores in APⅣ de-
veloped into normal embryo sacs following the normal
polygonum type pathway to give to mature embryo sacs,
each containing one egg and two synergids in the micro-
pylar pole end, two polar nuclei above the egg-apparatus,
and a group of antipodal at the chalazal end. The distribu-
tion and organization of MT (Figs.1-6) in the embryo sac
during megagametogenesis were similar to that seen in the
embryo sac of normal rice, such as IR36 (Xu et al., 2001b).
The other half of the functional megaspores in APⅣ de-
veloped along the pathway of the three variant polygonum
types, viz. “5-2-1”, “5-3-0” and “6-2-0” types (Liu et al.,
1996a; 1996b). In these variant polygonum types abnor-
mal behavior of nuclei and MT organizational patterns were
observed during the development of the embryo sacs.
Some of the major abnormal phenomena are described
below.
2.1 The “5-2-1” type
In the functional megaspores and the two-nucleate
embryo sacs of the “5-2-1” type the position of nuclei and
the distributional pattern of MT were not different from
those of the normal embryo sacs at the same stage of
development. In the early stage of four-nucleate embryo
sacs the distribution of nuclei also appeared to be quite
normal. But later, the distribution and movement of nuclei
in the four-nucleate embryo sac became different from the
normal. One of the nuclei belonged to the pair of nuclei
more closely located to the central region of the embryo
sac in the chalazal pole moved towards the micropylar end
of the embryo sac (Figs. 7, 7b, 8). Longitudinal MT bundles
existed between the two nuclei in the chalazal pole, and
also did between the two nuclei in the micropylar pole.
The long parallel MT (arrow) between the two sister nuclei
in the chalazal pole was a bit longer but less than that
situated between the nuclei in the micropylar end and the
MT organization around the nucleus (N) near the central
region of embryo sac was more dense than that around
another nucleus in the chalazal pole indicating that these
MT could play a role in the movement and positioning of
that nucleus (Figs. 7, 7a). As a result of the peculiar move-
ment of the nuclei a new four-nucleate embryo sac was
formed in which only one nucleus existed at the chalazal
end and three nuclei in the micropylar end (Fig.8). This is
one such peculiar feature that existed in the “5-2-1” em-
bryo sac, but as the embryo sac developed further more
peculiar feature appeared. In the eight-nucleate embryo
sac the development of the embryo sac from the eight-
nucleate stage to full maturity took a longer period of time.
During this prolonged developmental stage more complex
nuclei movement, in comparison with the other stages e.g.
the two-nucleate and four-nucleate stage, took place. In
order to facilitate description, the prolonged developmen-
tal stage at the eight-nucleate stage was divided into three
substages, viz.: early, mid and late stage of eight-nucleate
Figs.1-8. The micropylar end is at the bottom of photographs. Microtubule (MT) is shown in green, and chromosomal material and
nucleoli in red (N.B. The nucleoli show very strong fluorescence so they appear particularly outstanding in the photographs). A,
antipodal; E, egg; S, synergid. Arrows indicate the position of nuclei and/or nucleoli in the embryo sac if they are not given special
meaning. All photographs are projections from a series of optical sections obtained from whole embryo sacs sectioned at about 30 mm
to 50 mm in thickness. Scale bar = 10 mm. 1. Normal functional megaspore showing some randomly distributed MT (double arrow) in the
cytoplasm and nucleus(arrow). Note that the degenerated megaspores are un-specifically stained with PI so they also show some
fluorescence (star). 2. Normal functional megaspore at a later stage of development (i.e. mono-nucleate embryo sac) showing the
formation of a MT network (double arrow) distributed throughout the whole embryo sac. 3. Normal two-nucleate embryo sac showing
dense MT (double arrow) distributed around the nuclei. 4. Normal four-nucleate embryo sac showing a more sparsely distributed MT
network (double arrow) in the embryo sac. 5. Normal eight-nucleate embryo sac. Note the two nuclei (arrow heads) nearer to the central
region of the embryo sac have long MT (double arrow) associated with them. 6. Normal embryo sac near maturity. The egg-apparatus
(i.e. one egg (E) and two synergids (S)) is present at the micropylar pole (the position of the cells are indicated by the distribution of
nuclei, arrow). The two polar nuclei (arrow head) are situated above a network of microtubules surrounding the egg-apparatus. A group
of antipodal cells (A) is situated at the chalazal end. 7. Four-nucleate embryo sac from a “5-2-1” type. Note long parallel MT are present
between the two sister nuclei near the chalazal pole. Note also that the MT organization around the nucleus (N) near the central region
of embryo sac is more dense than that around another nucleus in the chalazal pole (see also Fig.7a), and the long parallel MT (arrow)
between the two sister nuclei in the chalazal pole is a bit longer but less than that situated between the nuclei in the micropylar end. 7a.
The same embryo sac as in Fig.7 but from a different sectional level showing the cytoplasm around the nucleus (N) near the central region
of embryo sac has segregated (arrow) from that of another nucleus in the chalazal end, i.e. this nucleus (N) separates and moves towards
the micropylar end. 7b. Another view of a four-nucleate embryo sac from the “5-2-1” type showing a nucleus (arrow) originally present
in the chalazal end is now moving towards the micropylar end resulting in the formation of three nuclei (Fig.8) in the micropylar pole of
the embryo sac. This embryo sac has been stained with eosin. 8. A four-nucleate embryo sac from the “5-2-1” type at a later stage of
development than Fig.7. Note there are three nuclei in the micropylar end (arrow) and only one in the chalazal pole (arrow head).
←
LIU Xiang-Dong et al.: Abnormal Behavior of Nuclei and Microtubule (MT) Organizational Changes During Embryo Sac
Development in the Poly-Egg Mutant, APⅣ of Rice 833
embryo sac development.
At the early stage of the eight-nucleate embryo sac
development in “5-2-1” type, only two nuclei at the cha-
lazal region and six nuclei at the micropylar region appeared
(Fig.9) (also see Liu et al., 1996b). Numerous densely
packed MT distributed around the nuclei in the chalazal
end and a spindle (arrow head) was observed among the
nuclei at the micropyle region. This pattern of MT distri-
bution was seldom seen in normal embryo sac. At the mid
stage of embryo sac development one nucleus from each
pole migrated towards the center of the embryo sac to
form polar nuclei. A trans-embryonic array of microtu-
bules appeared and became connected to the polar nuclei
similar to that seen in the embryo sac at the same stage of
development as the normal rice (unpublished data; see
also Xu et al., 2001a). When the eight-nucleate embryo
sac became mature it contained three eggs and two syner-
gids at the micropylar pole. The two polar nuclei at this
stage became situated above the poly egg-apparatus. A
group of antipodal cells aggregated at the chalazal end
(Liu et al., 1996a; 1996b). Reticulate MT distributed in the
eggs (Figs. 10, 10a) was similar to that observed in the egg
of normal rice indicating that the eggs formed in the “5-2-
1” type appeared to be normal.
2.2 The “5-3-0” type
In the “5-3-0” type the functional megaspore nucleus
after mitosis (Figs.11, 11a) produced a quite abnormal two-
nucleate embryo sac with one nucleus located to the mi-
cropylar end of the embryo sac surrounded by few
radiating MT (Fig.12). The other nucleus became posi-
tioned near the central region of the embryo sac away from
the micropyle. Some randomly distributed MT existed be-
tween the two nuclei (Fig.12). Often the megaspore abnor-
mally developed a vacuole at the chalazal end of the em-
bryo sac (Fig.11a). Later, the two nuclei divided again by
mitosis (Fig.13) to produce a four-nucleate embryo sac
(Figs.14,15). The distribution of the nuclei in this “5-3-0”
type of four-nucleate embryo sac was different to the four-
nucleate embryo sac of the normal (cf Fig.4) and the “5-2-
1” type (cf Fig.8). The development of the four-nucleate
embryo sac to the eight-nucleate embryo sac was also quite
different. The four-nuclei after mitosis became eight but
initially a group of two nuclei became located to the cen-
tral region of the embryo sac away from the micropyle while
another group of six nuclei aggregated together and lo-
cated to the micropylar region (Fig.16). One nucleus from
each group then migrated towards the central region of
the embryo sac to form the polar nuclei. Each polar nucleus
possessed a long array of MT bundles around it (Fig.16).
All the other nuclei were surrounded by reticulate
microtubules. When the embryo sac matured, three eggs
and two synergids (with the polar nuclei above the poly
egg-apparatus) developed. A group of antipodal became
closely attached to the polar nuclei, which invariably ad-
hered to the wall of the embryo sac near the micropylar
end (Liu et al., 1996a; 1996b; 1997). The overall arrange-
ment of the antipodal, polar nuclei and poly egg-appara-
tus in the “5-3-0” embryo sac were much closely packed
Figs.9-16. The micropylar end is at the bottom of photographs. Microtubule (MT) is shown in green, and chromosomal material and
nucleoli in red (N.B. The nucleoli show very strong fluorescence so they appear particularly outstanding in the photographs). E, egg; P,
polar nuclei; S, synergid; V, vacuole. Arrows indicate the position of nuclei and/or nucleoli in the embryo sac if they are not given special
meaning. All photographs are projections from a series of optical sections obtained from whole embryo sacs sectioned at about 30 mm
to 50 mm in thickness. Scale bar = 10 mm. 9. An eight-nucleate embryo sac from the “5-2-1” type at early stage of development. Note that
there are two nuclei at the chalazal end and six nuclei at the micropylar end. Arrowhead indicates the spindle. 10. A mature embryo sac
of the “5-2-1” type. There are three egg cells (E) and two synergids in the micropylar region (two egg cell (E) and polar nucleus (P) have
been indicated). Fig.10a. The same embryo sac as in Fig.10 but from a different sectional level. The third egg cell (E) and two synergids
(S) are shown. 11. A functional megaspore from the “5-3-0” type showing nucleus undergoing division. The position of division is much
nearer to the micropyle (arrow) than that seen in the normal embryo sac. 11a. A functional megaspore similar to Fig.11 showing the
presence of a vacuole (V) in the embryo sac. This is a highly abnormal feature. 12. A two-nucleate embryo sac from the “5-3-0” type. Note
that the MT (double arrow) between the two nuclei appears quite abnormal (cf Fig.3). Moreover, the two nuclei are much more closer to
each other in comparison with the normal embryo sac (cf Fig.3). 13. A two-nucleate embryo sac from the “5-3-0” type with both nuclei
undergoing mitosis to give rise to a four-nucleate embryo sac. Note the orientation of the spindle of the two dividing nuclei is different,
one is in parallel to the long axis of the embryo sac (arrow) and the other is in perpendicular to the long axis (arrowhead). 14. A four-
nucleate embryo sac from the “5-3-0”type. Two pairs of nuclei are present. Between sister nuclei there are long microtubule bundles
connecting them. Note that one of the nuclei (arrow head) will move to the micropylar pole. 15. A four-nucleate embryo sac from the “5-
3-0” type at late stage of development. Note that there are three nuclei (arrow) at the micropylar end and one (arrow head) near the
micropyle but not in the chalazal end (cf Fig.8). 16. An eight-nucleate embryo sac from the “5-3-0” type at middle stage of development.
The eight nuclei are shown. The two nuclei (arrow head) with high concentration of MT around them will become the polar nuclei. M
indicates some overlapping of the embryo sac with the nucellus.
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Acta Botanica Sinica 植物学报 Vol.46 No.7 2004834
LIU Xiang-Dong et al.: Abnormal Behavior of Nuclei and Microtubule (MT) Organizational Changes During Embryo Sac
Development in the Poly-Egg Mutant, APⅣ of Rice 835
than that seen in the normal or the “5-2-1” type embryo
sac. The distribution of the antipodal near the micropylar
end was not much different from that seen in the other
embryo sac types. MT distribution in the cells of the ma-
ture embryo sac of the “5-3-0” type appeared to be similar
to the normal and the “5-2-1” type embryo sac.
2.3 The “6-2-0” type
The functional megaspore nucleus in the “6-2-0” type
of embryo sac existed close to the micropyle. Numerous
MT concentrated around the nucleus (Fig.17). In the “6-2-
0” type the two-nucleate embryo sac was characterized by
having two nuclei positioned much closer to the micropy-
lar end. There were much cytoplasm and microtubules ag-
gregated around the two nuclei (Fig.18). In the four-nucle-
ate embryo sac the four nuclei formed remained in the mi-
cropylar end of the embryo sac. The nuclei were surrounded
by a very complex network of MT (Fig.19). When the eight-
nucleate embryo sac formed, all the eight nuclei remained
at the micropylar end of the embryo sac. The chalazal end
of the embryo sac contained a large vacuole. Numerous
reticulate MT were present in the cytoplasm near the mi-
cropylar end (Fig.20). At the middle stage of the eight-
nucleate embryo sac development, two nuclei situated
nearer to the central region of the embryo sac enlarged
and became the polar nuclei which were surrounded by
reticulate microtubules (Fig.21). Some of the nuclei devel-
oped into antipodal cells and they all aggregated near the
micropylar end of the embryo sac (Figs.21a; Liu et al.,
1996a). In the mature embryo sac of the “6-2-0” type, an-
tipodal cells, eggs as well as synergids all concentrated at
the micropylar region (Figs.22, 22a; Liu et al., 1996a; 1996b).
Network like MT was observed present in the synergids
(Fig.22) and this was not different from that seen in normal
rice.
3 Discussion
Liu et al. (1996a; 1996b) were the first to report that
there were three variant polygonum types of embryo sacs
present in the mutant rice, APⅣ (i.e. “5-2-1” type, “5-3-0”
type and “6-2-0” type). The main characteristics shown by
the different variant types in comparison with the normal
type were: abnormal positioning of the functional me-
gaspore nucleus, abnormal division of the functional me-
gaspore nucleus during mitosis, and non-synchronization
of sister-nuclei mitosis in the embryo sac. In this study, we
also found two abnormal features in APⅣ, which have
not been reported in detail before and they are: (1) Func-
tional megaspores formed with nuclei at or near the micro-
pylar pole (Figs.11, 11a, 17) and (2) The direction of divi-
sion in functional megaspore nucleus was perpendicular
to the micropylar-chalazal long axis instead of parallel to
the micropylar-chalazal long axis as seen in normal
development. These characteristics strongly showed that
abnormal positioning of functional megaspore nucleus and
the direction of division of the nucleus during mitosis are
important factors in contributing to the development of
the variant embryo sac types observed in APⅣ. Moreover,
during this study the existence of a new four-nucleate
embryo sac type was found (e.g. as seen in the “5-2-1”
type (Fig.8)) that contained one nucleus at the chalazal
Figs.17-22a. The micropylar end is at the bottom of photographs. MT is shown in green, and chromosomal material and nucleoli in
red (N.B. The nucleoli show very strong fluorescence so they appear particularly outstanding in the photographs). A, antipodal; E, egg;
P, polar nuclei; S, synergid; V, vacuole. Arrows indicate the position of nuclei and/or nucleoli in the embryo sac if they are not given
special meaning. All photographs are projections from a series of optical sections obtained from whole embryo sacs sectioned at about
30 mm to 50 mm in thickness. Scale bar = 10 mm. 17. A functional megaspore from the “6-2-0” type. Note that the nucleus is at the
micropylar pole of the embryo sac and it is surrounded by a high concentration of reticulate MT (double arrow). 18. A two nucleate
embryo sac from the “6-2-0” type. Note that the two nuclei are present in the micropylar end of the embryo sac. Both nuclei are
surrounded by a dense network of MT (double arrow). 19. A four-nucleate embryo sac from the “6-2-0” type. Note that there are four
nuclei present at the micropylar region of the embryo sac and the nuclei are surrounded by a very complicated array of MT (double
arrow). 20. An eight-nucleate embryo sac from the “6-2-0” type at early stage of development showing all eight nuclei at the micropylar
end of the embryo sac. The chalazal end of the embryo sac contains a large vacuole (V). Numerous reticulate MT are present in the
cytoplasm near the micropylar end. Note that the two nuclei (arrow head), which will become polar nuclei, near the central region of the
embryo sac have enlarged somewhat and MT associated with them is different (double arrow). 21. An eight-nucleate embryo sac from the
“6-2-0”type at middle stage of development. The two polar nuclei (arrow head) are located at the central region of the embryo sac.
Antipodals are concentrated more towards the micropylar region and some of them are undergoing division with the formation of
phragmoplasts (arrow). 21a. The same as Fig. 21 but at a different sectional level. Note the presence of antipodal (A) at the micropylar
end. The embryo sac has been stained with PI. 22. A mature embryo sac from the “6-2-0” type showing one synergid (S) and some
antipodals (A) near the micropylar region. Note there is a lot of longitudinally oriented MT (double arrow) in the synergid. Some
degenerated cell materials (arrow) have been unspecifically stained by PI. 22a. The same as Fig. 22 but at a different sectional level. Note
the egg cell (E) is abnormal because of the presence of antipodal (A) at the micropylar end.
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LIU Xiang-Dong et al.: Abnormal Behavior of Nuclei and Microtubule (MT) Organizational Changes During Embryo Sac
Development in the Poly-Egg Mutant, APⅣ of Rice 837
pole and three nuclei at the micropylar pole. The addi-
tional nucleus in the micropylar pole was derived from one
of the nuclei originally existed at the chalazal pole. It was
well known that additional eggs formed in the abnormal
embryo sacs were the result of movement of a nucleus
from the chalazal pole to the micropylar pole. Subsequent
mitosis of the nucleus and other nuclei at the micropylar
pole would result in poly egg-apparatus formation. The
polar distribution of nuclei during embryo sac develop-
ment clearly suggests that embryo sac or nuclei distribu-
tion polarity played an important part in embryo sac differ-
entiation and development. What is not clear is how polar-
ity in embryo sacs developed and how the direction of
mitotic division of nuclei in embryo sacs controlled. More
work is obviously needed to further clarify these points.
3.1 The relationship between abnormal MT organiza-
tion and behavior of embryo sac nuclei
Apart from the normal MT array often observed in the
polygonum type embryo sacs of normal rice, some abnor-
mal MT organizational patterns were also observed in the
variant polygonum type embryo sacs of APⅣ. The re-
sults showed that the abnormal behavior of nuclei was
often accompanied by abnormal pattern of MT distribu-
tion and organization. For example, at the early stage of
development of the four-nucleate embryo sac in the “5-2-
1” type, one of the nuclei near the chalazal pole separated
from its sister nucleus which then moved towards the mi-
cropylar end. Between the two sister nuclei long MT
bundles developed (Figs.7, 7a) and there are now plenty
of morphological evidence to show that the development
of long MT array are closely associated with the move-
ment of nuclei in cells (Xu et al., 1997; Zee and Ye, 2000).
Huang and Sheridan (1996) also observed similar MT or-
ganizational changes in the embryo sac of a maize indeter-
minate gametophytel mutant and they further showed that
the organization and distribution of MT were affected in a
very complex way in the mutant embryo sac.
3.2 The abnormal behavior of nuclei, changes in MT
organization and their controlling genes
The results in this study showed that there is a clear
relationship between abnormal nuclear behavior and
changes in MT organization in embryo sacs. These phe-
nomena are no doubt genetically controlled. Since the
embryo sacs in rice florets of a mutant, such as APⅣ, do
not produce nuclear and MT abnormality in all embryo
sacs. Only about 50% becomes abnormal. Thus it is un-
likely that a single or a set of genes is responsible for this
phenomenon. We postulate that it is likely that a transpos-
able element, active during embryo sac development, may
be involved. When the transposable element got inserted
into the genes responsible for embryo sac and/or microtu-
bule formation then the affected embryo sac will become a
mutant embryo sac. Moreover, because the nuclei and cells
in the embryo sac are all haploids, hence the mutant gene
(s) and its abnormal expression or behavior could easily
be recognized and studied. These visual markers could be
of great value to us in the analysis of the behavior of gene
and gene expression associated with embryo sac develop-
ment and they should also provide us with useful informa-
tion to show whether transposable element involvement
in mutant or normal embryo sac formation is indeed
possible.
Acknowledgements: We are grateful to Ms. YU Shu-
Hong and GUO Hai-Bin for some of the sectioning work.
The authors would like also to thank Professor TAN Zhi-
Yuan for assistance in the preparation of the manuscript.
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(Managing editor: WANG Wei)