Plant Cell Physiol. 44(11): 1192–1201 (2003)
JSPP © 2003
Behavior of Vacuoles during Microspore and Pollen Development in
Arabidopsis thaliana
Yoko Yamamoto 1, Mikio Nishimura 2, Ikuko Hara-Nishimura 3 and Tetsuko Noguchi 1, 4
1
Department of Biological Science, Nara Women’s University, Nara, 630-8506 Japan
Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585 Japan
3
Department of Biological Science, Graduate School of Science, Kyoto, University, Kitashirakawa, Sakyo-ku, Kyoto, 606-8502 Japan
2
;
Using the cryo-fixation/freeze-substitution method, we
studied the ultrastructural changes and behavior of vacuoles and related organelles (rER and Golgi bodies) during
microspore and pollen development, and pollen maturation
of Arabidopsis thaliana. In young microspores forming
exine (pollen outer cell wall), vacuoles looked like those of
somatic cells. In microspores during the formation of intine
(inner cell wall), a large vacuole appeared which was made
by fusion of pre-existing vacuoles and probably absorption
of solutions. In the young pollen grain after the first mitosis,
a large vacuole was divided into small vacuoles. The manner of division was not by binary fission and centripetally,
but by the invagination of tonoplasts from one side to the
opposite side of a vacuole. After the second mitosis, somatic
type vacuoles disappeared. In mature pollen grains just
before germination, membrane-bound structures containing fine fibrillar substances (MBFs) appeared. The MBFs
were considered to be storage vacuoles. In pollen grains
from flowers in bloom, MBFs changed to lysosomal structures with acid phosphatases (lytic vacuole). They gradually increased in number and volume, and decomposed the
cytoplasm. The autolysis of pollen grains is the first finding
in this study, which may contribute to the loss of ability of
pollen germination after anthesis.
Keywords: Arabidopsis thaliana — Autolysis — Golgi body
— Pollen — Vacuole.
Abbreviation: MBF, membrane-bound structure with fibrillar
substances; rER, rough endoplasmic reticulum.
Introduction
Pollen development takes place within the anther. Pollen
mother cells (meiocytes) produce a tetrad of haploid microspores after meiosis. The microspores become free from meiocyte cell wall after dissolution of the cell wall. During the
tetrad stage and microspore formation, the exines (outer pollen
walls) and intines (inner pollen walls) are synthesized around
the cells. Following the appearance of a large vacuole and the
migration of the nucleus toward the cell periphery, the asym4
metric division (microspore mitosis) occurs, producing a vegetative cell containing a generative cell. Then pollen grains
mature to accumulate substances which will be used for pollen
germination and early pollen tube elongation (for details, see
reviews, Bedinger 1992, McCormick 1993, Hesse 1995). These
drastic phenomena occurring during pollen development have
attracted the interest of botanists and ultrastructural studies on
this process have been carried out in various plant species. The
majority of these studies was carried out with conventional
chemical fixation for electron microscopy. In contrast, it was
shown that cryo-fixation/freeze substitution technique was
superior to conventional chemical fixation in its ability to preserve fine structures in pollen grains of Tradescantia (Noguchi
and Ueda 1990) and Brassica (Iwano et al. 1999), and pollen
tubes of Tradescantia (Noguchi 1990). This technique also has
progressed the research of the cytoskeleton system in pollen
tubes of Nicotiana (Lancelle et al. 1987) and Pyrus (Tiwari and
Polito 1988).
Arabidopsis thaliana whose complete DNA sequence is
now known, is used as an experimental material in various
fields of plant biology. Several ultrastructural studies of microspore and pollen development have been reported: exine formation (Paxson-Sowders et al. 1997); final maturation and rehydration (Van Aelst et al. 1993) and the development stages
(Owen and Makaroff 1995, Kuang and Musgrave 1996, Zhang
et al. 2002). All of these were carried out using chemical fixation methods. Therefore, we re-examined the ultrastructures of
pollen grains during developmental stages by cryo-fixation/
freeze substitution technique and have got many new findings.
In this paper, we focus our attention on vacuoles and related
organelles (the endoplasmic reticulum and Golgi bodies), and
report their behavior and ultrastructural changes during pollen
development and maturation.
Results
In the young microspores during exine formation, a
nucleus was located at the cell center, and plastids with small
amount of starch grains and thylakoids that did not stack to
grana occupied more than half of the cytoplasm (Fig. 1a, b).
Vacuoles were distributed throughout the cytoplasm and some
of them were dumbbell shaped. The electron density of con-
Corresponding author: E-mail, [email protected]; Fax, +81-742-20-3416.
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Vacuoles during pollen development in Arabidopsis
Fig. 1
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Vacuoles during pollen development in Arabidopsis
Fig. 2 Young pollen grains after generative cell formation. (a) Intine is still developing and vacuoles in various sizes are in clusters. Lipid bodies surround the generative cell. (b) The invaginations of tonoplast occur at several points and developed from one side of a vacuole. The exocytosis of small vacuole is clear (large arrow). (c) Lipid bodies surrounding the generative cell are stained with p-phenylene diamine. Chemical
fixation. (d) Dividing vacuoles, small vacuoles and lipids become more scattered than those in Fig. 2a. The plasma membrane is heavily convoluted (large arrow). EX, exine; G, Golgi body; GC, generative cell; GN, generative nucleus; M, mitochondrion; V, vacuole; VN, vegetative
nucleus; small arrow, invagination of tonoplast; asterisk, lipid body. Bar = 1 mm (a, c, d), 0.5 mm (b).
Fig. 1 Microspores during exine formation (a, b) and intine formation (c, d). (a) Vacuoles are distributed throughout the cytoplasm. (b) The electron density of vacuole is similar to that of the cytoplasm. Plastids contain starch grains and thylakoids that do not stack to grana. (c) A large vacuole locates in the cell periphery. Plastids markedly decreased in number and size. (d) The electron density of vacuoles is less than that of the
cytoplasm. Golgi bodies have increased in number and their cisternae. Golgi vesicles are not seen around the vacuole but near the plasma membrane. EX, exine; G, Golgi body; IN, intine; M, mitochondrion; N, nucleus; P, plastid; V, vacuole; arrow, dumbbell shaped vacuole; arrowhead,
Golgi vesicle; asterisk, lipid body. Bar = 1 mm (a–c), 0.5 mm (d).
Vacuoles during pollen development in Arabidopsis
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Fig. 3 Pollen grains after sperm cell formation. (a) Sperm cells are connected with each other. Numerous electron-dense structures are scattered
in the cytoplasm. The rER cisterna is dilated. (b1, b2) Two serial sections. The electron-dense structure connects with rER (arrow). G, Golgi body;
M, mitochondrion; P, plastid; SC, sperm cell; SN, sperm nucleus; VN, vegetative nucleus; asterisk, lipid body; star, electron-dense structure. Bar =
0.5 mm.
tents in vacuoles was similar to that of the cytoplasm. Golgi
bodies seemed to not actively produce vesicles. The rough
endoplasmic reticulum (rER) did not develop.
In microspores during the intine-formation, where nucleus
was located at the cell periphery and plastids markedly
decreased in number and size, a large vacuole appeared in addition to small vacuoles (Fig. 1c). The electron density of the
contents in the vacuoles was much less than those of cytoplasm (Fig. 1c, d). The rER and Golgi bodies were distributed
throughout the cytoplasm. Golgi bodies increased in number
and their cisternae, and Golgi vesicles seemed to migrate not to
the vacuole but to the cell surface (Fig. 1d).
After generative cell formation, a large vacuole started to
split into small vacuoles (Fig. 2a). The partition did not occur
by binary fission and centripetally, but by the invagination of
tonoplasts that occurred at several points and developed from
one side to the opposite side of a vacuole (Fig. 2a, b). Electrontransparent droplets without a clear membrane, which began to
appear before the first mitosis, increased in number and surrounded only the generative cells (Fig. 2a). As these droplets
were stained with p-phenylene diamine (Boshier et al. 1984)
(Fig. 2c), they were identified not as vacuoles but as lipid bodies. Small partite vacuoles accumulated globular materials,
<160 nm in diameter, with higher electron densities than those
of other inclusions. Sometimes, fusion of these small vacuoles
with the plasma membrane was observed (Fig. 2b, large arrow).
Golgi bodies were mainly located at the cell periphery, especially near the caving plasma membrane that separated the cell
from the intine (Fig. 2a). In the following stage (Fig. 2d), dividing vacuoles and small vacuoles dispersed throughout the cytoplasm, and the plasma membrane became intensely convoluted. Between the convoluted plasma membrane and the intine
there were many membrane-bound structures (profile in section). They seemed to be secreted globular materials and the
cross-sections of the convoluted plasma membrane.
The pollen of Arabidopsis is of the tri-cellular type, which
means the generative cell divides again to form two sperm cells
before pollen germination. After the second mitotic division,
vacuoles with the similar appearance to those in the previous
stage were no longer observed. Numerous electron-dense structures about 250 nm wide appeared (Fig. 3a). As the connection
between electron-dense structures and rER was observed (Fig.
3b), they were considered to be produced by ER cisternae. The
rER became more conspicuous, and its cisternae were dilated
and attached with many ribosomes on their surface.
In mature pollen grains in dehiscing anthers of flowers
before anthesis, whose stamens and pistil were the same height,
the electron-dense structures in the previous stage had disappeared (Fig. 4). Many membrane-bound structures with fibrillar
substances (MBFs) appeared. The MBFs occupied the central
part, more than half of the cytoplasm in many central sections
of the pollen grains (Fig. 4a). Highly dilated rER cisternae sur-
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Vacuoles during pollen development in Arabidopsis
Fig. 4 Mature pollen grains collected just before anthesis and rapidly frozen in liquid propane (a, c) and by a high pressure-freezing apparatus
(b). (a) Central section. (b, c) Many membrane-bound structures with fibrillar substances (MBFs) are observed. Highly dilated rER cisternae surround lipid bodies with thinner outlines than a unit-membrane. Golgi vesicles with 120–150 nm diameter are accumulated cell periphery (up-left
in c). G, Golgi body; M, mitochondrion; P, plastid; S, sperm cell; arrowhead, Golgi vesicle; asterisk, lipid body; star, MBF. Bar = 1 mm (a),
0.5 mm (b, c).
rounded spherical materials with thinner outlines than a unitmembrane (Fig. 4b, c). As these spherical materials reacted
with p-phenylene diamine (data not shown), they were identified not as vacuoles but lipid bodies. Golgi bodies increased in
number and seemed to actively produce vesicles, because many
Golgi vesicles 120–150 nm in diameter were observed throughout the cytoplasm, especially near the plasma membrane. These
vesicles did not fuse with the MBFs and were considered to be
Vacuoles during pollen development in Arabidopsis
1197
Discussion
Fig. 5 Neutral red treatment. (a) A mature pollen grain from a flower
just before anthesis. It corresponds to the pollen in Fig. 4a. (b) A geminated pollen. In the pollen grain, the base cytoplasm is more stained
than the proximal one. Bar = 10 mm.
P-particles (polysaccharide particles) appeared in mature pollen
grains and pollen tubes of various plants (Weber 1988). When
pollen grains were treated with 0.07% neutral red, a dye that is
taken up selectively by vacuoles (Mahlberg 1972, Ehara et al.
1996), they became red (Fig. 5a). The redness was not homogeneous within the cells, and the neutral-red-positive area coincided with the location of MBFs (compare Fig. 5a with Fig.
4a). Just after pollen germination, the cytoplasm at the bases of
the pollen grains was stained more readily than the proximal
cytoplasm near the pollen tube (Fig. 5b); this also coincided
with the distribution of MBFs in the germinated pollen but not
pollen tubes (data not shown).
In the pollen grains harvested from flowers in bloom
whose stamen became higher than the pistil, the cytoplasm
showed various profiles (Fig. 6). Some were similar to those of
mature pollen grains from flowers before anthesis (data not
shown). The first structural change occurred within the MBFs.
They began to contain osmiophilic globoids <90 nm in diameter and decrease in volume (Fig. 6a). In some pollen grains,
both MBFs with osmiophilic globoids and electron-transparent
membrane-bound structures with osmiophilic globoids were
observed. In other pollen grains, only the latter were observed,
and their globoids increased in diameter, to <150 nm (Fig. 6b).
The electron-transparent membrane-bound structures fused to
each other (Fig. 6b large arrow), became larger, and contained
several osmiophilic globoids and tubular and vesicular structures (Fig. 6c, d). Their image liked as lysosomes observed in
animal cells, therefore we performed a cytochemical detection
test for acid phosphatase (a marker enzyme of lysosomes) to
determine the chemical nature of the structure. Because products of reactions involving acid phosphatases were detected in
the structures (Fig. 6e), we considered that the structures were
lysosomes. With the appearance of lysosomal structures, the
lipid droplets surrounded by rER disappeared (Fig. 6c, d). The
ratio of the total lysosome volume to the cell volume in a pollen grain varied. In pollen grains in which more than half of the
cytoplasm had decomposed, MBFs and the lipid droplets surrounded by rER had completely disappeared, and the rER,
Golgi body cisternae and mitochondria were intensively swollen (Fig. 6d).
The transformation of vacuoles during the development of
microspores and pollen, as observed in the present investigation, is summarized in Fig. 7.
The appearance of a large vacuole before generative cell
formation (first mitosis) and its degradation into many small
vacuoles are generally observed in pollen of various plant species. Owen and Makaroff (1995) reported that the enlargement
of vacuoles depends on the fusion of pre-existing small vacuoles in A. thaliana. One of our results supports it, because
dumbbell-shaped vacuoles observed before the appearance of a
large vacuole (Fig. 1a) are considered as the fusion process
rather than the division process of vacuole. In addition, our
result that the electron density of contents in the large vacuole
decreased than those of pre-existing small vacuoles (compare
Fig. 1c, d to Fig. 1a, b) indicates the absorption of some solutions into vacuoles. The microspores also increased in volume
(about 1.2 times) during the period in spite of inactive appearance of rER and Golgi bodies. Probably, the enlargement of
vacuole occurs by both the fusion of pre-existing small vacuoles and the absorption of some solutions.
Until now, the information about the manner of vacuole
degradation was limited not only in A. thaliana but also in
other plant species. This was probably because the tonoplasts
of vacuoles were not smooth but wavy in pollen prepared by
chemical fixation, which did not give enough structural resolution for determining the manner of division. Our data clearly
showed that the vacuoles divide not by binary fission and not
centripetally like plastids and mitochondria (Kuroiwa et al.
1998) but by the invagination of tonoplast that occurred at several points and developed from one side to the opposite side of
a vacuole (Fig. 2a, b). We observed the fusion of divided vacuoles with the plasma membrane (Fig. 2b). Vacuoles (both lytic
and storage) usually do not exocytose in somatic cells, but exocytosis seems to occur in pollen, because the vacuoles disappeared completely from the pollen grains at the next stage. If
the large vacuole (the origin of small vacuoles) developed by
the absorption of solutions, then the partite small vacuoles
would be expected to dilute the lytic abilities and never disturb
the surface environment of the cells, even if they secreted their
inclusion by exocytosis.
The behavior of vacuoles during and just after sperm cell
formation in pollen grains of A. thaliana, varied in different
reports. Regan and Moffatt (1990) reported that, according to
the neutral red staining, vacuoles disappeared during the first or
second mitotic divisions. In contrast, the existence of vacuoles
during these periods was reported in studies using electron
microscopy (Owen and Makaroff 1995, Kuang and Musgrave
1996). In our study, the typical vacuoles that are similar in
appearance to those observed in young pollen grains (Fig. 1, 2)
were not observed in the pollen grains just after sperm formation (Fig. 3). During these periods, electron-dense structures
were actively produced from the rER cisterna. Although, we
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Vacuoles during pollen development in Arabidopsis
Fig. 6 Mature pollen grains from flowers after anthesis. (a) MBFs contain osmiophilic globoids. (b) Electron-transparent membrane-bound
structures with osmiophilic globoids but without fibrillar substances as like MBFs have appeared. Highly dilated rER surrounding lipid bodies is
still observed. (c) A lysosomal structure containing osmiophilic globoids and tubular structures is observed in addition to the electron-transparent
ones with the globoids. (d) The cytoplasm is occupied by lysosomal structures. Highly dilated rER surrounding lipid bodies have disappeared. The
rER, Golgi body cisternae and mitochondria are intensely swollen. (e) Reaction products of acid phosphatases are observed in the lysosomal
structure. EX, exine; G, Golgi body; M, mitochondrion; L, lysosomal structure; arrowhead, Golgi vesicle; large arrow, dumbbell-shaped membrane-bound structure; small arrow, osmiophilic globoid; asterisk, lipid body; double arrowheads, reaction products of acid phosphatases; star,
MBF. Bar = 0.5 mm.
Vacuoles during pollen development in Arabidopsis
1199
Fig. 7 Schematic representation of the transformation of vacuoles during various stages of pollen development. N, nucleus; G, generative cell; S,
sperm cell.
have not identified whether it is a kind of vacuole, it is clear
that the continuity between the electron-dense structure and the
vacuoles in young pollen grains does not exist, because the
former is newly produced from the rER cisterna in these periods.
In mature pollen grains of A. thaliana prepared by chemical fixation, many vacuoles with an average diameter of
300 nm have been reported (Van Aelst et al. 1993, Kuang and
Musgrave 1996). However, their identification as vacuoles
probably depended on their appearance: circular membranebound structures in electron microscopic sections. In our study,
MBFs appeared only in the mature pollen grains from flowers
just before anthesis (Fig. 4). The profiles of MBFs are very different from those of vacuoles observed by chemical fixation;
the former contained fine fibrils but the inclusions in the latter
are transparent. Therefore, we identified the MBFs as a kind of
vacuoles for the following reasons. Regan and Moffatt (1990)
reported that neutral red positively stained mature pollen grains
of A. thaliana, and we ourselves confirmed the stainability of
mature pollen grains by neutral red (Fig. 5). In both experiments, the neutral-red-positive area coincided with the location
of MBFs. Further, the redness was not homogeneous within the
cells. Therefore, we consider that the neutral red had entered
sub-cellular compartments. However, we could not find vacuolelike structures other than MBFs in mature pollen grains. The
specific appearance of MBFs in only mature pollen grains suggests that they are storage vacuoles. It has already been
reported that neutral red stains not only vegetative vacuoles but
also protein-storage vacuoles isolated from maturing and germinating pumpkin seeds (Hara-Nishimura et al. 1987). In addition, we already have supporting data: expression of the gene
for the b-vacuolar processing enzyme (b-VPE) that is involved
in the maturation of seed proteins in protein storage vacuoles
(Kinoshita et al. 1999) was detected in mature pollen grains
harvested from flowers just before anthesis of Arabidopsis
plants transformed with pbVPE::gus genes (unpublished data).
The origin and detailed behavior of the MBFs will be reported
elsewhere.
It is thought that mature pollen grains usually become desiccate and enter a phase of stasis. After pollination, the pollen
grain undergoes a period of rehydration and its metabolic
machinery is reactivated (Heslop-Harrison 1987, Van Aelst et
al. 1993). In contrast, we observed the autolysis of mature pollen by lysosomal structures with acid phosphatases. Our results
suggest that the appearance of osmiophilic globoids in MBFs is
the start of pollen autolysis. However, similar electron-dense
globoids in mature pollen grains in Chlorophytum elatum were
considered to be a calcium pool that might be utilized in the
course of pollen germination (Butowt et al. 1997). It is not
clear whether the osmiophilic globoids in A. thaliana include
calcium or not. We prefer to consider that they come from the
inclusions of MBFs, because the osmiophilic globoids
increased in size with the decrease of the fibrillar substances
and the size of MBFs. If the MBFs are storage vacuoles containing materials for pollen germination, as we assumed, then it
would be reasonable to hypothesize that the MBFs are decomposed when the mature pollen grains are not able to germinate.
Taken together, our results suggest that the process of mature
pollen autolysis is as follows. MBFs decompose their contents
and transform into the electron-transparent membrane-bound
structures with osmiophilic globoids. Then they fuse with each
other, increase in volume, and acquire a lysosomal nature
through the addition of acid phosphatases. As the lysosomal
structures increase, the highly dilated rER surrounding lipid
bodies (one of the prominent structures in mature pollen
grains) disappears, and then the rER, Golgi bodies, and mitochondria are decomposed after swelling. The autolysis of
mature pollen grain is the first finding not only in A. thaliana
but also other plant species. Therefore, we examined many pollen grains by electron microscopy samples made at different
times and concluded that they are not artifacts but a real phenomenon in A. thaliana. The following facts may support our
conclusion. (1) In A. thaliana, the height of stamen is same as
that of pistil in the flowers just before anthesis; in contrast, the
pistil became higher than pistils after anthesis (Fig. 7). (2) The
germination ratio of pollen in vitro was highest in pollen grains
from flowers just before anthesis and declined after anthesis of
the flowers (unpublished data). (3) The cytoplasm of pollen just
after germination looks more like those of pollen grains har-
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Vacuoles during pollen development in Arabidopsis
vested from flowers just before anthesis than that of pollen
grains from flowers in bloom. Pickert (1988) reported that
pollen of A. thaliana has a longevity of less than 3 d, which
is much shorter than that of Nicotiana tabacum pollen. N.
tabacum pollen keeps the germination ability more than 3 d in
room temperature (Noguchi and Morré 1991) and more than 1
year in the refrigerator. In A. thaliana, the pollen grains are
gradually decomposed by lysosomal structures after anthesis
and may lose their ability to germinate in a short time. The
autolysis of mature pollen grains may contribute to the selfpollination in A. thaliana.
Material and Methods
Plant materials and growth conditions
Seeds of A. thaliana L. (Heynh) ecotype Wassilewskija (WS)
were sown on vermiculite mixed with pumice grains (1 : 1 by volume)
and grown at 22°C under continuous fluorescent light at a photon flux
density of 45–55 mmol m–2 s–1.
Cryo-fixation and freeze-substitution
Microspores and immature pollen grains in non-dehiscing
anthers were extruded onto aluminum foil 4 mm square and rapidly
frozen in liquid propane at –190°C. Most of the mature pollen grains
in dehiscing anthers were attached to formvar films mounted on wire
loops 8 mm in diameter and then frozen. Some pollen grains were
frozen by a high-pressure freezing apparatus (BAL-TEC HPM010).
The frozen cells were transferred to cooled acetone at –85°C containing 4% osmium tetroxide and 0.2% uranyl acetate. After substitution
for 48 h at –85°C, the cells were washed with acetone and embedded
in Spurr resin. Sections were stained with lead citrate and examined
under an electron microscope (Hitachi H-7000).
Cytochemical detection of acid phosphatase activity
Pollen grains were fixed with 3% glutaradehyde buffered with
0.05 M Na-cacodylic acid (pH 7.2) for 10 min at 4°C. The cells were
washed for 30 min with distilled water and then incubated for 2 h at
37°C in reaction medium containing 0.15 g of Na2-b-glycerophosphate
as substrate, 0.012 g of lead nitrate, 0.11 g of potassium sodium tartrate, 0.004 g of manganese chloride and 10 ml of 0.05 M acetate
buffer (pH 5.2), with a transfer to fresh medium every 30 min. The
cells were then washed successively with distilled water, 1.5% acetic
acid and distilled water again, and were immersed in 1% ammonium
sulfide solution for 2 min. The cells were washed with distilled water,
and then post-fixed with a 1% osmium tetroxide solution buffered with
0.05 M Na-cacodylic acid (pH 7.2) for 2 h, washed with water, dehydrated with acetone, and embedded in Spurr resin (for details, see
Noguchi 1976).
Lipid staining
Pollen grains were pre-fixed with 4% glutaraldehyde and postfixed in 1% osmium tetroxide. During dehydration with serial dilutions
of ethanol, 0.1% p-phenylene diamine was added to 70% ethanol to
stain the lipids (Boshier et al. 1984).
Neutral red treatment
Pollen grains were soaked in 0.075% neutral red for 60 min for
differentiation of vacuoles.
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(Received May 6, 2003; Accepted August 29, 2003)