Journal of Experimental Botany, Vol. 50, No. 334, pp. 581–594, May 1999
Unique actin and microtubule arrays co-ordinate the
differentiation of microspores to mature pollen in
Nicotiana tabacum
Laura Zonia1,2,3, Jaroslav Tupý2 and Christopher J. Staiger1
1 Purdue University, Department of Biological Sciences, 1392 Lilly Hall, W. Lafayette, IN 47907–1392, USA
2 Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Na Pernikářce 15, Prague 6,
16000, Czech Republic
Received 8 September 1998; Accepted 3 December 1998
Abstract
The complex cellular events that occur during development of the male gametophyte of higher plants suggest
a role for the cytoskeleton. This investigation has
revealed that unique microtubule arrays mediate
events that occur during microspore development;
both actin and microtubule arrays have important roles
during the asymmetrical microspore mitosis and
unique actin arrays mediate events that occur during
early pollen development. Migration of the nucleus to
the generative pole during cellular polarization of the
microspore is mediated by a microtubule cage that
encloses the nucleus. Nuclear position at the generative pole is maintained by an actin net that tethers it
to the pole prior to the asymmetrical mitosis. During
entry into mitosis, the microtubule cage becomes
modified and transforms into the asymmetrical mitotic
spindle. Actin is localized within the region of the
mitotic spindle and in the phragmoplast. Following
mitosis, actin networks enclose first the generative
cell and then the vegetative nucleus. These actin networks function during migration of the generative cell
and vegetative nucleus toward the centre of the pollen
grain. Mature pollen contains a dense cortical
actin meshwork and a disc-shaped microtubule array
enclosing the generative cell. The functional importance of the unique actin and microtubule arrays is
verified by their targeted disruption with specific cytoskeletal inhibitors, which disrupt normal development
and cellular morphology. In summary, these data provide evidence that the co-ordinated reorganization of
unique actin and microtubule arrays is an essential
determinant of microspore and pollen development.
Key words: Microspore development, pollen development,
actin cytoskeleton, microtubule cytoskeleton, cytoskeletal
inhibitors.
Introduction
Formation of the male gametophyte of higher plants from
the microspore stage to mature pollen proceeds through
a complex developmental pathway that has been the
subject of several excellent reviews (McCormick, 1993;
Bedinger et al., 1994; Touraev et al., 1997). The present
summary focuses specifically on development of Nicotiana
tabacum pollen, although the general features of development are similar in other species. Meiosis occurs within
the microspore mother cells and produces four haploid
cells that are enclosed within a callose sac, called a tetrad.
Enzymatic digestion of this sac releases the free microspores into the anther locule. The free microspores
undergo a period of rapid growth, cellular polarization,
and the cell shape changes from spherical to elliptical. In
early microspores the nucleus is centrally located, but
then undergoes a first migration to the cell perimeter. By
the late microspore stage the nucleus has migrated along
the cell perimeter to the generative pole in preparation
for the asymmetrical mitosis. This asymmetrical mitosis
produces the pollen grain that contains two cells with
very different sizes, nuclear morphologies and developmental fates. The generative cell is smaller, its nucleus
contains very condensed chromatin, and it will undergo
a mitotic division to produce the two sperm cells. The
timing of the generative cell mitosis is species-specific: in
many species, including tobacco, it occurs within the
pollen tube after pollen germination; in species with
3 To whom correspondence should be addressed at: Fax: +420 2 3119412. E-mail: [email protected]
© Oxford University Press 1999
582 Zonia et al.
tricellular pollen it occurs before the final stage of maturation. The vegetative cell is larger, its nucleus contains
very decondensed chromatin and is transcriptionally
active. Upon germination the vegetative cell produces the
pollen tube that functions to penetrate the style and
deliver the non-motile sperm cells to the ovule. Following
the asymmetrical microspore mitosis, the vegetative cell
undergoes a period of growth and the cell shape changes
from elliptical to spherical. Concurrently, the generative
cell and vegetative nucleus migrate toward the centre of
the pollen grain, and the generative cell becomes completely enclosed within the vegetative cell cytoplasm.
During the final stage of tobacco pollen maturation, the
generative cell becomes strongly disc-shaped and the
pollen grain prepares for anthesis by becoming metabolically quiescent and by a dehydration process that
removes up to 90% of the total water content.
The cytoskeleton has a particularly important role in
the plant cell cycle. Cortical and cytoplasmic microtubule
and actin arrays are present during interphase ( Traas et
al., 1987; Hepler et al., 1993; Hush et al., 1994; Vesk et
al., 1996). As the cells enter G , the cortical microtubule
2
array is reorganized into the preprophase band (PPB)
that marks the position where the cell plate will intersect
the parental plasma membrane during cytokinesis ( Wick
and Duniec, 1983; Palevitz, 1986; Mineyuki et al., 1988;
Wick, 1991). Actin remains in the cortex during this stage
but is also localized in the PPB and appears to regulate
nuclear position with respect to the PPB ( Kakimoto and
Shibaoka, 1987; Lloyd and Traas, 1988; McCurdy and
Gunning, 1990; Mineyuki and Palevitz, 1990) as well as
nuclear migration to the division plane (Miyake et al.,
1997; Kennard and Cleary, 1997). Concurrently with PPB
formation, a radial microtubule array forms around the
nucleus that eventually transforms into the mitotic spindle
(Mineyuki et al., 1991). Late anaphase is marked by the
appearance of the phragmoplast, the specialized structure
that directs synthesis of the wall dividing the two daughter
cells (for review, see Staehelin and Hepler, 1996). The
phragmoplast is composed of both microtubules and
F-actin (Gunning, 1982; Palevitz, 1987; Kakimoto and
Shibaoka, 1987; Zhang et al., 1993).
Cytoskeletal elements in the later stages of pollen
development encompassing germination and pollen tube
growth have been extensively described (for reviews, see
Cai et al., 1997; Taylor and Hepler, 1997). However, few
studies have investigated these structures in the earlier
stages of microspore development. In the monocots Lilium
and Tradescantia, a cortical microtubule array is present
at the tetrad and early microspore stages (Dickinson and
Sheldon, 1984; Sheldon and Dickinson, 1986; Tiwari,
1988). Developing Tradescantia microspores undergo two
nuclear displacements, first from a central to a poleward
position and subsequently along the cell perimeter back
toward the middle of the cell. The first displacement
occurs concurrently with the formation of a large vacuole,
while the second is dependent on the accumulation of
cytoplasmic microtubules ( Terasaka and Niitsu, 1990). A
unique microtubule array exists in orchid microspores,
which undergo the microspore mitosis within persistent
tetrads. This generative pole microtubule system
assembles at the pole, predicting the site of the asymmetrical mitosis (Brown and Lemmon, 1991, 1992a, b). In
the dicot Brassica, no microtubule networks were
observed during microspore nuclear migration (Hause et
al., 1991, 1993). In several different species, the mitotic
apparatus of the asymmetrical microspore mitosis has the
unusual feature of being cone-shaped at one pole and
barrel-shaped at the other pole (Brumfeld, 1941; HeslopHarrison, 1968; Burgess, 1970; Brown and Lemmon,
1994; Terasaka and Niitsu, 1995). Two groups have
performed experiments to test the effect of a microtubule
inhibitor on pollen development. Both groups performed
long-term cultures of late microspores in the presence of
colchicine. Colchicine at 1.25 mM blocked the second
nuclear migration in Tradescantia microspores, but had
no effect on the first nuclear migration ( Terasaka and
Niitsu, 1990). In Nicotiana tabacum microspores,
1.25 mM colchicine effectively blocked the asymmetrical
microspore mitosis, while low levels caused 1–3% of the
cells to undergo symmetrical mitosis ( Eady et al., 1995).
However, neither of these groups demonstrated a disruption of the microtubule cytoskeleton in the presence of
colchicine. Very limited data exist regarding the actin
cytoskeleton during the development of microspores to
mature pollen. Cytoplasmic arrays have been observed in
the tetrads of Lilium, Gasteria and Brassica (Sheldon and
Hawes, 1988; Van Lammeren et al., 1989; Gervais et al.,
1994). Actin has also been observed in the phragmoplast
of mitotic microspores of Brassica (Hause et al., 1991;
Gervais et al., 1994). Additionally, a dense cortical meshwork of actin bundles exists in mature Gasteria and
Brassica pollen prior to anthesis ( Van Lammeren et al.,
1989; Hause et al., 1991; Gervais et al., 1994).
The morphological changes that occur during development of microspores to mature pollen are precisely controlled in a temporal and spatial manner and suggest a
role for the cytoskeleton. The authors wanted to define
the mechanisms that control microspore and pollen development in the model system Nicotiana tabacum. In this
report, it is shown that unique microtubule arrays function during microspore development and the asymmetrical mitosis, whereas unique actin arrays function during
the asymmetrical mitosis and during early pollen differentiation. Furthermore, it is shown that targeted disruption
of these unique arrays perturbs developmental progession
at specific points, thus providing evidence that the
observed arrays are essential for specific developmental
events and function synergistically in the production of
the mature pollen grain.
Cytoskeleton in tobacco microspores 583
Materials and methods
Plant material and developmental staging
Nicotiana tabacum cv. Samsun plants were grown in a
glasshouse. The developmental stage of microspores and pollen
was determined by examining nuclear position and cell morphology and was assessed prior to the start of a series of experiments by staining with 5 mg ml−1 4,6-diamidino-2-phenylindole
(DAPI ) in 30% ethanol. It should be noted that the population
of microspores and pollen within a single flower is not
synchronous, and several closely related stages can co-exist even
within a single anther. This caveat is especially important when
quantifying the results of the in vitro culture studies described
below.
Isolation of microspores and pollen
One flower bud for each developmental stage was used for
studies analysing the actin cytoskeleton. For studies analysing
the microtubule cytoskeleton, three flower buds at equivalent
developmental stages were used due to the losses encountered
during sample processing. Anthers were removed to 16×100 mm
glass tubes containing 300 ml of M1 minimal medium (0.5 M
sucrose, 1% [w/v] lactalbumin hydrolysate, 10 mM KNO ,
3
1 mM MgSO .7H O, 0.16 mM H BO , 3 mM glutamine, 1 mM
4
2
3 3
uridine, 0.5 mM cytidine, and 1 mM phosphate buffer, pH 7)
(Tupý et al., 1991). Microspores or pollen were gently pressed
out of the anthers with a glass rod and then transferred to a
1.5 ml centrifuge tube. The pressed anthers were washed twice
with 300 ml M1 minimal medium and the rinsate was then
combined with the microspore suspension. Cells were sedimented
by centrifugation at 150 g for 1 min, supernatant was removed,
and the cells were washed twice with 800 ml M1 minimal
medium. For direct cytoskeletal analysis, cells were finally
resuspended in 300 ml M1 minimal medium and processed as
described below.
Pharmacological studies on developmental progression during
in vitro culture
For pharmacological studies requiring in vitro culture, all
isolation procedures were carried out aseptically in a sterile
flow box. Flower buds were sterilized in 70% ethanol for 5 min
and then allowed to air-dry. Anthers were dissected out and all
subsequent steps were identical as described above, except that
the final resuspension was 30 ml M1 minimal medium. Cells
were cultured in Nunc (Roskilde, Denmark) 4-well culture
dishes (1.5 ml well volume) in a dark humid chamber. For
each developmental stage to be tested, equal volumes of the
isolated and resuspended cells were added to 235 ml M1
medium±inhibitor. A range of concentrations was tested for
each inhibitor: 20 nM–10 mM cytochalasin D; 1 pM–1 mM
latrunculin B; 1 mM–30 mM oryzalin. At the highest concentrations, there was complete inhibition of all further development;
at the lowest concentrations development was either slightly
perturbed or there was essentially no effect. It was decided to
run the experiments at the lowest concentrations that still
clearly disrupted development. The inhibitors cytochalasin D,
latrunculin B, and oryzalin were all suspended in dimethylsulfoxide (DMSO) (Sigma, St Louis, MO) and stored at −20 °C.
The final culture concentrations of 1 mM cytochalasin D, 5 nM
latrunculin B, 25 mM oryzalin represent DMSO concentrations
of 0.1% to 0.25% [w/v]. These DMSO concentrations had no
effect on development or on the normal cytoskeletal arrays
(data not shown). Cultures were incubated at 24 °C for
approximately 8 h before labelling cytoskeletal arrays as
described below. Developmental progression of the cells in M1
versus M1+inhibitor was assessed by removing 15 ml of the
culture to a microscope slide containing 15 ml of 10 mg ml−1
DAPI in 30% ethanol, placing a coverslip, and viewing with
fluorescence microscopy as described below. For each experiment, 300–500 cells were counted for each treatment.
The values reported are the means of three separate
experiments±standard error. Development in the control
cultures normally varied by 5–10%. The absolute values for
each test were analysed by paired t-test to determine the
statistical significance of the inhibitor effects on development.
These P-values are given in the Results.
Actin labelling
Microspores and pollen were isolated and/or cultured as
described above and then transferred to Superfrost Plus
microscope slides (Fisher Scientific) that had been coated with
poly-L-lysine (Sigma). Cells were allowed to adhere to the slides
for 10 min before pouring off the medium. A method was
developed for fixation of the actin cytoskeleton in developing
microspores and pollen that is based on previously published
protocols for other plant tissues (Traas et al., 1987; Sonobe
and Shibaoka, 1989; Goodbody and Lloyd, 1990). Cells were
permeabilized and fixed in 300 mM m-maleimidobenzoyl-Nhydroxysuccinimide ester (MBS) (Pierce, Rockford, IL) in
MTSB buffer (50 mM PIPES, 5 mM EGTA, 5 mM MgSO )
4
containing 0.05% [w/v] NP-40 detergent and 1.5 mg ml−1 DAPI
to counterstain nucleic acids. Slides were incubated in a dark
humid chamber for 10 min before pouring off the fixative
solution. Then the actin cytoskeleton was labelled in a fresh
aliquot of the same solution to which had been added a final
concentration of 94 nM rhodamine-phalloidin (Molecular
Probes, Eugene, OR). Slides were incubated in a dark humid
chamber for 45 min. The cells were then rinsed with MTSB
before mounting with a solution containing 1 mg ml−1
1,4-diazabicyclo (2,2,2) octane (DABCO) (Sigma) in 951
glycerol:PBS. Cells were viewed as described below and the
results were documented immediately.
Microtubule labelling
Microspores and pollen were isolated and/or cultured as
described above and then transferred to new 1.5 ml centrifuge
tubes. Cells were sedimented by centrifugation at 150 g for
1 min, supernatant was removed, and cells were resuspended
in 300 ml of a cell wall-digesting solution. This solution was
prepared with modifications according to the protocol of
Goodbody and Lloyd (1994) and contained 1.5% [w/v] cellulase
R-5 ( Yakult, Tokyo, Japan) and 1% [w/v] each of pectolyase
Y-23 (Seishin, Japan), hemicellulase and Driselase (Sigma), and
Novozyme (InterSpex Products, Foster City, CA), in MTSB
supplemented with 0.5 M sucrose. The enzymes were allowed
to dissolve on ice and then insolubles were removed by
centrifugation at 600 g for 5 min. The cleared supernatant was
transferred to new 1.5 ml centrifuge tubes before adding a final
dilution of 15200 protease inhibitor mix (1.6 mg ml−1 benzamidine HCl, 0.1 mg ml−1 phenanthroline, 1 mg ml−1 each aprotinin,
leupeptin, pepstatin A, all Sigma, dissolved in ethanol and
stored at −20 °C ). Cell walls were digested for 45–60 min on
an orbital platform shaker with 30 strokes min−1 at 23 °C. Then
cells were sedimented by centrifugation at 140 g for 40 s, washed
once for 5 min with MTSB+0.5 M sucrose, sedimented, and
resuspended in 500 ml of a fixing solution that contained 4%
[w/v] paraformaldehyde (Sigma), 0.5 M sucrose, 0.1% [w/v]
NP-40, in MTSB. Cells were fixed for 60 min at 23 °C on an
orbital platform shaker with 30 strokes min−1. Cells were then
sedimented as before, resuspended in 500 ml 1% [w/v] Triton
584 Zonia et al.
X-100 in MTSB+0.5 M sucrose, and permeabilized for 10 min.
Cells were then washed with 800 ml volumes of each of the
following solutions: once for 10 min with MTSB+0.5 M
sucrose; once for 10 min with MTSB; and twice for 15 min with
phosphate-buffered saline (PBS ). Finally, cells were resuspended
in 300 ml PBS and transferred to Superfrost Plus microscope
slides coated with poly-L-lysine and allowed to air-dry almost
to completion before addition of a 15200 dilution of a mouse
monoclonal anti-b-tubulin (Amersham) in PBS containing 1%
[w/v] bovine serum albumin (Fraction V, Sigma). The slides
were incubated for 12 h at room temperature in a dark humid
chamber and then washed three times for 10 min each with
PBS before adding a 15200 dilution of FITC-conjugated goatanti-mouse IgG (Sigma) in PBS. The slides were incubated for
3 h in a dark humid chamber at room temperature and then
washed once for 10 min with PBS, once for 15 min with PBS
containing 2.5 mg ml−1 DAPI to counterstain nucleic acids, and
finally once for 5 min with PBS. The slides were mounted as
described above for actin labelling.
Microscopy and image capture
Cells were observed with a Nikon-Microphot SA microscope
fitted with a 100 W epifluorescence light source, a 100×1.4 NA
Plan-Apo objective, and standard filter block sets. Images were
transmitted through a 10× projection lens, recorded onto
Kodak T-Max P3200 film and subsequently transformed to a
digital format using a Nikon Coolscanner. Images were contrast-enhanced using Adobe Photoshop 4.0 (San Jose, CA)
and printed on a Tektronix dye-sublimation printer. (Note:
The microspore and pollen exine is autofluorescent at the
UV-wavelengths used in these studies. Hence, the exine layer
appears as a bright outline around or along the cells.)
Reagents
Rhodamine-phalloidin was from Molecular Probes (Eugene,
OR). MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester)
was from Pierce (Rockford, IL). Anti-b-tubulin (N 357) was
from Amersham. FITC-conjugated goat anti-mouse IgG was
from Sigma. Latrunculin B was from Calbiochem, cytochalasin
D was from Sigma, and oryzalin was a generous gift from
Dow-Elanco, Indianapolis, IN. All other chemicals were reagent
or plant cell culture grade and were from Sigma or Calbiochem.
Results
The actin cytoskeleton undergoes dramatic reorganization
prior to the start of and during the asymmetrical mitosis,
and during early pollen development
Prominent cellular events during development of microspores to mature pollen include changes in nuclear position and cell morphology. The nature of these changes
led us to investigate whether the actin cytoskeleton had
a fundamental role in these events. These results are
shown in Fig. 1. The investigation was started at the
tetrad stage, when the four haploid cells are enclosed
within a callose sac ( Fig. 1B). At this stage the cells have
a large, centrally located nucleus and actin filaments are
organized in an irregular radial array between the nucleus
and the cell perimeter (Fig. 1A). The callose sac is enzymatically digested to release the free microspores, which
undergo a period of rapid growth. The nucleus migrates
from a central position to the cell perimeter, and subsequently toward one of the poles of the elliptical cell
( Fig. 1D). During this stage actin filaments are absent or
extremely sparse ( Fig. 1C ). However, once the nucleus is
located at the generative pole ( Fig. 1F ), an actin net
surrounds the nucleus, apparently tethering it to the pole
with shorter filaments ( Fig. 1E). The asymmetrical
mitosis rapidly ensues. Actin filaments are localized within
the region of the mitotic spindle at metaphase (Fig. 1G,
H ), and with the phragmoplast during telophase (Fig. 1I,
J ). The products of the asymmetrical mitosis are well
differentiated in early binucleate pollen, which contains
the smaller, condensed generative nucleus and the larger,
diffuse vegetative nucleus ( Fig. 1L). At this stage actin
filaments are asymmetrically localized in a ring around
the generative cell that also has shorter projections
extending into the vegetative cell cytoplasm (Fig. 1K ).
During subsequent growth and development the vegetative cell becomes spherical ( Fig. 1N ), the generative cell
and vegetative nucleus migrate toward the centre of the
pollen grain, and the generative cell becomes completely
enclosed within the vegetative cell cytoplasm (Fig. 1P).
Prior to the start of the migration actin filaments are
organized into interconnected rings enclosing both the
generative cell and the vegetative nucleus, and also as
longer filaments projecting into the cytoplasm of the
vegetative cell (Fig. 1M ). After migration is complete the
actin ring around the generative cell disappears, a dense
actin patch forms around the vegetative nucleus, and
there is a concurrent decrease in cytoplasmic filaments
and an increase in cortical filaments ( Fig. 1O). During
the final stage of pollen maturation, the generative cell
becomes disc-shaped (Fig. 1R) and actin filaments form
a dense cortical meshwork in the vegetative cell (Fig. 1Q).
Thus, it was observed that actin arrays are organized into
prominent and unique structures at critical developmental transitions: just prior to the asymmetrical mitosis
( Fig. 1E), during mitosis and cytokinesis (Fig. 1G, I ), in
early binucleate pollen ( Fig. 1K ), and during the
co-ordinated migration of the generative cell and vegetative nucleus toward the centre of the pollen grain
( Fig. 1M ).
Actin cytoskeleton inhibitors induce specific developmental
aberrations
To assess the extent to which actin arrays have an essential
role in developmental progression, the effect on development that results from disruption of actin arrays during
in vitro culture in the presence of the toxins cytochalasin
D and latrunculin B was quantified. Cytochalasins cap
the barbed-ends of filaments and prevent dynamic actin
assembly and disassembly (Cooper, 1987). Cytochalasins
have been shown rapidly to inhibit cytoplasmic streaming
in pollen tubes (Picton and Steer, 1981) by disrupting the
Cytoskeleton in tobacco microspores 585
Fig. 1. Reorganization of the actin cytoskeleton during development of the microspore to mature pollen. All cells were double-labelled with
rhodamine-phalloidin to visualize the actin cytoskeleton (top row: A, C, E, G, I, K, M, O, Q) and DAPI to visualize the nucleus (bottom row: B,
D, F, H, J, L, N, P, R). All cells, magnification ×1000. (A, B) Tetrads with an irregular radial array of actin between each nucleus and the cell
perimeter. (C, D) Early to mid-microspores lack pronounced actin arrays. ( E, F ) Late microspores contain an actin net that tethers the nucleus at
the generative pole in preparation for the asymmetrical mitosis. (G, H ) Actin filaments are localized within the region of the mitotic spindle during
metaphase. (I, J ) Actin filaments are prominently localized in the phragmoplast, the site of formation of the generative cell wall. ( K, L) Early
binucleate pollen contain an actin ring only around the generative cell. (M, N ) Mid-bicellular pollen have interconnected actin rings around both
the generative cell and vegetative nucleus, with longer filaments extending into the vegetative cell cytoplasm. (O, P) Late bicellular pollen have lost
the actin ring around the generative cell, have an extensive actin patch around the vegetative nucleus, decreased cytoplasmic filaments, and increased
cortical filaments. (Q, R) Mature pollen has an extensive meshwork of cortical actin filaments.
586 Zonia et al.
organization of the actin cytoskeleton (Perdue and
Parthasarathy, 1985; Lancelle and Hepler, 1988).
Latrunculins inhibit actin assembly by binding to monomeric actin (Spector et al., 1983; Coué et al., 1987).
Latrunculins have been shown to disrupt the actin cytoskeleton in Fucus and Pelvetia zygotes during polar axis
fixation (Love et al., 1997; Hable and Kropf, 1998).
Recent work has demonstrated that latrunculin B binds
to maize pollen monomeric actin (G-actin) and inhibits
in vitro polymerization (BC Gibbon and CJ Staiger,
unpublished results). Both cytochalasin D (1 mM ) and
latrunculin B (5 nM ) perturbed development at specific
stages while having little or no effect at other stages, as
shown in Fig. 2. During early microspore growth and
nuclear migration, 91% (P=0.129) of cells underwent
normal development in the presence of cytochalasin D,
while 88% (P=0.291) of cells underwent normal development in the presence of latrunculin B. Thus, development
in the presence of the inhibitors was not significantly
different than controls. These results are consistent with
the observation that there are no prominent actin arrays
present at this developmental stage (Fig. 1C ).
Development at the late microspore stage is significantly
disrupted by these actin inhibitors, with only 33% (P=
0.003) or 54% (P=0.0002) of cells undergoing normal
developmental progression in the presence of cytochalasin
D or latrunculin B, respectively. When the position of
the nucleus was assessed, it was found that the inhibitors
did not block movement of the nucleus to the pole but
rather interfered with the maintenance of the nucleus at
the pole, resulting in a large proportion of cells with a
more centrally located nucleus (see following section).
Progression through the asymmetrical mitosis was also
significantly disrupted by the inhibitors, with only 54%
(P=0.003) or 62% (P=0.048) of cells maintaining mitotic
fidelity in the presence of cytochalasin D or latrunculin
B, respectively. Early development of binuclear pollen is
also significantly disrupted by these actin inhibitors. In
the presence of cytochalasin D or latrunculin B, only 54%
(P=0.008) or 61% (P=0.007) of cells underwent normal
development, respectively. Many of the cells failed to
change shape and showed mispositioning of both the
vegetative nucleus and generative cell. Taken together,
these results demonstrate that actin arrays perform
important functions that are critical for developmental
progression just prior to and during the asymmetrical
mitosis, and also during early pollen development.
Furthermore, the inhibitors are not generally cytologically
Fig. 2. Effects on development caused by the actin cytoskeletal inhibitors cytochalasin D and latrunculin B. Microspores and pollen were isolated
from one flower bud (containing the mixed starting populations as shown along the x-axis) for each developmental stage and cultured in maturation
medium (M1) or M1 spiked with the actin inhibitors 1 mM cytochalasin D (cyt D) or 5 nM latrunculin B ( lat B). After 8 h the cultures were
assessed for developmental progression through the stage listed above the graph as described in Materials and methods. The number of cells that
showed developmental progression in the M1 control cultures were reported as 100%, and those that showed progression in the presence of the
inhibitors were reported as x% relative to the controls. The values are the means±standard error for three separate experiments.
Cytoskeleton in tobacco microspores 587
toxic because early microspore development was essentially unaffected.
Cytoskeletal defects caused by the actin inhibitors reveal
that specific actin arrays mediate specific cellular events
In order to identify specific arrays that are essential for
development, an examination was made of actin arrays
in cells that had been exposed to the actin inhibitors at
different stages of development during in vitro culture. It
was first verified that culturing the cells did not perturb
normal actin arrays (data not shown). Figure 3 shows
that the earliest inhibitor effects are observed at the late
microspore stage. In fully polarized cells, an actin tether
normally forms around the nucleus after it has reached
the pole (Fig. 1E, F ). In the presence of cytochalasin D
or latrunculin B this tether does not form ( Fig. 3A, G)
and the nucleus drifts away from the pole and loses its
condensed structure ( Fig. 3B, H ). During normal development, actin arrays are localized within the region of
the asymmetrical mitotic spindle and in the phragmoplast
( Fig. 1G, I ). However, in the presence of cytochalasin D,
the phragmoplast is frequently aberrant in shape and
position (Fig. 3C, D). In the presence of latrunculin B,
reorganization of actin filaments into the region of the
mitotic spindle is generally completely disrupted ( Fig. 3I,
J ). The normal actin arrays observed in early binucleate
pollen following the asymmetrical mitosis (Fig. 1K, M )
are severely disrupted by cytochalasin D. The cell wall
Fig. 3. Cytoskeletal and morphological defects caused by the actin inhibitors cytochalasin D and latrunculin B. Cells were exposed to 1 mM
cytochalasin D (A–F ) or 5 nM latrunculin B (G–L) at different developmental stages, as defined by the associated legend. All cells were doublelabelled with rhodamine-phalloidin to visualize the actin cytoskeleton (top row: A, C, E, G, I, K ) and DAPI to visualize the nucleus (bottom row:
B, D, F, H, J, L). All cells, magnification ×1000. (A, B) Cytochalasin D inhibits formation of the actin tether that normally occurs at the late
microspore stage, and as a result the nucleus drifts away from the pole and loses its condensed structure. (C, D) The direction and shape of the
phragmoplast are disrupted by cytochalasin D. ( E, F ) Cytochalasin D severely disrupts formation of the phragmoplast/generative cell wall, the
actin array around the vegetative nucleus, and pollen development following the asymmetrical mitosis, thus causing mispositioning and aberrant
morphology of both the generative cell and vegetative nucleus. (G, H ) Latrunculin B blocks formation of the actin tether around the nucleus at
the late microspore stage, and the nucleus drifts away from the pole and becomes more diffuse. (I, J ) Displacement of actin from the region of the
mitotic spindle by latrunculin B also disrupts positioning of the condensed chromatin. ( K, L) Latrunculin B induces an aberrant phragmoplast
formation around the generative cell and blocks formation of the actin ring around the vegetative nucleus, resulting in vegetative nucleus and
generative cell mispositioning.
588 Zonia et al.
(or phragmoplast remnant) curves away from the pole
and fails to encircle the generative cell ( Fig. 3E). The
shape of the generative cell nucleus typically parallels the
shape of the phragmoplast remnant and the nucleus does
not migrate toward the centre of the pollen grain
(Fig. 3F ). The vegetative nucleus array fails to form
(Fig. 3E), and the vegetative nucleus generally drifts to
the opposite pole of the cell (Fig. 3F ). Similar effects are
observed in cells that have been exposed to latrunculin B
(Fig. 3K, L). Exposure to either actin inhibitor causes a
disorganized structure and position of the vegetative
nucleus, and fewer cytoplasmic or cortical filaments. Also,
the cells generally remain elliptically shaped (Fig. 3K, L)
and fail to become spherical. Taken together, the results
of these studies demonstrate the functional role of actin
arrays during maintenance of the polarized nuclear position in the microspore, during the asymmetrical mitosis
and cytokinesis, and during migration of the generative
cell and vegetative nucleus to the centre of the pollen
grain.
The microtubule cytoskeleton is organized into structures
associated with microspore nuclear migration, the
asymmetrical mitosis and the generative cell in mature
pollen
Microtubule arrays were localized during microspore and
pollen development in order to clarify a potentially different role for the microtubule cytoskeleton. These results
are shown in Fig. 4. At the tetrad stage, when the four
haploid meiotic products are enclosed within a callose
sac (Fig. 4B), microtubules are organized in an irregular
radial array between each centrally located nucleus
and the cell perimeter (Fig. 4A). In early microspores
(Fig. 4D), microtubules are localized at punctate sites
around the centrally located nucleus ( Fig. 4C ). These
sites later become connected by longer bundles ( Fig. 4E)
that eventually form a dense basket enclosing the nucleus
(Fig. 4G). This structure is asymmetrical; one end is
flattened while the other is more elongated and typically
contains a tether that connects to a point on the cell
perimeter. This tether appears to first connect with the
wall toward which the nucleus migrates (Fig. 4G, H ) and
subsequently connects with the pole in the late microspore
stage (Fig. 4I, J ). When the nucleus is located at the pole
(Fig. 4L), the microtubule array loses the tether and the
structure becomes modified ( Fig. 4K ), eventually being
transformed into the mitotic spindle. This spindle has an
asymmetrical shape, with one pointed end and one flat
end ( Fig. 4M ). After the asymmetrical mitosis, microtubule structures do not again become prominent until the
late bicellular phase, when they are observed as a ring
enclosing the generative cell (Fig. 4O, P). As pollen
reaches maturity, this microtubule array persists in association with the generative cell and attains an elongated
structure that apparently directs the shape of the generative cell ( Fig. 4Q, R). Microtubules also form a dense
cortical network from the mid-bicellular stage through to
pollen maturation (data not shown). In summary, it was
observed that microtubule arrays are organized into
prominent and unique structures associated with microspore nuclear migration (Fig. 4C, E, G), with the asymmetrical microspore mitosis ( Fig. 4M ), and with the
generative cell of mature pollen (Fig. 4R).
Oryzalin blocks microspore nuclear migration and the
asymmetrical mitosis
To assess the extent to which microtubule arrays have an
essential role in developmental progression, the effects on
development caused by exposure to the microtubule inhibitor oryzalin during in vitro culture at different developmental stages were tested. Oryzalin is a dinitroaniline
herbicide that directly inhibits dynamic microtubule
assembly and disassembly and promotes the depolymerization of existing arrays (Bajer and Molé-Bajer, 1986;
Morejohn et al., 1987). Like the actin inhibitors, oryzalin
disrupted development only at certain stages and had
little or no effect at other stages, as shown in Fig. 5.
During early microspore growth and nuclear migration,
only 55% (P=0.01) of the cells underwent normal developmental progression in the presence of 25 mM oryzalin.
Oryzalin significantly blocked the early phase of the
asymmetrical mitosis, with only 38% (P=0.026) of cells
entering mitosis. Progression through mitosis was also
significantly perturbed, with only 50% (P=0.015) of cells
maintaining mitotic fidelity. However, during early pollen
development, oryzalin had essentially no effect on the
migration of the generative cell and vegetative nucleus
and the change in vegetative cell shape from elliptical to
spherical (P=0.222). This result is consistent with the
observation that no prominent microtubule arrays are
present at this stage (data not shown). Taken together,
these results indicate that microtubule arrays perform
important functions during microspore nuclear migration
and during the asymmetrical mitosis.
Microtubule cytoskeleton defects caused by oryzalin reveal
the function of microtubules in nuclear migration and the
asymmetrical mitosis
In order to demonstrate a functional role for the observed
microtubule arrays, the arrays in cells that had been
exposed to 25 mM oryzalin at different stages of development during in vitro culture were examined next. It was
first verified that culturing the cells did not perturb normal
microtubule arrays (data not shown). As shown in Fig. 6,
oryzalin effects are apparent at the early microspore stage.
In early microspores, oryzalin blocks localization of microtubules at punctate nucleation sites around the nucleus
( Fig. 6A, B). Exposure of more advanced microspores to
Cytoskeleton in tobacco microspores 589
Fig. 4. Reorganization of the microtubule cytoskeleton during development of the microspore to mature pollen. All cells were double-labelled with
anti-b-tubulin/FITC-conjugated secondary antibody to visualize the microtubule cytoskeleton (top row: A, C, E, G, I, K, M, O, Q) and DAPI to
visualize the nucleus (bottom row: B, D, F, H, J, L, N, P, R). ( E), (G) and (M ) are projected stacks of images from two different focal planes.
All cells, magnification ×1000. (A, B) Tetrad with an irregular radial microtubule array around each nucleus. (C, D) Early microspores have
microtubules localized at punctate sites surrounding the nucleus. ( E, F ) The punctate nucleation sites are interconnected with longer microtubule
strands while still in the early microspore stage. (G, H ) Microtubules enclose the nucleus in early to mid-microspores to form a cage that connects
to the cell wall by a longer microtubule tether. (I, J ) The microtubule tether connects to the generative pole during migration of the nucleus to that
site. ( K, L) In late microspores, the microtubule tether is lost and the nuclear cage becomes modified prior to the asymmetrical mitosis. (M, N )
The mitotic spindle of the asymmetrical mitosis is also asymmetrical, with one end cone-shaped and one end barrel-shaped. (O, P) A microtubule
ring forms around the generative cell in a mid-bicellular pollen protoplast. (Q, R) The microtubule ring associated with the generative cell begins
to elongate and flatten in late bicellular pollen.
590 Zonia et al.
Fig. 5. Effects on development caused by the microtubule cytoskeletal inhibitor oryzalin. Microspores and pollen were isolated from one flower
bud (containing the starting population as shown along the x-axis) for each developmental stage and cultured in maturation medium (M1) or M1
spiked with the microtubule inhibitor 25 mM oryzalin. After 8 h the cultures were assessed for developmental progression through the stage listed
above the graph as described in Materials and methods. The number of cells that showed developmental progression in the M1 control cultures
were reported as 100%, and those that showed progression in the presence of oryzalin were reported as x% relative to the controls. The values are
the means±standard error for three separate experiments.
oryzalin blocks elaboration of the microtubule cage that
encloses the nucleus ( Fig. 6C, D, E, F ). Disruption of
the mitotic spindle causes displacement of the condensed
chromosomes ( Fig. 6G, H ) and the metaphase array
(Fig. 6I, J ). When mid-bicellular pollen are exposed to
oryzalin, the microtubule array around the generative cell
is severely fragmented and the cell typically remains
spherical ( Fig. 6K, L). Taken together, the results of these
studies demonstrate the functional role of microtubule
arrays during microspore nuclear migration, during the
asymmetrical mitosis, and during development of the
generative cell in mature pollen.
Discussion
Cellular differentiation is initiated by the temporal and
spatial propagation of specific signals. These signals target
certain cellular machinery that then mediates the appropriate response. Development of the microspore to mature
pollen is an excellent example of cellular differentiation
in higher plants. The authors long-term goal is to understand the mechanisms that control development. In this
report, it is shown that the cytoskeleton plays a fundamental role during development of the microspore to
mature pollen. In tetrads, actin and microtubule networks
are organized as irregular radial arrays that extend
between the centrally-located nucleus and the cell perimeter (Figs 1A, 4A). However, these networks have
greatly different localization and function during all subsequent microsporogenesis and differentiation of the male
gametophyte.
Microtubule arrays are dominant during microspore
development. A primary event at this stage is cellular
polarization, and includes migration of the nucleus to the
generative pole. Microtubules are localized at discrete
sites around the nucleus of early microspores ( Fig. 4C ).
These sites are then interconnected by longer bundles that
eventually form a microtubule cage that encircles the
nucleus (Fig. 4E, G). Migration of the nucleus to the cell
perimeter and then to the generative pole appears to be
mediated by a microtubule tether that connects the cage
with the target site and provides the framework for this
process (Fig. 4G, I ). The microtubule motor proteins
kinesin and dynein have been identified in extracts of
tobacco pollen tubes (Liu and Palevitz, 1996; Moscatelli
et al., 1996), and microtubule-based motors are
known to function during plant cell division (Asada and
Collings, 1997). Further evidence for the importance of
Cytoskeleton in tobacco microspores 591
Fig. 6. Cytoskeletal and morphological defects caused by the microtubule inhibitor oryzalin. Cells were exposed to 25 mM oryzalin at different
stages of development, as defined by the associated legend. All cells were double-labelled with anti-b-tubulin/FITC-conjugated secondary antibody
to visualize the microtubule cytoskeleton (top row: A, C, E, G, I, K ) and DAPI to visualize the nucleus (bottom row: B, D, F, H, J, L). All cells,
magnification ×1000. (A, B) Oryzalin blocks the localization of microtubules at discrete nucleation sites around the nucleus in early microspores.
(C, D) The microtubule nucleation sites around the nucleus in early microspores do not become interconnected when the cells are exposed to
oryzalin. ( E, F ) Elaboration of the microtubule cage around the nucleus in mid- to late microspores is blocked in the presence of oryzalin, and the
nucleus fails to migrate to the generative pole prior to the asymmetrical mitosis. (G, H, I, J ) The asymmetrical mitosis is severely perturbed when
oryzalin blocks formation of the mitotic spindle. ( K, L) The normal microtubule array around the generative cell in late bicellular pollen becomes
severely fragmented in the presence of oryzalin, and the generative cell fails to become disc-shaped.
microtubule arrays during microspore development is
that nuclear migration is perturbed in microspores cultured in the presence of the microtubule inhibitor oryzalin
(Fig. 5). Oryzalin blocks formation of the microtubule
cage that encloses the nucleus during this migration
(Fig. 6C, E). It is also significant that actin arrays are
largely absent at this stage (Fig. 1C ) and that the actin
inhibitors cytochalasin D and latrunculin B have essentially no effect on nuclear migration (Fig. 2). The microtubule cage around the nucleus is a highly unique structure
and there appear to be no previous reports of it in the
literature. In microspores of the dicot Brassica, no microtubule arrays were observed during the nuclear migration
(Hause et al., 1991, 1993). In the monocot Tradescantia,
cytoplasmic microtubules were observed to accumulate
only after the first nuclear migration to the cell perimeter
( Terasaka and Niitsu, 1990). Furthermore, the generative
pole microtubule system of the persistent tetrad microspores of orchids only contacts the nucleus after it has
attained its position near the generative pole (Brown and
Lemmon, 1991, 1992b).
Both actin and microtubule arrays have important
functions during the asymmetrical mitosis. Once the nucleus attains its position at the generative pole in late
microspores, an actin array forms that surrounds the
nucleus and has shorter projections between the nucleus
and the pole (Fig. 1E). This actin array appears to tether
the nucleus to the pole, in that disruption of the array
with cytochalasin D or latrunculin B results in nuclear
mispositioning, and the nucleus loses its condensed structure (Fig. 3A, B, G, H ). The microtubule cage that
mediated nuclear migration becomes modified and
592 Zonia et al.
transforms into the mitotic spindle (Fig. 4K, M ). The
mitotic apparatus of the asymmetrical microspore mitosis
is also asymmetrical, with one end cone-shaped and one
end barrel-shaped (Fig. 4M ). There have been previous
reports of an asymmetrical mitotic spindle during asymmetrical mitoses (Brumfeld, 1941; Heslop-Harrison, 1968;
Burgess, 1970; Brown and Lemmon, 1994; Terasaka and
Niitsu, 1995; Zhang et al., 1995). The present report
provides further evidence that asymmetrical mitoses are
mediated by asymmetrical mitotic spindles. Actin is also
present within the region of the asymmetrical mitotic
spindle ( Fig. 1G) and in the phragmoplast (Fig. 1I ). This
observation is significant in that although actin has been
observed in the presumed spindle during meiosis of the
microspore mother cells ( Van Lammeren et al., 1989), it
was not previously observed in the spindle during the
asymmetrical microspore mitosis ( Van Lammeren et al.,
1989; Hause et al., 1991, 1993; Gervais et al., 1994). Both
actin and microtubule arrays have important functions
during the asymmetrical mitosis in that disruption of
either array with specific inhibitors perturbs the start of
or progression through mitosis (Figs 2, 5).
Actin arrays are dominant during pollen development
following the asymmetrical mitosis. Immediately after
mitosis, an actin ring forms only around the generative
cell that parallels the site of synthesis of the generative
cell wall ( Fig. 1K ). As the two cells differentiate further,
an actin ring forms also around the vegetative nucleus,
the two rings are interconnected by shorter filaments, and
there are longer filaments that extend into the cytoplasm
of the vegetative cell (Fig. 1M ). These actin arrays function during the migration of the generative cell and
vegetative nucleus toward the centre of the pollen grain,
in that disruption of these arrays with actin inhibitors
causes a severe perturbation of pollen development
(Fig. 2). In the presence of cytochalasin or latrunculin,
the generative cell array is aberrant and formation of the
vegetative nucleus array is blocked ( Fig. 3E, K ). As a
result, the generative cell fails to migrate from the pole,
the vegetative nucleus becomes mispositioned within the
cell, and both nuclei have aberrant structures (Fig. 3F,
L). Myosin has been identified in pollen tubes of Nicotiana
alata and Lilium longiflorum, and the actin filament
network provides a framework for the active movement
of myosin-coated organelles and vesicles (Miller et al.,
1995; Asada and Collings, 1997). It is also significant
that microtubule arrays are absent during this stage of
pollen development (data not shown) and exposure to
oryzalin has essentially no effect on development (Fig. 5).
Once the vegetative nucleus and generative cell attain a
central position, the actin ring on the generative cell
disappears and actin forms a dense patch around the
vegetative nucleus ( Fig. 1O). Around this time, a microtubule array forms around the generative cell that persists
through pollen maturation (Fig. 4O). This microtubule
array appears to be essential for the generative cell shape
change from spherical to disc-shaped (Fig. 4Q). During
the mid- to late-stages of pollen maturation, there is a
decrease in cytoplasmic actin filaments and an increase
in cortical actin ( Fig. 1Q) and microtubule (data not
shown) arrays that ultimately form a dense cortical
meshwork.
The extensive cytoskeletal reorganizations that are
implicated by these data are notable for several reasons.
First, the critical events that occur during the transition
from microspore to pollen take place within several days,
and progression through mitosis can be completed in as
little as several hours. Therefore, the cytoskeletal reorganizations that occur during these developmental transitions
must be precisely timed and executed. Second, these
reorganizations are demonstrated to be an essential determinant of cellular morphology and developmental progression. Furthermore, the extent of this co-ordinated
reorganization of unique actin and microtubule arrays
has not been previously documented within a single plant
cell. At the present time, little is known of the mechanisms
that are involved in restructuring the cytoskeleton in
plant cells. Future work will be undertaken to characterize
upstream elements that signal the cytoskeletal rearrangements that occur during development of microspores to
mature pollen.
Acknowledgements
This work was funded by a United States Department of
Agriculture—National Research Initiative Competitive Grants
Program award (No. 95–37304–2293) to LZ. Supplementary
support was from a US–Czech Science and Technology Program
grant (No. JF95022) to LZ and JT.
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