Download Actin Filaments of Guard Cells Are Reorganized

Plant Physiol. (1997) 11 5: 1491-1 498
Actin Filaments of Guard Cells Are Reorganized in Response
to Light and Abscisic Acid’
Soon-Ok Eun and Youngsook Lee*
Department of Life Science (S.-O.E., Y.L.), School of Environmental Engineering (Y.L.), Pohang University of
Science and Technology, Pohang, 790-784, Korea; and lnstitute of Molecular Biology, Academia Sinica,
Nankang, Taipei, Taiwan 11 529, Republic of China (Y.L.)
We recently showed that treatment with actin antagonists perturbed stomatal behavior in Commelina communis 1. leaf epidermis
and therefore suggested that dynamic changes in actin are necessary for signal responses in guard cells (M. Kim, P.K. Hepler, S.O.
Eun, K . 4 . Ha, Y. Lee [1995]Plant Physiol 109: 1077-1084). Here
we show that actin filaments of guard cells, visualized by immunofluorescence microscopy, change their distribution in response to
physiological stimuli. When stomata were open under white-light
illumination, actin filaments were localized in the cortex of guard
cells, arranged in a pattern that radiates from the stomatal pore. In
marked contrast, for guard cells of stomata closed by darkness or by
abscisic acid, the actin organization was characterized by short
fragments randomly oriented and diffusely labeled along the pore
site. Upon abscisic acid treatment, the radial pattern of actin arrays
in the illuminated guard cells began to disintegrate within a few
minutes and was completely disintegrated in the majority of labeled
guard cells by 60 min. Unlike actin filaments, microtubules of guard
cells retained an unaltered organization under all conditions tested.
These results further support the involvement of actin filaments in
signal transduction pathways of guard cells.
A set of guard cells surrounding stomata of terrestrial
plants function much like sliding doors in a building, opening to allow the CO, uptake required for photosynthesis
and closing to reduce water loss during periods of water
deficit. Such regulation is initiated by sensing environmental and interna1 stimuli such as light, humidity, CO,, and
the plant-stress hormone ABA, and is accomplished by
osmotic volume changes of the cells. Previous studies have
implicated heterotrimeric G-proteins, the H+ pump, and
the movement of various ions regulated by ion channels in
these processes (for review, see Assmann, 1993). Thus,
guard cells provide an ideal system in which to examine
whether other molecules, including cytoskeletal elements,
take part in plant signaling and, if so, how they interact
with better-characterized ones.
Actin filaments and microtubules are dynamic cellular
components; they disassemble into their building units,
This research was supported by the Basic Science Research
Fund of Pohang University of Science and Technology and the
Science and Engineering Foundation of Korea (grant no. 61-0507056-2 awarded to Y.L.) and by a postdoctoral fellowship from
Korea Science and Engineering Foundation awarded to 5-O.E.
* Corresponding author; e-mail [email protected]; fax 82-562279-2199.
actin monomers and tubulin dimers, respectively, and reassemble at spatially defined sites of the cell. Traditionally,
they have been known to participate in diverse processes
such as mitosis, cytokinesis, cytoplasmic streaming, intraand intercellular transport, and cell shaping by providing a
framework in animal cells and by directing wall deposition
in plant cells. Recently, it has become evident that they also
function as a signal transducer; upon the perception of
extracellular stimuli the cytoskeleton is rapidly reorganized, and these structural changes in turn affect activities
of other signal-mediating molecules. This has been well
demonstrated in animal cells (Cantiello et al., 1991;
Schwiebert et al., 1994; Carraway and Carraway, 1995;
Diakonova et al., 1995; Prat et al., 1996). Actin in yeasts
serves a similar function in the relay of pheromonestimulated responses (Leeuw et al., 1995). In plants actin
filaments provide a matrix for components of the phosphatidylinositol cycle, a major pathway in transducing
extracellular signals across the plasma membrane. For
example, phosphatidylinositol 4-kinase (Xu et al., 1992),
phosphoinositide-specific phospholipase C (Huang et al.,
1997), and diacylglycerol kinase (Tan and Boss, 1992) are
associated with the detergent-extracted cytoskeleton. The
phospholipase C activity of broad bean (Viciafaba L.) leaves
is inhibited by an actin-binding protein, profilin (Drabak et
al., 1994). Thus, the state of actin polymerization in plant
cells can be critica1 in activating and recruiting signal molecules to a site where they interact, as in other cells.
We previously showed the presence of actin filaments in
mature guard cells and demonstrated that application of
actin antagonists perturbed stomatal behavior (Kim et al.,
1995). From these observations we suggested that the dynamic feature of actin filaments may be important in guard
cell signaling . Since the organization of actin filaments
typically changes when they are involved in cell signaling,
we investigated the possibility that the distribution of actin
filaments in guard cells actually changes during normal
stomatal movements. We report that actin filaments of
illuminated guard cells are radially organized at the cell
cortex, and that the radial arrays are rapidly disassembled
when stomatal closing is induced by ABA. Likewise, the
radial pattern of cortical actin filaments are abolished in
guard cells of dark-closed stomata.
Abbreviations: FITC, fluorescein isothiocyanate; TRITC, tetramethyl rhodamine isothiocyanate.
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Eun and Lee
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MATERIALS A N D METHODS
Plant Material
Commelina communis L. plants were grown in a greenhouse at the controlled temperature of 18 to 22°C. The light
period was 13 to 16 h with the maximum light intensity of
1000 pmol m-'s-l at noon. We used the youngest fully
expanded leaves of 4- to 5-week-old plants.
Conditions for Opening or Closing of Stomata
To compare the organization of cytoskeletal elements in
guard cells of open and closed stomata, plant materials
were treated under various conditions before fixation.
Guard cells of open stomata were obtained under two
different conditions: 2 to 3 h before the beginning of the
light period from well-watered plants in water-saturated
(100% RH) air and 3 h after the beginning of the light
period under 300 to 400 pmol m-' s-l of white light.
Guard cells of closed stomata were obtained in three different ways: 2 to 3 h before the beginning of the light
period in drier air (RH <70%), in darkness 3 h after the
beginning of the normal light period, and ABA treatment
of leaf epidermal peels approximately 3 h after the beginning of the light period under white light of about 300
pmol m-' s-'. In each experiment the open or closed state
of the stomatal aperture was confirmed microscopically
before fixation.
For analysis of the effect of ABA on the state of actin over
time, epidermal peels were floated on a buffer containing
10 mM K+-Mes (pH 6.1), 0.5 mM EGTA, and 50 mM KC1.
When stomata were wide open, ABA was added to a final
concentration of 10 p ~Multiple
.
samples were taken at 3,
6, 9, 12, 30, and 60 min after the onset of ABA treatment.
Severa1 samples from each time were examined for the
stomatal size, and the rest were fixed immediately for immunolocalization.
Visualization of Actin Filaments and Microtubules
Actin filaments were immunolocalized as described previously (Kim et al., 1995) with the following modifications.
The abaxial epidermis was peeled from mature leaves and
fixed at room temperature or 37°C for 1 h with mmaleimidobenzoyl N-hydroxysuccinimide ester dissolved
to 200 p~ in 50 miv phosphate buffer (pH 6.8) containing 5
mM EGTA, 0.05% (v/v) Triton X-100, 0.3 mM phenylmethylsulfonic acid, 1.5 p~ aprotinin, 2 p~ pepstatin, and 20
piv leupeptin. The epidermal peels were frozen in liquid
nitrogen and then cracked into small pieces with prechilled
metal blocks. They were further fractured if necessary by
tapping gently with the tips of forceps. They were extracted for 2 h with 1%(v/v) Triton X-100 in the buffer
used for fixation and then rinsed with PBS. The separate
blocking step was omitted. The small epidermal fragments
were sequentially incubated with a 1:20 dilution of monoclonal actin antibody, a 1 : l O O dilution of biotinylated goat
anti-mouse IgM, and a 1:40 dilution of streptavidin-FITC.
Antibodies (Amersham) were diluted with PBS containing
Plant Physiol. Vol. 11 5, 1997
1%BSA and 0.05% (v/v) Triton X-100. A11 incubations were
carried out at 37°C. The duration was 45 min for each of the
primary and the secondary antibodies and 30 min with
streptavidin-FITC. The labeled samples were mounted
with the medium containing 90% glycerin, 10% PBS (pH 8.9
adjusted with NaHCO,), and 0.1% p-phenylenediamine.
They were observed and recorded under an epifluorescence microscope equipped with Automatic-2 exposure system (Zeiss) using T-Max 400 film (Kodak).
The sample-handling procedures for labeling microtubules of leaf epidermis were very similar to those given for
localization of actin filaments except for some differences
during fixation and incubation with antibodies: epidermal
tissue was fixed at room temperature with 3.7% paraformaldehyde, 0.2% picric acid, 0.05% (v/v) Triton X-100, and
5 mM EGTA, prepared in 50 miv phosphate buffer (pH 6.8);
anti-/3-tubulin (Amersham) and FITC-conjugated antimouse IgG (Sigma) were diluted to 1:50 and 1:200 and used
as primary and secondary antibodies, respectively.
For double labeling of actin filaments and microtubules
in the same cells, leaf epidermis was processed as described above for the preservation of actin filaments until
they were incubated with biotinylated secondary antibodies. They were further incubated with anti-p-tubulin and
then with a mixture of 1:40 streptavidin-FITC and 1:80
TRITC-anti-mouse IgG (Sigma). Fluorescence from FITC
and TRITC was observed using a fluorescence microscope
(Optiphot-2, Nikon) equipped with filter blocks of a narrow band pass (for FITC: excitation 465-495 nm, barrier
BA515-555; for TRITC: excitation 540/25 nm, barrier
BA605/55). Images were recorded on T-Max 400 film using
a Microflex UFX-DX photographic attachment (Nikon).
Since the aim of this study was to examine cytoskeletal
distributions in guard cells in relation to stomatal regulation, it would be ideal to use a fixative that maintains both
stomatal size and the integrity of cytoskeletal structure. To
our knowledge, a fixation procedure that can retain actin
filaments in fully open or completely closed stomata has
not yet been reported. In our hands, m-maleimidobenzoyl
N-hydroxysuccinimide ester, a protein cross-linker that requires detergents to get into the cell, is the best fixative for
preservation of actin filaments in guard cells, as in other
types of plant cells. Fixing the cells in detergent-containing
solutions inevitably destroys the intactness of the plasma
membrane, and therefore it entails the collapse of the stomata1 aperture. However, the clear differences that are
observed between labeled guard cells of open versus closed
stomata suggest that the fixation procedures themselves do
not alter the integrity of the cytoskeletal structure.
RESULTS
Actin Filaments in Cuard Cells of Open Stomata
When stomata were opened by illumination, actin in
guard cells was in its filamentous form and localized at the
cortex in a radial pattern: the filaments were closely spaced
at the center of the ventral side, the side of guard cells near
the stomatal pore, and spread out toward the opposite side,
the dorsal side (Fig. 1). Some of the filaments were
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Stimulus-Dependent Actin Reorganization in Guard Cells
1493
cortical near the dorsal side of the cell but became partially
cytoplasmic near the nucleus, which was often located
close to the stomatal pore side and also stained brightly
with actin antibodies.
Stomatal opening is typically promoted by high plant
water potential and high ambient humidity (Assmann,
1993; Kearns and Assmann, 1993). Stomata of C. communis
leaves under these conditions were sometimes wide open,
even in the absence of light, approximately 2 to 3 h before
the beginning of the light period. In this case, actin was also
localized in a radial pattern indistinguishable from that
observed in the guard cells swollen by illumination (data
not shown).
Actin Filaments in Guard Cells of Dark-Closed Stomata
Figure I. Actin filaments in guard cells of stomata open under light.
The original stomatal aperture was not maintained during the fixation. Fine actin filaments are localized at the cortex of the guard cells.
Some microfilaments appear to be branched (arrows). The radial
pattern of actin filaments are apparent in the guard cells, where they
are in focus. Diffuse staining from the nucleus (n) is seen at the center
of the guard cells near the stomatal pore. The microfilaments in some
parts of the cell on the right side are out of focus because of the
convexity of guard cells in the epidermal surface. Bar represents
10 /Jim.
branched in the direction of the dorsal side. Although the
continuity of the actin filaments along the paradermal surface of guard cells was evident when focusing through the
cell depth, the cortical actin filaments in guard cells could
rarely all be focused in a single plane. This observation
indicates that the radial actin arrays are located very close
to the plasma membrane, which is highly convex because
of the pronounced three-dimensional shape of guard cells.
In some guard cells a few actin filaments appeared to be
connected to the nuclear envelope; therefore, they were
When plants were kept under dark conditions with moderate humidity, stomata most often remained completely
closed 2 to 3 h before and after the onset of the usual light
period. There was no difference in actin labeling in the
guard cells of dark-closed stomata at these two different
times of the day. Furthermore, actin labeling in these cells
was entirely distinct from that observed in guard cells of
open stomata: fluorescence was either diffuse (Fig. 2, A and
B) or shown as randomly distributed fragments (Fig. 2C).
The diffuse staining was seen through the depth of the cells
and more concentrated near the ventral side of the guard
cells (Fig. 2, A and B). On rare occasions, long filaments
running parallel to the long axis of the guard cell were
labeled in the subcortical cytoplasm (Fig. 2D).
ABA Effects on Actin Filaments
To determine whether actin organization in the guard
cells of stomata closed by ABA is distinct from that observed in the guard cells of dark-closed stomata and to
examine dynamics of actin reorganization in guard cells,
we performed time-scale experiments with ABA. Different
Figure 2. Actin filaments in guard cells of dark-closed stomata. Various patterns of actin labeling are shown in three different
sets of guard cells. Right-side cell of each pair in A to C and both cells in D are labeled. Diffuse staining shown on the ventral
side of the guard cells throughout the cell depth (focal plane was at the cortex in A and the nucleus in B), and randomly
oriented short cortical fragments (C) were the most common patterns. Relatively long filaments along the length of the cell
(D) were occasionally observed.
Bar represents
10 August
/j.m.
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Copyright © 1997 American Society of Plant Biologists. All rights reserved.
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Plant Physiol. Vol. 115, 1997
Eun and Lee
100
0
10
20
30
40
50
60 v
Time in ABA (min)
Figure 3. Left, Cortical actin patterns in permeabilized ABA-treated guard cells. A, Long radial filaments, spanning most of
the cell width. B, Shorter filaments but still in the radial pattern. C, Fragments in a random orientation or diffuse labeling.
Right, Time plot of stomatal size (A) and the percentage of guard cells with the radial actin filaments (A and B patterns, •)
after the onset of ABA treatment at time 0. Bar in A represents 10 /xm.
Microtubules in Guard Cells
from samples that had been kept for hours under either
stomatal opening or closing conditions, epidermal pieces
Actin filaments and microtubules in plant cells are often
taken in the process of ABA treatment showed large variintimately associated in the cell cortex, and their stability is
ations in stomatal size and actin filament patterns. Thus,
interdependent. We localized microtubules to investigate
we measured stomatal size and categorized cortical actin
whether microtubules in guard cells are also redistributed
patterns into three groups: (a) long radial filaments spanin response to stimuli that alter actin distribution and
ning most of the cell width, (b) shorter filaments still in the
whether changes in one cytoskeletal element influence the
radial direction, and (c) fragments in a random orientation
polymerization state of the other.
or diffuse labeling (Fig. 3; Table I). Before ABA treatment
Cortical microtubules were arranged in a radial pattern
the dominant pattern was the radial arrays; guard cells
similar to that of actin filaments in guard cells of open
showing long or short radial filaments consisted of 90% of
stomata (Fig. 4). However, compared with actin filaments,
the total guard cells labeled. After 3 min in ABA, although
microtubules appeared denser, with smaller angles bethe actin pattern in the majority of guard cells was still the
tween individual arrays. In addition, microtubule arrays
radial one, the population of guard cells with this orgafrom one side of a guard cell reached the other side without
nized pattern was substantially reduced compared to that
branching (Fig. 4). Another difference was that there was
before ABA treatment. At the same time, the stomatal size
little labeling of tubulin in the subcortical cytoplasm and
was also reduced. During the extended period of ABA
around the nuclear envelope. Thus, most microtubules in
treatment, diffuse or spotty labeling lacking any radial
arrays of actin, the pattern similar to that observed in the
guard cells of dark-closed stomata, became more prevalent,
Table I. Time course of ABA effects on actin filaments in guard
reaching 85 and 98% after 30 and 60 min, respectively. The
cells and stomatal aperture
gradual but steady disassembly of actin filaments during
Pattern of Actin
Stomatal Size
Filaments as Shown
ABA-induced stomatal closing was observed in all timeLabeled
Time in
± SE
in
Figure
3
Cell
No.
course experiments we performed (n = 4). A similar trend
ABA
(n = 40)
of actin depolymerization with a faster time course was
A
B
C
observed under the conditions of rapid stomatal closure,
%
of
total
labeled
cells
j*m
min
addition of 10 /XM ABA in 30 mM KC1 and 10 mM K+-Mes
12.9 ± 0.3
10
118
67
0
23
(pH 6.1), without EGTA (data not shown). Whereas actin
11.8 ± 0.3
99
24
23
3
53
11.2 ± 0.2
filaments at the cortex disappeared, in the subcortical area,
60
62
19
21
6
10.5 ± 0.3
65
93
23
12
9
where not many actin filaments were localized in open
8.1 ± 0.4
27
55
33
18
12
guard cells, long filaments similar to those shown in Figure
97
5.8 ± 0.3
85
2
13
30
2D were observed frequently in guard cells treated with
3.2 ± 0.2
81
98
0
2
60
ABA for 60 min.
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Stimulus-Dependent Actin Reorganization in Guard Cells
1495
Figure 4. Microtubules in guard cells. Microlubules are distributed in a radial pattern in the illuminated guard cells (A) and
in the guard cells treated with ABA for 3 min (B) and 30 min (C and D). C and D show intact microtubules in the two
peridermal regions of the same pair of guard cells. Bar represents 10 urn.
(Cho and Wick, 1990; Cleary, 1995), interaction with fungus
(Kobayashi et al., 1994), and phototropic responses (Meske
and Hartmann, 1995; Mineyuki et al., 1995). Although the
turnover rate of actin filaments in plant cells has not been
determined, interphase microtubule dynamics in plant
cells was demonstrated to be faster than that in animal cells
(Hush et al., 1994). The response of actin filaments to ABA
we observed in guard cells is faster than any hormonal
reorganization of cytoskeleton demonstrated to date in
plant cells. Another particular characteristic of actin filaments in guard cells is that the changes in the structure are
reversible in response to cyclic changes of environmental
conditions. These qualities make actin in guard cells suitDISCUSSION
able for a signal-mediating component, like its counterpart
We previously suggested that actin is an essential comin animal cells.
ponent in signal transduction pathways of guard cells
Guard cells of stomata that opened under two different
based on the data that the actin antagonists cytochalasin D
conditions showed the same radial pattern of actin organiand phalloidin, which resulted in a net decrease and inzation. Similarly, both darkness and ABA caused stomatal
crease in actin filaments in guard cells, respectively, interclosure, resulted in the disappearance of cortical filaments,
fered with stomatal movements (Kim et al., 1995). The
and led to almost identical diffuse/spotty patterns of actin.
results presented in this paper further support the hypothThese results imply that structural changes in actin are not
esis by clearly demonstrating fast reorganization of actin in
limited to a particular signal but are common to physioguard cells in response to physiological stimuli.
logical signals that open or close stomata. Our temporal
During the last decade accumulating evidence has
studies with ABA demonstrated that disintegration of cortical actin filaments in guard cells occurs in parallel with
shown that actin filaments, microtubules, and the proteins
that bind to the cytoskeletal elements are functionally asclosing of stomata; both changes were apparent within 3
sociated with signal transducers. In this regard, the degree
min after the onset of ABA treatment and progressed furand the location of cytoskeletal assembly are critical for
ther along with time. More advanced techniques that allow
inducing proper responses of the cell. In animal cells actin
ABA treatment of guard cells that have been loaded with
filaments are readily reorganized by chemotactic stimuli
labeled phalloidin or tagged actin may clarify whether
(Howard and Meyer, 1984; Condeelis, 1993), growth factors
actin reorganization precedes stomatal closing.
(Rijken et al., 1991; Ridley et al., 1992; Nobes et al., 1995),
Therefore, does actin depolymerization always favor
and extracellular matrix (Hartwig, 1992). Furthermore,
closing of stomata? Our previous data do not support this
when cytoskeletal reorganization experiences interference,
idea, since cytochalasin D, which abolished the radial actin
cellular responses to the stimuli are interrupted (Ridley
filaments in guard cells, enhanced stomatal opening. Fusiand Hall, 1992; Tominaga et al., 1993; Peppelenbosch et al.,
coccin, another fungal toxin that enhances stomatal open1995; Takaishi et al., 1995). Actin responses to growth
ing, also caused actin depolymerization under light (S.-O.
substances in plant cells are not well documented. HowEun and Y. Lee, unpublished data). Moreover, both openever, actin filaments in plant cells do change their organiing and closing movements are inhibited by phalloidin,
zation; for example, during the cell cycle (Seagull et al.,
which increases the number of the filamentous form of
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1987; Cleary et al., 1992; Zhang et al.,
1993), differentiation
actin
filaments
in guard cells (Kim et al., 1995). Therefore,
guard cells appeared to be localized in the cortex of the
cells. Double labeling of actin filaments and microtubules
in the same guard cells confirmed these differences in the
organization of the two cytoskeletal elements (Fig. 5, A-D).
However, the most striking difference between microtubules and actin filaments was their response to stimuli. The
circumferential organization of microtubules in illuminated guard cells (Fig. 4A) was not affected by incubation
of epidermis with ABA (Fig. 4, B-D) or under darkness
(Fig. 5D), whereas these conditions caused depolymerization of actin filaments (Figs. 2, A-C and 5B).
Copyright © 1997 American Society of Plant Biologists. All rights reserved.
1496
Eun and Lee
Plant Physiol. Vol. 115, 1997
affected by dynamic changes in actin structure. The physical state of actin in animal cells affects activities of lipidhydrolyzing enzymes, receptor- and nonreceptor-protein
kinases, G-proteins, and their modulators (Carraway and
Carraway, 1995), as well as ion channels (Cantiello et al.,
1991; Schwiebert et al., 1994; Prat et al., 1996). Our patchclamping data showed that activities of K + channels in
guard cells are certainly influenced by application of actin
antagonists (Hwang et al., 1997). We are currently investigating other possible target molecules of actin in guard
cells. Equally important is elucidating the signaling pathways that reside upstream of actin in guard cells. Actin
polymerization is modulated by numerous actin-binding
proteins. However, few of them have been characterized in
guard cells to date. Thus, an understanding of the regulation of polymerization and depolymerization of actin in
guard cells awaits more detailed information concerning
characteristics of these actin-binding proteins. Intracellular
Ca 2+ levels are also known to affect actin filaments, although these effects may be indirect by activating a group
of actin-severing proteins such as gelsolin, as has been
suggested in mammalian cells. It is interesting that ABA
provokes both a cytosolic [Ca 2+ ] increase (Irving et al.,
1992; McAinsh et al., 1992) and actin depolymerization
(Table I) within several minutes in guard cells of C. communis. It would be informative to understand how these
two are related to each other in guard cell signaling. In a
different manner, a subfamily of small GTP-binding protein, Rho, functions as a molecular switch in the regulation
of actin in animal and fungal systems. A full-length Rho
cDNA isolated from pea seedlings (Yang and Watson,
1993) and partially characterized genes in several different
plants (Lee and Lee, 1996) suggest its universal presence in
higher plants. We have preliminary results implicating the
existence of Rho proteins in C. communis guard cells and its
participation in stomatal movements. These investigations
will likely unravel important new aspects of signal transduction in plant cells.
It has been shown that actin filaments and microtubules
in plant cells are often closely aligned, and alteration in one
results in reorganization of the other. Disruption of actin
filaments with cytochalasin D interfered with reorganization and/or stability of microtubules in onion mitotic cells
(Eleftheriou and Palevitz, 1992) and during the development of cotton fibers (Seagull, 1990) and wheat mesophyll
cells (Wernicke and Jung, 1992). However, we do not consider the role of actin filaments in guard cells in such a
connection because the changing state of actin filaments
did not affect the well-organized microtubules in guard
Figure 5. Organization of actin and microtubules in guard cells.
Double labeling of actin (A and B) and microtubules (C and D) in
cells. In fact, the radial pattern of microtubules remained
guard cells of open (A and C) and closed (B and D) stomata under
intact in guard cells regardless of the size of their stomata.
light and darkness, respectively. E and F, Schematic drawing of
Whether microtubules change their distributions and
cytoskeletal organization in guard cells (actin in E and microtubules
play a role in stomatal movements is equivocal. Our studin F). Bar represents 10 /xm.
ies showed no changes in microtubules in guard cells of C.
communis in response to darkness or ABA, and treatment of
C. communis leaf epidermis with the microtubule antagowe propose that depolymerization of actin allows changes
nists taxol or oryzalin did not affect stomatal aperture (data
in stomatal aperture but does not determine the direction
not shown), suggesting that microtubules are not involved
of change.
in stomatal movements. In broad bean guard cells as well,
For better understanding of roles of actin cytoskeleton in
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that are
Copyright © 1997 American Society of Plant Biologists. All rights reserved.
Stimulus-Dependent Actin Reorganization in Guard Cells
fere w i t h stomatal behavior (Jiang e t al., 1996). However,
stomatal opening in Tradescantia virginiana was inhibited
by colchicine, a microtubule-depolymerizing alkaloid
(Couot-Gastelier and Louguet, 1992), and microtubules in
broad bean g u a r d cells of open stomata became fragmented
when stomata were closed by ABA (Jiang e t al., 1996).
Further investigations a r e necessary t o clearly understand
whether t h e differences between these reports and w h a t we
observed a r e due to differences i n t h e plant materials or in
t h e techniques used.
In conclusion, actin cytoskeleton of guard cells undergoes changes i n their organization i n response t o changes
i n physiologically important stimuli. These results are consistent w i t h our earlier pharmacological d a t a and provide a
foundation t o claim actin as a component i n g u a r d cell
signal transduction pathways.
ACKNOWLEDCMENTS
We thank Drs. Virginia S. Berg and Richard C. Crain for their
helpful comments concerning the manuscript, Mr. Shi-In Kim for
the management of plants, and Ms. Jae-Ung Hwang for assistance
with the time-course experiments and many helpful discussions.
Received May 19, 1997; accepted September 7, 1997.
Copyright Clearance Center: 0032-0889/97/ 115/1491/OS.
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