-
Plant Physiol. (1 997) 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 per-
turbed stomatal behavior in Commelina communis 1. leaf epidermis
and therefore suggested that dynamic changes in actin are neces-
sary 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 immuno-
fluorescence 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, open- ing 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 environmen- tal 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. 61-0507- 056-2 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 82-562-
279-2199.
actin monomers and tubulin dimers, respectively, and re-
assemble at spatially defined sites of the cell. Traditionally,
they have been known to participate in diverse processes such as
mitosis, cytokinesis, cytoplasmic streaming, intra- and
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 reorga- nized, 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 pheromone- stimulated responses (Leeuw et
al., 1995). In plants actin filaments provide a matrix for
components of the phos- phatidylinositol 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 mol- ecules 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 dy- namic 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, tetra-
methyl rhodamine isothiocyanate.
1491
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1492 Eun and Lee Plant Physiol. Vol. 11 5, 1997
MATERIALS A N D METHODS
Plant Material
Commelina communis L. plants were grown in a green- house 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 dif- ferent ways: 2
to 3 h before the beginning of the light period in drier air
(RH
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Stimulus-Dependent Actin Reorganization in Guard Cells 1493
Figure I. Actin filaments in guard cells of stomata open under
light.The original stomatal aperture was not maintained during the
fixa-tion. Fine actin filaments are localized at the cortex of the
guard cells.Some microfilaments appear to be branched (arrows). The
radialpattern of actin filaments are apparent in the guard cells,
where theyare in focus. Diffuse staining from the nucleus (n) is
seen at the centerof the guard cells near the stomatal pore. The
microfilaments in someparts of the cell on the right side are out
of focus because of theconvexity of guard cells in the epidermal
surface. Bar represents10 /Jim.
branched in the direction of the dorsal side. Although
thecontinuity of the actin filaments along the paradermal sur-face
of guard cells was evident when focusing through thecell depth, the
cortical actin filaments in guard cells couldrarely all be focused
in a single plane. This observationindicates that the radial actin
arrays are located very closeto the plasma membrane, which is
highly convex becauseof the pronounced three-dimensional shape of
guard cells.In some guard cells a few actin filaments appeared to
beconnected to the nuclear envelope; therefore, they were
cortical near the dorsal side of the cell but became
partiallycytoplasmic near the nucleus, which was often locatedclose
to the stomatal pore side and also stained brightlywith actin
antibodies.
Stomatal opening is typically promoted by high plantwater
potential and high ambient humidity (Assmann,1993; Kearns and
Assmann, 1993). Stomata of C. communisleaves under these conditions
were sometimes wide open,even in the absence of light,
approximately 2 to 3 h beforethe beginning of the light period. In
this case, actin was alsolocalized in a radial pattern
indistinguishable from thatobserved in the guard cells swollen by
illumination (datanot shown).
Actin Filaments in Guard Cells of Dark-Closed Stomata
When plants were kept under dark conditions with mod-erate
humidity, stomata most often remained completelyclosed 2 to 3 h
before and after the onset of the usual lightperiod. There was no
difference in actin labeling in theguard cells of dark-closed
stomata at these two differenttimes of the day. Furthermore, actin
labeling in these cellswas entirely distinct from that observed in
guard cells ofopen stomata: fluorescence was either diffuse (Fig.
2, A andB) or shown as randomly distributed fragments (Fig. 2C).The
diffuse staining was seen through the depth of the cellsand more
concentrated near the ventral side of the guardcells (Fig. 2, A and
B). On rare occasions, long filamentsrunning parallel to the long
axis of the guard cell werelabeled in the subcortical cytoplasm
(Fig. 2D).
ABA Effects on Actin Filaments
To determine whether actin organization in the guardcells of
stomata closed by ABA is distinct from that ob-served in the guard
cells of dark-closed stomata and toexamine 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 differentsets
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 ventralside
of the guard cells throughout the cell depth (focal plane was at
the cortex in A and the nucleus in B), and randomlyoriented 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 /j.m.
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1494 Eun and Lee Plant Physiol. Vol. 115, 1997
100
0 10 20 30 40 50 60v
Time in ABA (min)
Figure 3. Left, Cortical actin patterns in permeabilized
ABA-treated guard cells. A, Long radial filaments, spanning most
ofthe 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.
from samples that had been kept for hours under eitherstomatal
opening or closing conditions, epidermal piecestaken in the process
of ABA treatment showed large vari-ations in stomatal size and
actin filament patterns. Thus,we measured stomatal size and
categorized cortical actinpatterns into three groups: (a) long
radial filaments span-ning most of the cell width, (b) shorter
filaments still in theradial direction, and (c) fragments in a
random orientationor diffuse labeling (Fig. 3; Table I). Before ABA
treatmentthe dominant pattern was the radial arrays; guard
cellsshowing long or short radial filaments consisted of 90% ofthe
total guard cells labeled. After 3 min in ABA, althoughthe actin
pattern in the majority of guard cells was still theradial one, the
population of guard cells with this orga-nized pattern was
substantially reduced compared to thatbefore ABA treatment. At the
same time, the stomatal sizewas also reduced. During the extended
period of ABAtreatment, diffuse or spotty labeling lacking any
radialarrays of actin, the pattern similar to that observed in
theguard cells of dark-closed stomata, became more
prevalent,reaching 85 and 98% after 30 and 60 min, respectively.
Thegradual but steady disassembly of actin filaments
duringABA-induced stomatal closing was observed in all time-course
experiments we performed (n = 4). A similar trendof actin
depolymerization with a faster time course wasobserved under the
conditions of rapid stomatal closure,addition of 10 /XM ABA in 30
mM KC1 and 10 mM K+-Mes(pH 6.1), without EGTA (data not shown).
Whereas actinfilaments at the cortex disappeared, in the
subcortical area,where not many actin filaments were localized in
openguard cells, long filaments similar to those shown in Figure2D
were observed frequently in guard cells treated withABA for 60
min.
Microtubules in Guard Cells
Actin filaments and microtubules in plant cells are
oftenintimately associated in the cell cortex, and their stability
isinterdependent. We localized microtubules to investigatewhether
microtubules in guard cells are also redistributedin response to
stimuli that alter actin distribution andwhether changes in one
cytoskeletal element influence thepolymerization state of the
other.
Cortical microtubules were arranged in a radial patternsimilar
to that of actin filaments in guard cells of openstomata (Fig. 4).
However, compared with actin filaments,microtubules appeared
denser, with smaller angles be-tween individual arrays. In
addition, microtubule arraysfrom one side of a guard cell reached
the other side withoutbranching (Fig. 4). Another difference was
that there waslittle labeling of tubulin in the subcortical
cytoplasm andaround the nuclear envelope. Thus, most microtubules
in
Table I. Time course of ABA effects on actin filaments in
guardcells and stomatal aperture
Time inABA
min
0369
123060
A
Pattern of ActinFilaments as Shown
in Figure 3
B C
LabeledCell No.
Stomatal Size± SE
(n = 40)
% of total labeled cells j*m
675319121820
2324212327132
10236065558598
118996293339781
12.9 ± 0.311.8 ± 0.311.2 ± 0.210.5 ± 0.38.1 ± 0.45.8 ± 0.33.2 ±
0.2
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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)
andin the guard cells treated with ABA for 3 min (B) and 30 min (C
and D). C and D show intact microtubules in the twoperidermal
regions of the same pair of guard cells. Bar represents 10 urn.
guard cells appeared to be localized in the cortex of thecells.
Double labeling of actin filaments and microtubulesin the same
guard cells confirmed these differences in theorganization of the
two cytoskeletal elements (Fig. 5, A-D).However, the most striking
difference between microtu-bules and actin filaments was their
response to stimuli. Thecircumferential organization of
microtubules in illumi-nated guard cells (Fig. 4A) was not affected
by incubationof epidermis with ABA (Fig. 4, B-D) or under
darkness(Fig. 5D), whereas these conditions caused
depolymeriza-tion of actin filaments (Figs. 2, A-C and 5B).
DISCUSSION
We previously suggested that actin is an essential com-ponent in
signal transduction pathways of guard cellsbased on the data that
the actin antagonists cytochalasin Dand phalloidin, which resulted
in a net decrease and in-crease in actin filaments in guard cells,
respectively, inter-fered with stomatal movements (Kim et al.,
1995). Theresults presented in this paper further support the
hypoth-esis by clearly demonstrating fast reorganization of actin
inguard cells in response to physiological stimuli.
During the last decade accumulating evidence hasshown that actin
filaments, microtubules, and the proteinsthat bind to the
cytoskeletal elements are functionally as-sociated with signal
transducers. In this regard, the degreeand the location of
cytoskeletal assembly are critical forinducing proper responses of
the cell. In animal cells actinfilaments are readily reorganized by
chemotactic stimuli(Howard and Meyer, 1984; Condeelis, 1993),
growth factors(Rijken et al., 1991; Ridley et al., 1992; Nobes et
al., 1995),and extracellular matrix (Hartwig, 1992).
Furthermore,when cytoskeletal reorganization experiences
interference,cellular responses to the stimuli are interrupted
(Ridleyand Hall, 1992; Tominaga et al., 1993; Peppelenbosch et
al.,1995; Takaishi et al., 1995). Actin responses to
growthsubstances in plant cells are not well documented. How-ever,
actin filaments in plant cells do change their organi-zation; for
example, during the cell cycle (Seagull et al.,1987; Cleary et al.,
1992; Zhang et al., 1993), differentiation
(Cho and Wick, 1990; Cleary, 1995), interaction with
fungus(Kobayashi et al., 1994), and phototropic responses (Meskeand
Hartmann, 1995; Mineyuki et al., 1995). Although theturnover rate
of actin filaments in plant cells has not beendetermined,
interphase microtubule dynamics in plantcells was demonstrated to
be faster than that in animal cells(Hush et al., 1994). The
response of actin filaments to ABAwe observed in guard cells is
faster than any hormonalreorganization of cytoskeleton demonstrated
to date inplant cells. Another particular characteristic of actin
fila-ments in guard cells is that the changes in the structure
arereversible in response to cyclic changes of
environmentalconditions. These qualities make actin in guard cells
suit-able for a signal-mediating component, like its counterpartin
animal cells.
Guard cells of stomata that opened under two differentconditions
showed the same radial pattern of actin organi-zation. Similarly,
both darkness and ABA caused stomatalclosure, resulted in the
disappearance of cortical filaments,and led to almost identical
diffuse/spotty patterns of actin.These results imply that
structural changes in actin are notlimited to a particular signal
but are common to physio-logical signals that open or close
stomata. Our temporalstudies with ABA demonstrated that
disintegration of cor-tical actin filaments in guard cells occurs
in parallel withclosing of stomata; both changes were apparent
within 3min after the onset of ABA treatment and progressed
fur-ther along with time. More advanced techniques that allowABA
treatment of guard cells that have been loaded withlabeled
phalloidin or tagged actin may clarify whetheractin reorganization
precedes stomatal closing.
Therefore, does actin depolymerization always favorclosing of
stomata? Our previous data do not support thisidea, since
cytochalasin D, which abolished the radial actinfilaments in guard
cells, enhanced stomatal opening. Fusi-coccin, another fungal toxin
that enhances stomatal open-ing, also caused actin depolymerization
under light (S.-O.Eun and Y. Lee, unpublished data). Moreover, both
open-ing and closing movements are inhibited by phalloidin,which
increases the number of the filamentous form ofactin filaments in
guard cells (Kim et al., 1995). Therefore,
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1496 Eun and Lee Plant Physiol. Vol. 115, 1997
Figure 5. Organization of actin and microtubules in guard
cells.Double labeling of actin (A and B) and microtubules (C and D)
inguard cells of open (A and C) and closed (B and D) stomata
underlight and darkness, respectively. E and F, Schematic drawing
ofcytoskeletal organization in guard cells (actin in E and
microtubulesin F). Bar represents 10 /xm.
we propose that depolymerization of actin allows changesin
stomatal aperture but does not determine the directionof
change.
For better understanding of roles of actin cytoskeleton inguard
cells, it is necessary to identify molecules that are
affected by dynamic changes in actin structure. The phys-ical
state of actin in animal cells affects activities of
lipid-hydrolyzing enzymes, receptor- and
nonreceptor-proteinkinases, G-proteins, and their modulators
(Carraway andCarraway, 1995), as well as ion channels (Cantiello et
al.,1991; Schwiebert et al., 1994; Prat et al., 1996). Our
patch-clamping data showed that activities of K+ channels inguard
cells are certainly influenced by application of actinantagonists
(Hwang et al., 1997). We are currently investi-gating other
possible target molecules of actin in guardcells. Equally important
is elucidating the signaling path-ways that reside upstream of
actin in guard cells. Actinpolymerization is modulated by numerous
actin-bindingproteins. However, few of them have been characterized
inguard cells to date. Thus, an understanding of the regula-tion of
polymerization and depolymerization of actin inguard cells awaits
more detailed information concerningcharacteristics of these
actin-binding proteins. IntracellularCa2+ levels are also known to
affect actin filaments, al-though these effects may be indirect by
activating a groupof actin-severing proteins such as gelsolin, as
has beensuggested in mammalian cells. It is interesting that
ABAprovokes both a cytosolic [Ca2+] increase (Irving et al.,1992;
McAinsh et al., 1992) and actin depolymerization(Table I) within
several minutes in guard cells of C. com-munis. It would be
informative to understand how thesetwo are related to each other in
guard cell signaling. In adifferent manner, a subfamily of small
GTP-binding pro-tein, Rho, functions as a molecular switch in the
regulationof actin in animal and fungal systems. A full-length
RhocDNA isolated from pea seedlings (Yang and Watson,1993) and
partially characterized genes in several differentplants (Lee and
Lee, 1996) suggest its universal presence inhigher plants. We have
preliminary results implicating theexistence of Rho proteins in C.
communis guard cells and itsparticipation in stomatal movements.
These investigationswill likely unravel important new aspects of
signal trans-duction in plant cells.
It has been shown that actin filaments and microtubulesin plant
cells are often closely aligned, and alteration in oneresults in
reorganization of the other. Disruption of actinfilaments with
cytochalasin D interfered with reorganiza-tion and/or stability of
microtubules in onion mitotic cells(Eleftheriou and Palevitz, 1992)
and during the develop-ment of cotton fibers (Seagull, 1990) and
wheat mesophyllcells (Wernicke and Jung, 1992). However, we do not
con-sider the role of actin filaments in guard cells in such
aconnection because the changing state of actin filamentsdid not
affect the well-organized microtubules in guardcells. In fact, the
radial pattern of microtubules remainedintact in guard cells
regardless of the size of their stomata.
Whether microtubules change their distributions andplay a role
in stomatal movements is equivocal. Our stud-ies showed no changes
in microtubules in guard cells of C.communis in response to
darkness or ABA, and treatment ofC. communis leaf epidermis with
the microtubule antago-nists taxol or oryzalin did not affect
stomatal aperture (datanot shown), suggesting that microtubules are
not involvedin stomatal movements. In broad bean guard cells as
well,pharmacological disruption of microtubules did not inter-
-
Stimulus-Dependent Actin Reorganization in Guard Cells 1497
fere with 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 guard cells of
open stomata became fragmented when stomata were closed by ABA
(Jiang e t al., 1996). Further investigations are necessary to
clearly understand whether the differences between these reports
and what we observed are due to differences i n the plant materials
or in the techniques used.
In conclusion, actin cytoskeleton of guard cells under- goes
changes i n their organization i n response to changes in
physiologically important stimuli. These results are con- sistent
wi th our earlier pharmacological data and provide a foundation to
claim actin as a component i n guard 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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