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 ylee@postech.ac.kr; 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.

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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

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.

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

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,

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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