actin filaments in mature guard cells are radially ... · cyanate-phalloidin into living guard...

Plant Physiol. (1 995) 109: 1077-1 084

Actin Filaments in Mature Guard Cells Are Radially Distributed and lnvolved in Stomatal Movement'

Moonjeong Kim, Peter K. Hepler, Soon-Ok Eun, Kwon Soo Ha, and Youngsook Lee*

Department of Life Science, Pohang University of Science and Technology, Pohang, Korea (M.K., S.-O.E., Y.L.); Department of Biology, University of Massachusetts, Amherst, Massachusetts (P.K.H.); and Biomolecule Research

Group, Korean Basic Science Center, Yusungku, Taejon, Korea (K.S.H.)

Stomatal movements, which regulate gas exchange in plants, involve pronounced changes in the shape and volume of the guard cell. To test whether the changes are regulated by actin filaments, we visualized microfilaments in mature guard cells and examined the effects of actin antagonists on stomatal movements. Immunolo- calization on fixed cells and microinjection of fluorescein isothiocyanate-phalloidin into living guard cells of Commelina communis L. showed that cortical microfilaments were radially distributed, fanning out from the stomatal pore site, resembling the known pattern of microtubules. Treatment of epidermal peels with phalloidin prior to stabilizing microfilaments with m-maleimidobenzoyl N-hydroxysuccimimide caused dense packing of radial microfilaments and an accumulation of actin around many organelles. 60th stomatal closing induced by abscisic acid and opening under light were inhibited. Treatment of guard cells with cytochalasin D abol- ished the radial pattern of microfilaments; generated sparse, poorly oriented arrays; and caused partia1 opening of dark-closed stomata. These results suggest that microfilaments participate in stomatal aperture regulation.

Regulation of stomatal movement is important for the uptake of CO, required for photosynthesis and the preven- tion of water loss by transpiration. Various environmental and interna1 signals, including light, CO,, and hormones such as ABA, working through different signal transduction pathways, are known to control the process of stomatal opening and closing (reviewed by Assmann, 1993). In this study we addressed the question of whether the cytoskeleton, in particular actin microfilaments, participates in the signal transduction process.

Recently, cytoskeletal elements have been shown to participate in signal transmission processes in various cell types (Payrastre et al., 1991; Horvath et al., 1992; Akiba et al., 1993). The signaling molecules, such as receptor and effector molecules, can be associated with cytoskeletal ele-

'This work was supported by a nondirected research fund, Korea Research Foundation, 1993, a grant from the Basic Science Research Institute Program, Ministry of Education of Korea, 1994, project No. BSRI-94-4435 awarded to Y.L., and by Korean Minis- try of Science and Technology grant No. PNOOOll to K.S.H. Sup- port was also provided by the U.S. National Science Foundation, grant No. MCB-9304953 to P.K.H.

* Corresponding author; e-mail ylee8vision.postech.ac.kr; fax 82-562-279-2199.

ments, and disruption of the cytoskeleton impairs the activities of effector molecules (Payrastre et al., 1991; Vaziri and Downes, 1992). Upon stimulation, a Tyr kinase asso- ciates with the cytoskeleton (Horvath et al., 1992) and the G a protein dissociates (Sarndahl et al., 1993). The PI cycle, an important signaling pathway, is also probably regulated by actin-binding proteins (Forscher, 1989; Vaziri and Downes, 1992; Sohn et al., 1994). In plant systems as well, there are many experimental results suggesting that actin filaments are important in signal transduction (Putnam- Evans et al., 1989; Tan and Boss, 1992; Xu et al., 1992; Yang and Boss, 1994). An actin-binding protein, profilin, was found in plants (Valenta et al., 1991; Staiger et al., 1993) and shown to bind PIP, and inhibit PLC activity (Drnbak et al., 1994).

Although guard cells have served as one of the most frequently used model system to study signal transduction and cell shape determination in plants, virtually a11 of our knowledge about the cytoskeleton relates to the latter process of differentiation and the acquisition of the mature shape. From these studies it was established that during the early stage of guard cell differentiation in grasses, microtubules become radially aligned (Cho and Wick, 1989; Cleary and Hardham, 1989; Marc et al., 1989), whereas the microfilaments are longitudinally oriented (Cho and Wick, 1990); similar studies have not been performed on fully mature guard cells with which the signal transduction studies have been conducted.

An obvious reason for this shortcoming stems from the difficulty of introducing the appropriate probes, e.g. antibodies or peptide stains, through the heavily thickened wall of the mature guard cell. We have overcome this problem in two ways: (a) through microinjection of fluorescent phalloidin into living cells and (b) through the application of immunoprocedures on frozen, cracked cells. Briefly, the results reveal that microfilaments in the mature guard cells fan out radially from the pore site. Our results also show that modulation of this actin organization impairs the ability of the stomata to respond to environmental cues.

Abbreviations: CB, cytochalasin B; CD, cytochalasin D; 3-D, three dimensional; FITC, fluorescein isothiocyanate; MBS, m-maleimidobenzoyl N-hydroxysuccinimide ester; PI, phosphatidylinositol; PIP,, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C.

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1078 Kim et al. Plant Physiol. Vol. 109, 1995

MATERIALS AND METHODS

Plant Materials

Commelina communis L. and Vicia faba L. were grown in a greenhouse or a growth chamber in cycles of 24/19 and 19/17"C, respectively, with a 16-h photoperiod. We used fully expanded leaves of 3- to 4-week-old plants.

lmmunofluorescence Microscopy of Guard Cell Protoplasts

The procedure of guard cell protoplast isolation from V . faba has been described (Kruse et al., 1989). The protoplasts were suspended in a bathing medium containing 0.35 M

mannitol, 0.1 mM CaCI,, 10 mM KCI, 0.5 mM ascorbic acid, and 1 mM Mes at pH 6.0. The protoplast suspension was mixed with 2.5% low-temperature-gelling agarose and spread on a coverglass that was coated with 0.5% agarose. A11 of the following procedures were performed on ice except where indicated otherwise. Protoplasts were perme- abilized and fixed in buffer (50 mM Pipes, 1 mM MgSO,) containing 0.3 mM PMSF, 50 pg/mL leupeptin, 5 mM EGTA, 0.1% phenylenediamine, 0.1% Triton X-100, 0.05% Nonidet P-40, and 100 p~ MBS (Pierce) for 1 h. Samples were washed with the buffer and blocked with 20% goat serum for 30 min. Ten percent goat serum was included during the incubation with 11500 monoclonal antibody against chicken gizzard actin (Amersham) for 3 h at room temperature. The control was incubated with normal mouse serum. Incubations with 1 /200 biotinylated goat anti-mouse IgM (Amersham) and then with 1 /50 streptavidin-FITC (Amersham) followed for 2 h at 4°C and for 30 to 60 min at room temperature, respectively. Samples were examined using a fluorescence microscope (Zeiss, BPF 510 filter) and photographed with Zeiss photomicrographic equipment (Automatic-2) using Kodak gold I1 film. Autofluorescence of chloroplasts was reduced using an Oriel (Stratford, CT) filter (No. 57377) in the emission path that blocked all light longer than 600 nm.

Microiniection of FITC-Phalloidin into Living Guard Cells

The abaxial epidermal tissue was stripped from the leaf of C. communis, and the peel was secured onto the bottom of a culture chamber using medical adhesive (Factor 11, Inc., Lakeside, AZ). The peel was then flooded with an injection bath containing 10 mM K+-Mes (pH 6.1), 30 mM KCI, and 0.1 mM CaC1,. FITC-phalloidin (Molecular Probes, Inc., Eugene, OR) was dissolved in 50 mM K+- citrate to 13.2 p~ and was backfilled into the pipette. Pressure injection and the observation of fluorescent label- ing with a confocal laser scanning microscope were carried out as described elsewhere (Zhang et al., 1990). Neutra1 density filters were used to minimize photobleaching and damage of cells. In these examples, red autofluorescence of the chloroplasts was blocked with a Corion (Holliston, MA) filter (No. LS-600-F-H991), which also effectively blocked transmission of light longer than 600 nm.

lmmunofluorercence Microscopy of Cuard Cells in lntact Epidermis

To observe the distribution of microfilaments in guard cells of open stomata, leaves of C. communis were floated on water and irradiated with 500 pmol m-' s-l white light for 2 to 3 h. Pieces of the abaxial epidermis were peeled from the leaves, fixed, and cracked using a method modified from that of U'asteneys et al. (1996). Briefly, the epidermal pieces were treated for 1 h with 200 p~ MBS, which was dissolved in 5 mM Hepes, 2.5 mM K,HPO,, 0.2 mM CaCl,, 0.5% Triton X- 100, 0.3 mM PMSF, and 10 pg / mL leupciptin. After the epidermal pieces were washed with a solution containing 15 rnM Hepes, 2 mM K,HPO,, 2 m M MgSO,, and 2 mM EGTA, they were frozen in liquid nitrogen and physically cracked using prechilled metal blocks. They were further extracted in PBS containing 1% Triton X-100 for 2 h and washed with PBS containing 0.1% Gly. Ten percent goat serum was used as a blocking solution. [ncu- bations with antibodies and with streptavidin-FITC were performed as in immunolabeling of guard cell protoplasts, except that 1 / 100 actin antibody, 1 / 100 biotinylated goat anti-mouse IgM, and 1 / 40 streptavidin-FITC were used. The samples were mounted using 50 mM Tris (pH 9.5), 50% glycerol, and 0.1% p-phenylenediamine and observed with a confocal laser scanning microscope (Zeiss) coupled to an inverted microscope (Axiovert 135M, Zeiss) using a high numerical aperture oil-immersion objective (Zeiss l'lan- Apocromat, X63, 1.4 numerical aperture). A series of optical sections were scanned in the z axis and then 3-D images were obtained using LSM software (Zeiss, version 3.6).

Test of Drug Effects on Stomatal Movements and Microfilament Organization in Guard Cells

For experiments testing closing movement, the initial open state of stomata was obtained by floating fully expanded leaves of C. communis on water and irradiating them with 500 pmol mp2 s-' white light. The abaxial epidermis was peeled, cut into small pieces, and floated on 10 mM K+-Mes (pH 6.1) containing 30 mM KCI. To test the effects of drugs that modulate the actin state, 0.1 mM phalloidin, 10 kg/mL CB, or 20 PM CD was added to this bathing mediuin. Thirty minutes after the addition of a drug, 10 PM ABA was added to induce stomatal closing. In the case of opening experiments, epidermal peels were prepared before the beginning of the light period, floated on 10 mM Kt-Mes (pH 6.1) containing 30 mM KCl, with or without the test chemical, and illuminated with 500 pmol m-2 s-l white light or maintained in the dark. Stomatal apertures were measured using an eyepiece micrometer. To reduce the variation in stomatal responses to externa1 stimuli due to differences in individual plants and cheinge in season, we used epidermal peels from a single lealf in each experiment and completed a set of experiments within a few weeks. Averages and SES from more than three separate experiments are presented. DMSO or ethanol used to dissolve the drugs did not exceed 0.5%, and control samples were always treated with the same concentratiions of the solvent.

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Actin Filaments in Mature Guard Cells 1079

To test drug effects on distribution of microfilaments,epidermal pieces were peeled before the beginning of thelight period, floated on a solution containing 10 HIM K+-Mes (pH 6.1), 30 mM KC1, and the drug, and illuminatedwith 500 jumol m~2 s"1 white light. After 90 min, immu-nohistochemistry procedures were performed as describedabove.

RESULTS

Distribution of Actin Filaments in Guard Cell Protoplasts

The thick cell wall of mature guard cells is a formidablebarrier and hindered visualization of microfilaments bypreventing actin antibodies and fluorescent phalloidinfrom penetrating to the cytoplasm. These led to our initialstudies, in which we digested the cell wall with cellulaseand produced guard cell protoplasts. Microfilaments werevisible only when the nuclei of isolated protoplasts main-tained normal shape, which indicated that the protoplastswere in a healthy condition. Two sets of microfilamentswere labeled with the actin antibody (Fig. 1): cortical mi-crofilaments, which formed a fine meshwork (Fig. 1A), andsubcortical microfilaments, which were thick and sparse(Fig. IB). Some of the microfilaments appeared to be con-tinuous from the nucleus to the plasma membrane byoptical sectioning. When normal mouse serum was used inplace of the primary antibody, no fluorescence wasobserved.

Distribution of Actin Filaments in Guard Cells

Even though protoplasts were derived from matureguard cells, because they lack a cell wall and thus any hintof polarized cell shape, they are not appropriate objects fordeciphering the spatial distribution and organization of thecytoskeletal elements in a functioning guard cell. Accord-ingly, we turned our attention to intact guard cells, apply-ing at first the method of intracellular microinjection ofFITC-phalloidin as a probe for microfilaments. Again thethickness of the cell wall and also the high turgor pressurewere difficult to overcome. However, in a few instances we

were successful and observed that the microfilaments ra-diated outward from the stomatal pore site (Fig. 2), thusclosely resembling the pattern of microtubules in develop-ing guard cells (Cho and Wick, 1989; Cleary and Hardham,1989; Marc et al., 1989). The nuclear area was also stainedintensely as in protoplasts (Fig. 2). Unfortunately, becauseof rapid photobleaching, we were unable to perform 3-Dimage reconstruction or to examine these cells throughcycles of opening and closing.

To confirm or reject the findings obtained on living cellswe applied immunofluorescence methods for detection ofmicrofilaments. However, those procedures that had beensuccessful for young guard cells (Cho and Wick, 1990) werenot suitable for the thick-walled mature guard cells. Long-term incubation of epidermal peels with cellulase, suffi-cient to permit entry of the antibodies, appeared to damagethe cells in ways that rendered them unsuitable for furtherstudy. Therefore, we rapidly froze the guard cells andphysically cracked them in the frozen state. Upon thawing,the interior of the cracked cells was freely permeable toactin antibodies (Fig. 3). The result confirmed the fan-shaped organization of microfilaments observed in the liv-ing guard cells microinjected with FITC-phalloidin. Rela-tively stable immunolabeling of microfilaments allowed usto obtain a series of optical sections in the z axis using theconfocal microscope. 3-D reconstruction from the series(Fig. 3) revealed that the fan-shaped array of microfila-ments was located close to the plasmalemma, and themicrofilaments were continuous all around the circumfer-ence of the guard cells, forming hoop-like structures. Nu-cleus, debris of chloroplasts, and other organelles remainedafter extraction and were also labeled with actin antibodies.Controls incubated with normal mouse serum did notshow any staining (data not shown). Subcortical microfila-ments observed in guard cell protoplasts were not clearlyshown by immunolocalization; most likely they were re-moved during the preparative procedures through thelarge cracks made for introduction of antibody. The loss ofthe subcortical microfilaments may explain the higher de-gree of radial directionality of microfilaments observed inthe immunostained guard cells than in microinjected ones.

Figure 1. Distribution of actin filaments in guard cell protoplasts of V. faba L. The protoplasts were immunostained withmouse actin antibody. In A the focus is on the cortical region, whereas in B it is on nucleus. The bar is 10 jim and appliesto A and B. www.plantphysiol.orgon August 18, 2020 - Published by Downloaded from



1080 Kirn et al. Plant Physiol. Vol. 109, 1995

Figure 2. Distribution of actin filaments in a living guard cell of C. communis L. microinjected with FITC-phalloidin. Theguard cell on the right was injected with FITC-phalloidin. The cell was photographed by bright-field (A) and fluorescence(B) microscopy. The arrow points to the injection site. The bar is 10 /im and applies to A and B.

Effects of Phalloidin and Cytochalasins on StomatalMovement

To determine whether guard cell microfilaments are in-volved in the regulation of stomatal opening and closing,we treated the epidermal tissue with the actin antagonistsphalloidin (Coluccio and Tilney, 1984) and CB or CD(Brown and Spudich, 1979; Flanagan and Lin, 1980; Lan-celle and Hepler, 1988) and examined the effect of eachdrug by measuring the size of the stomatal aperture.

Phalloidin inhibited stomatal closing induced by 10 JAMABA (Fig. 4A). The effect increased with increasing con-centrations of phalloidin and saturated at 0.1 mM. Thestomatal apertures 90 min after ABA treatment were 147%wider in 0.01 mM phalloidin, 184% wider in 0.1 mM phal-loidin, and 192% wider in 1 mM phalloidin, when com-pared with controls exposed to ABA alone (Fig. 4B). Whenthe epidermis was treated with phalloidin and then illumi-nated with white light, stomatal opening was inhibited(Fig. 5). At 150 min after phalloidin treatment, the stomatalaperture was 66% of that of the controls illuminated in theabsence of phalloidin.

CB (10 /j,g/mL), in contrast to phalloidin, increased light-induced stomatal opening to 165% compared to controlsafter 90 min. Even in the dark, stomata opened wide whentreated with CB (Fig. 6A). The effects of CD were verysimilar to those of CB; during 90 min of incubation in 200nM or higher CD, stomata opened to a 2-fold greater aper-ture size than the controls (Fig. 6B). CB and CD did notaffect the stomatal closing induced by ABA; stomata in thesample pretreated with Cytochalasins closed in 90 min, to adegree similar to those samples treated with ABA alone(data not shown).

Distribution of Actin Filaments after Drug Treatments

Immunofluorescence microscopy showed that phalloidintreatment led to the formation of regularly spaced densemicrofilaments (Fig. 7A). Chloroplasts and nuclei wereoften clearly delimited by brightly stained microfilaments.Indeed, because of the heavy staining, it was not possible,with the 3-D reconstructed images, to clearly discern finecytoplasmic microfilaments (Fig. 7B). CD treatment greatly

Figure 3. Distribution of actin filaments in guard eel Is of C. commun/sthat had been frozen and cracked. The actin filamentsin guard cells were stained by indirect immunofluorescence microscopy using an antibody to actin from chicken gizzard.The bar is 10 ju,m and applies to both photographs. www.plantphysiol.orgon August 18, 2020 - Published by Downloaded from



Actin Filaments in Mature Guard Cells

m

5 2 - t j -

O

1081

I - I - I ' I -

f- ............. .................. f

......... ...... .I 2.. ........i.." ... ......

O 30 60 90 120 t t

Phalloidin ABA Time (min)

Figure 4. Effect of phalloidin on ABA-induced stomatal closing. The solution that floated the epidermal pieces either contained or did not contain 0.1 mM phalloidin (A) and various concentrations of phalloidin (8). A: O, Control; O, ABA; O, phalloidin; B, ABA plus phalloidin. B: O, Control; O, ABA; O, 0.01 mM phalloidin plus ABA; A, 0.1 mM phalloidin plus ABA; O , 1 mM phalloidin plus ABA. Other experimental procedures are described in "Materials and Methods." Phalloidin plus ABA samples were different from ABA samples at P < 0.01 at t = 90 min (Student's t test).

O 4 60 120 180 240 300 Light

Time (min) Phalloidin

Figure 5. Effect of phalloidin on stomatal opening under white light. The solution that floated the epidermal tissue pieces contained either 0.1 m M phalloidin or ethanol (control), which was used to dissolve phalloidin. The final concentration of ethanol did not exceed 0.5%. O, Control; O, phalloidin. Other experimental procedures are described in "Materials and Methods." The phalloidin sample was different from the control at P < 0.01 at time = 150 and 270 min (Student's t test).

O

o ; . . I . . I . . I . . I

O 30 60 90 120 4 CD Time (min)

Figure 6. Effect of cytochalasins on stomatal aperture under dark- ness. The solution that floated the epidermal pieces contained either 10 pg/mL CB (A) or various concentrations of CD (B) or DMSO, which was used to dissolve cytochalasins. The final concentration of DMSO did not exceed 0.5%. The dark condition was maintained during the experiments. A: O, DMSO control; O, CB. B: O, DMSO control; O, 0.2 p~ CD; B, 2 p~ CD; O, 20 p~ CD. Other experimental procedures are described in "Materials and Methods." CB or CD samples were different from the control (DMSO) at P < 0.01 (Student's t test).

reduced the number of microfilaments, and when some remained, their regularity in the radial direction was reduced (Fig. 8A). The perinuclear region stained brightly with actin antibody, and sometimes there were a few, relatively thick microfilaments radiating out from the nuclear membrane (Fig. 88).

D I SC U SSI O N

The results reveal that mature guard cells of Commelina contain cortical microfilaments that radiate outward from the pore site. The strength of that conclusion derives from observations made by confocal laser scanning microscopy on two different cell preparations: (a) live, mature guard cells that had been microinjected with fluorescent phalloidin and (b) mature guard cells fixed with MBS, frozen, and physically cracked open to permit actin antibody staining. The similarity of the observations from these two indepen- dent methods gives us confidence that the radial distribution of microfilaments reflects the in vivo status of these elements in functioning guard cells. That these elements participate in the signal transduction events that control stomatal opening is revealed by the experiments with




Figure 7. Distribution of actin filaments in a guard cell of C. communis treated with 100 JJ.M phalloidin. A shows clearcortical arrays of actin filaments. B, The 3-D projection of this cell. The bar is 10 /im and applies to A and B.

agents that modify microfilament function. Both phalloi-din, which binds to F-actin and stabilizes these structures(Coluccio and Tilney, 1984), and cytochalasin, which insome systems may depolymerize F-actin (Brown and Spu-dich, 1979; Flanagan and Lin, 1980), whereas in otherscauses the formation of large aggregates of microfilaments(Lancelle and Hepler, 1988), clearly modify the ability ofthe guard cell to respond to environmental and hormonalcues. Thus, phalloidin inhibited ABA-induced closing andlight-induced opening, whereas cytochalasin stimulatedopening in both the light and dark. The partially oppositeeffects of the two agents support the specific effects ofphalloidin on microfilaments, unlike many general meta-bolic inhibitors and poisons, which also inhibit both open-ing and closing of stomata (Karlsson and Schwartz, 1988).When the structural and physiological data are consideredtogether, it seems clear that microfilaments participate inthe signal transduction processes that control stomatalaperture.

A matter that needs to be addressed concerns the use ofphalloidin as an indicator of microfilament distribution, onthe one hand, and its use as a modulator of microfilamentfunction on the other. The problem can be resolved whenconsidering the concentrations used. For microinjection,we estimate that the final concentration of phalloidin maybe 20 to 50 nM; in parallel studies of dividing cells, thisconcentration is too low to block dynamic rearrangementsof microfilaments or vital cellular processes such as celldivision and cytoplasmic streaming (Cleary et al., 1992;Zhang et al., 1993). Although the intracellular concentra-tion is not known in the studies using phalloidin as aninhibitor, with the cell being soaked in 0.1 mM, it is rea-sonable to suspect that concentrations well in excess of 50nM are attained. Under these conditions, it seems likelythat microfilaments are stabilized and that their ability tofunction normally is reduced.

One might have expected that microfilaments are radi-ally organized in guard cells given the fact that microtu-

Figure 8. Distribution of actin filaments in two pairs of guard cells of C. communis treated with 20 JJ.M CD. A, Only a sparsearray of actin filaments is evident in this 3-D projection. B, Examination of the nuclear area reveals an array of perinuclearactin filaments. The bars are 10 /urn. www.plantphysiol.orgon August 18, 2020 - Published by Downloaded from



Actin Filaments in Mature Guard Cells 1083

bules are known to be distributed in this manner (Cho and Wick, 1989; Cleary and Hardham, 1989; Marc et al., 1989) and of the likely possibility that microtubules and microfilaments co-align. However, microfilament patterns in rye guard cells, during the early stage of differentiation when they still assume a kidney shape, are distinct from those of microtubules; microfilaments are mainly parallel to the long axis of the cell, with an additional meshwork seen, whereas microtubules are already radially distributed (Cho and Wick, 1990). If this holds true in young Commelina guard cells as well, then microfilaments must be reorga- nized during differentiation to acquire the pattern found in mature guard cells. Microfilaments in guard cells, as in other cell types, may function to stabilize the specific pattern of microtubules (Kobayashi et al., 1988; refs. in Shibaoka and Nagai, 1994). In this way, microfilaments may participate together with cortical microtubules to control the orientation of cellulose deposition and, thus, during development modulate the processes that create the specialized shape of the mature guard cell.

Although it is likely that microfilaments participate in the differentiation of stomatal guard cells, the results herein support another role of microfilaments: the regulation of stomatal aperture. The questions then are how do they work and what are their specific functions? Given the highly reinforced cell wall of the mature guard cell, it seems unlikely that microfilaments are able to directly control the cell shape change during stomatal opening and closing and that abolishing microfilaments or destroying their ability to act would have any significant consequence on the operation of the stomata.

We favor the idea that microfilaments, presumably through actin-binding proteins, can directly modulate ion channels or modulate the signal transduction complexes, e.g. elements of the PI cycle, which in turn can control ion channels and the movement of solutes that underlies the control of the stomatal aperture. In both plant and animal cells, profilin, an actin monomer-binding protein, has high affinity for PIP,, a substrate of PLC, and inhibits the hy- drolysis of PIP, (Forscher, 1989; Drerbak et al., 1994). Thus, the actin-binding protein may regulate PI metabolism and the intracellular Ca2+ concentration, which in turn modu- lates many physiological phenomena in the cell. PLC is activated in guard cells exposed to ABA, suggesting that actin may regulate the stomatal movements using a mech- anism similar to that found in animal cells (Y.B. Choi, R.C. Crain, S.M. Assmann, and Y. Lee, unpublished results). In addition, microfilaments may directly regulate ion channels in the plasma membrane of guard cells. Supporting this possibility, the voltage-dependent inward K+ channel in guard cell plasma membrane was activated by cytochalasin treatment (J.-U. Hwang and Y. Lee, unpublished results). This change in ion channel activities would tend to increase the accumulation of Kt inside the cell, which fits well with the opening effect of cytochalasin (Fig. 6). There- fore, microfilaments may regulate stomatal movements by regulating ion channel activities, which has been shown in animal cells during cell volume regulation (Cantiello et al., 1993; Cornet et al., 1993). The fact that actin filaments

participate in the regulation of ion channel function is also shown by studies of caulonemata of the moss Funauia. Under normal conditions cytokinin, the natural effector of bud formation, causes inward currents to shift in position from the nuclear region to the dista1 end of the cell where the bud will form. However, CB prevents this shift in location of inward current, suggesting that microfilaments may participate in the control of distribution of plasma membrane-associated ion channels (Saunders, 1986).

Guard cell protoplasts have been widely used as a model system for investigation of the roles of ion transport in signal transduction of plants. Only healthy guard cell protoplasts, and none of the damaged ones, showed microfilaments. Moreover, the organization of the microfilaments in protoplasts was very different from that in intact cells (Figs. 1 and 3). This difference in the pattern of microfilaments may be a factor that caused the different ion transport characteristics observed in studies using guard cell protoplasts versus intact guard cells (Blatt, 1990).

In conclusion, it seems likely that microfilaments in guard cells interact with components that control ion movements. These may be direct or involve a signal transduction pathway, such as the PI cycle. The identification of these pathways and interacting factors will provide new clues for understanding signal transduction and/ or plant cell shape determination.

ACKNOWLEDCMENTS

We thank Dr. G. Wasteneys for the protocol for freeze-cracking plant tissue for immnnofluorescence study, Mr. Shi-In Kim for the management of plants, Ms. Jae-Ung Hwang for the graphs, and Mr. 11 Woo Lee for printing the pictures.

Received May 1, 1995; accepted July 27, 1995. Copyright Clearance Center: 0032-0889/95/ 109/1077/08.

LITERATURE CITED

Akiba S, Sato T, Fujii T (1993) Evidence for an increase in the association of cytosolic phospholipase A, with the cytoskeleton of stimulated rabbit platelets. J Biochem 113: 4-6

Assmann SM (1993) Signal transduction in guard cells. Annu Rev Cell Biol 9: 345-375

Blatt MR (1990) Potassium channel currents in intact stomatal guard cells: rapid enhancement by abscisic acid. Planta 180: 445455

Brown SS, Spudich JA (1979) Cytochalasin inhibits the rate of elongation of actin filament fragments. J Cell Biol 83: 657-662

Cantiello HF, Prat AG, Bonventre JV, Cunningham CC, Hartwig JH, Ausiello DA (1993) Actin-binding protein contributes to cell volume regulatory ion channel activation in melanoma cells. J Biol Chem 268: 4596-4599

Cho SO, Wick SM (1989) Microtubule orientation during stomatal differentiation in grasses. J Cell Sci 92: 581-594

Cho SO, Wick SM (1990) Distribution and function of actin in the developing stomatal complex of winter rye (Secale cerede cv. Puma). Protoplasma 157: 154-164

Cleary AL, Gunning BES, Wasteneys GO, Hepler PK (1992) Microtubule and F-actin dynamics at the division site in living Tradescantia stamen hair cells. J Cell Sci 103: 977-988

Cleary AL, Hardham AR (1989) Microtubule organization during development of stomatal complexes in Lolitlm rigidum. Proto- plasma 149: 67-81




Coluccio LM, Tilney LG (1984) Phalloidin enhances actin assem- bly by preventing monomer dissociation. J Cell Biol 99: 529-535

Cornet M, Lambert IH, Hoffman EK (1993) Relation between cytoskeleton, hypo-osmotic treatment and volume regulation in Ehrlich ascites tumor cells. J Membr Biol 131: 55-66

Drebak BK, Watkins PAC, Valenta R, Dove SK, Lloyd CW, Staiger CJ (1994) Inhibition of plant plasma membrane phosphoinositide phospholipase C by the actin-binding protein, profilin. Plant J 6: 389400

Flanagan MD, Lin S (1980) Cytochalasin block actin filament elongation by binding to high affinity sites associated with F-actin. J Biol Chem 255: 835-838

Forscher P (1989) Calcium and polyphosphoinositide control of cytoskeletal dynamics. Trends Neurosci 12: 468-474

Horvath AR, Muszbek L, Kellie S (1992) Translocation of pp6OC-"" to the cytoskeleton during platelet aggregation. EMBO J 11:

Karlsson PE, Schwartz A (1988) Characterization of the effects of metabolic inhibitors, ATPase inhibitors and a potassium-channel blockers on stomatal opening and closing in isolated epidermis of Commelina communis L. Plant Cell Environ 11: 165-172

Kobayashi H, Fukuda H, Shibaoka H (1988) Interrelation between the spatial deposition of actin filaments and microtubules during the differentiation of tracheary elements in cultured Zinnia cells. Protoplasma 143: 29-37

Kruse T, Tallman G, Zeiger E (1989) Jsolation of guard cell protoplasts from mechanically prepared epidermis of Vicia faba leaves. Plant Physiol 90: 1382-1386

Lancelle SA, Hepler PK (1988) Cytochalasin-induced ultrastruc- tural alterations in Nicotiana pollen tubes. Protoplasma S2: 65-75

Marc J, Mineyuki Y, Palevitz BA (1989) The generation and consolidation of a radial array of cortical microtubules in developing guard cells of Allium cepa L. Planta 179: 516-529

Payrastre B, Henegouwen PMPVBE, Breton M, Hartigh JC, Plan- tavid M, Verkleij AJ, Boonstra J (1991) Phosphoinositide kinase, diacylglycerol kinase, and phospholipase C activities associated to the cytoskeleton: effect of epidermal growth factor. J Cell Biol 115: 121-128

Putnam-Evans C, Harmon A, Palevitz BA, Fechheimer M, Cormier MJ (1989) Calcium-dependent protein kinase is local- ized with F-actin in plant cells. Cell Motil Cytoskel 12: 12-24

Sarndahl E, Bokoch GM, Stendahl O, Andersson T (1993) Stim- ulus-induced dissociation of a subunit of heterotrimeric GTP-

855-861

binding proteins from the cytoskeleton of human neutrophils. Proc Natl Acad Sci USA 90: 6552-6556

Saunders MJ (1.986) Cytokinin activation and redistribution of plasma membrane ion channels in Funaria. Planta 167: 402-409

Shibaoka H, Nagai R (1994) The plant cytoskeleton. Curr Opin Cell Biol 6: 10-15

Sohn RH, Goldschmidt-Clermont PJ (1994) Profilin: at the cross- roads of signal transduction and the actin cytoskeleton. 13ioes- says 16: 465472

Staiger CJ, Goodbody KC, Hussey PJ, Valenta RD, Drebalk BK, Lloyd CW (1993) The profilin multigene family of maize: differ- ential expression of three isoforms. Plant J 4: 631-641

Tan Z, Boss WF (1992) Association of phosphatidylinositol kinase, phosphatidylinositol monophosphate kinase, and diacylglycerol kinase with the cytoskeleton and F-actin fractions of carrot (Daucus carotu L.) cells grown in suspension culture. Plant Physiol 100: 2116-2120

Valenta R, Duchene M, Pettenburger K, Sillaber C, Valtmt P, Bettelheim P, Breitenbach M, Rumpold H, Kraft D, Scheiner O (1991) Identification of profilin as a nove1 pollen allergen; IgE autoreactivity in sensitized individuals. Science 109:

Vaziri C, Downes CP (1992) Association of a receptor artd G- protein-regulated phospholipase C with the cytoskeleton. J Biol Chem 267: 22973-22981

Wasteneys GO, Collings DA, Gunnings BES, Hepler PK, Menzel D (1996) Actin in living and fixed characean internodal cells: identification of a cortical array of fine actin strands and chlo- roplast actin rings. Protoplasma 190: (in press)

Xu P, Lloyd CW, Staiger CJ, Drebak BK (1992) Association of phosphatidylinositol4-kinase with the plant cytoskeleton. Plant Cell 4: 941-951

Yang W, Boss WF (1994) Regulation of phosphatidylinositol 4-kinase by the protein activator PIK-A49. J Biol Chem 269: 3852- 3857

Zhang DH, Wadsworth P, Hepler PK (1990) Microtubule dynamics in living dividing plant cells: confocal imaging of microinjected fluorescent brain tubulin. Proc Natl Acad Sci US.4 87:

Zhang DH, Wadsworth P, Hepler PK (1993) Dynamics of microfilaments are similar, but distinct from microtubules during cytokinesis in living, dividing plant cells. Cell Motil Cytoskele- ton 24: 151-155

619-626

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actin filaments in mature guard cells are radially ... · cyanate-phalloidin into living guard...

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