Thermotolerant guard cell protoplasts of tree tobacco do not require exogenous hormones to survive in culture and are blocked from reentering the cell cycle at the G1-to-S transition

When guard cell protoplasts (GCPs) of tree tobacco [Nicotiana glauca (Graham)] are cultured at 32 degrees C with an auxin (1-napthaleneacetic acid) and a cytokinin (6-benzylaminopurine), they reenter the cell cycle, dedifferentiate, and divide. GCPs cultured similarly but at 38 degrees C and with 0.1 micro M +/- -cis,trans-abscisic acid (ABA) remain differentiated. GCPs cultured at 38 degrees C without ABA dedifferentiate partially but do not divide. Cell survival after 1 week is 70% to 80% under all of these conditions. In this study, we show that GCPs cultured for 12 to 24 h at 38 degrees C accumulate heat shock protein 70 and develop a thermotolerance that, upon transfer of cells to 32 degrees C, enhances cell survival but inhibits cell cycle reentry, dedifferentiation, and division. GCPs dedifferentiating at 32 degrees C require both 1-napthaleneacetic acid and 6-benzylaminopurine to survive, but thermotolerant GCPs cultured at 38 degrees C +/- ABA do not require either hormone for survival. Pulse-labeling experiments using 5-bromo-2-deoxyuridine indicate that culture at 38 degrees C +/- ABA prevents dedifferentiation of GCPs by blocking cell cycle reentry at G1/S. Cell cycle reentry at 32 degrees C is accompanied by loss of a 41-kD polypeptide that cross-reacts with antibodies to rat (Rattus norvegicus) extracellular signal-regulated kinase 1; thermotolerant GCPs retain this polypeptide. A number of polypeptides unique to thermotolerant cells have been uncovered by Boolean analysis of two-dimensional gels and are targets for further analysis. GCPs of tree tobacco can be isolated in sufficient numbers and with the purity required to study plant cell thermotolerance and its relationship to plant cell survival, growth, dedifferentiation, and division in vitro.     

The effect of Cl- upon the sensitivity of starch-containing and starch-deficient stomata and guard cell protoplasts towards potassium ions,fusicoccin and abscisic acid

Chloride ions are necessary to compensate for the positively charged potassium ions imported into guard cells of Allium cepa L. during stomatal opening. Therefore an external Cl^- supply of intact Allium plants is important. But high levels of chloride have been found to reduce the sensitivity of the starch-lacking stomata and isolated guard cell protoplasts (GCPs) from Allium to potassium ions, fusicoccin and abscisic acid. Furthermore, with high levels of chloride, malate anions disappear from the guard cells of Allium, a finding which contrasts with situation in Vicia where the stomatal sensitivity to K⁺ ions, fusicoccin and ABA is not influenced by Cl^- ions and malate levels are unaffected. It is suggested that the absence of malate as a proton yielding primer inhibits the mechanism of H⁺/K⁺ exchange in Allium.Abbreviations ABA abscisic acid - FC fusicoccin - GCPs guard cell protoplasts     

Heat reduces nitric oxide production required for auxin-mediated gene expression and fate determination in tree tobacco guard cell protoplasts

Tree tobacco (Nicotiana glauca) is an equatorial perennial with a high basal thermotolerance. Cultured tree tobacco guard cell protoplasts (GCPs) are useful for studying the effects of heat stress on fate-determining hormonal signaling. At lower temperatures (32°C or less), exogenous auxin (1-naphthalene acetic acid) and cytokinin (6-benzylaminopurine) cause GCPs to expand 20- to 30-fold, regenerate cell walls, dedifferentiate, reenter the cell cycle, and divide. At higher temperatures (34°C or greater), GCPs expand only 5- to 6-fold; they do not regenerate walls, dedifferentiate, reenter the cell cycle, or divide. Heat (38°C) suppresses activation of the BA auxin-responsive transgene promoter in tree tobacco GCPs, suggesting that inhibition of cell expansion and cell cycle reentry at high temperatures is due to suppressed auxin signaling. Nitric oxide (NO) has been implicated in auxin signaling in other plant systems. Here, we show that heat inhibits NO accumulation by GCPs and that l-N(G)-monomethyl arginine, an inhibitor of NO production in animals and plants, mimics the effects of heat by limiting cell expansion and preventing cell wall regeneration; inhibiting cell cycle reentry, dedifferentiation, and cell division; and suppressing activation of the BA auxin-responsive promoter. We also show that heat and l-N(G)-monomethyl arginine reduce the mitotic indices of primary root meristems and inhibit lateral root elongation similarly. These data link reduced NO levels to suppressed auxin signaling in heat-stressed cells and seedlings of thermotolerant plants and suggest that even plants that have evolved to withstand sustained high temperatures may still be negatively impacted by heat stress.     

Responses of Commelina communis L. Guard Cell Protoplasts to Abscisic Acid

Fitzsimons, P. J. and Weyers, J. D. B. 1987. Responses of Commelinacommunis L. guard cell protoplasts to abscisic acid.—J.exp. Bot. 38: 992–1001. Guard cell protoplasts (GCPs) isolated from the leaf epidermisof Commelina communis L. responded to abscisic acid (ABA) ina manner which was qualitatively and quantitatively similarto that of intact stomata. ABA inhibited swelling of GCPs underlow-CO₂ conditions and swollen GCPs responded to the hormoneby shrinking. Both the absolute volume decrease and the initialrate of shrinking were commensurate with the extent and ratesof solute loss computed for ABA-treated intact, open stomata.This indicates that GCPs represent a suitable experimental systemfor studies of ABA-mediated solute fluxes. A radiotracer equilibrationmethod was developed for the rapid estimation of GCP osmoticvolume changes. Using this technique it was found that, on average,82% of the reduction in solute content caused by ABA treatmentwas due to the loss of K⁺. It is envisaged that electroneutralitymight be maintained during ABA-induced shrinkage of GCPs bynet inward proton movement leading to acidification of the vacuole. Key words: Abscisic acid, Commelina communis L., guard cells, protoplasts     

Immunohistochemical detection of blue light-induced phosphorylation of the plasma membrane H+-ATPase in stomatal guard cells

Blue light (BL) receptor phototropins activate the plasma membrane H(+)-ATPase in guard cells through phosphorylation of a penultimate threonine and subsequent binding of the 14-3-3 protein to the phosphorylated C-terminus of H?-ATPase, mediating stomatal opening. To date, detection of the phosphorylation level of the guard cell H?-ATPase has been performed biochemically using guard cell protoplasts (GCPs). However, preparation of GCPs from Arabidopsis for this purpose requires >5,000 rosette leaves and takes >8 h. Here, we show that BL-induced phosphorylation of guard cell H?-ATPase is detected in the epidermis from a single Arabidopsis rosette leaf via an immunohistochemical method using a specific antibody against the phosphorylated penultimate threonine of H?-ATPase. BL-induced phosphorylation of the H?-ATPase was detected immunohistochemically in the wild type, but not in a phot1-5 phot2-1 double mutant. Moreover, we found that physiological concentrations of the phytohormone ABA completely inhibited BL-induced phosphorylation of guard cell H?-ATPase in the epidermis, and that inhibition by ABA in the epidermis is more sensitive than in GCPs. These results indicate that this immunohistochemical method is very useful for detecting the phosphorylation status of guard cell H?-ATPase. Thus, we applied this technique to ABA-insensitive mutants (abi1-1, abi2-1 and ost1-2) and found that ABA had no effect on BL-induced phosphorylation in these mutants. These results indicate that inhibition of BL-induced phosphorylation of guard cell H?-ATPase by ABA is regulated by ABI1, ABI2 and OST1, which are known to be early ABA signaling components for a wide range of ABA responses in plants.     

The Investigation of Stomatal Ionic Relations using Guard Cell Protoplasts: II. OSMOTIC RELATIONS OF GUARD CELL PROTOPLASTS IN SHORT AND LONG-TERM INCUBATION

Clint, G. M. 1985. The investigation of stomatal ionic relationsusing guard cell protoplasts. II. Osmotic relations of guardcell protoplasts in short and long-term incubation.—J.exp. Bot 36: 1739–1748 Measurements were made of the volume changes exhibited by isolatedguard cell protoplasts (GCPs) of Commelina communis L, whenexposed to a range of concentrations of external osmotica Inshort-term incubation, GCPs behaved as osmometers and showedrapid volume changes in response to changing external osmoticpressure (₀). In long-term incubation, GCPs prepared and incubatedwith added external KCl showed further slow changes in volume,in a manner suggesting that regulation of volume occurred. Protoplastsprepared and incubated without added external KCl had smallervolumes for a given value of ₀, and their ability to regulatevolume in long-term incubation was reduced or absent. Treatment with fusicoccin caused an increase in both the volumeand the K⁺ content of GCPs. The increase in volume continuingafter the increase in K⁺ content had ceased, in a manner similarto that observed in walled guard cells in epidermal strips. Key words: Guard cell protoplasts, volume regulation, Commelina communis     

High Respiratory Activity of Guard Cell Protoplasts from Vicia faba L.

The rate of O₂ uptake was about 29 times higher in guard cellprotoplasts (GCPs) than in mesophyll protoplasts (MGPs) on aChi basis. The O₂ uptake was inhibited by respiratory inhibitors,but stimulated by respiratory uncouplers. On a Chi basis, theactivities of Cyt c oxidase and NADH-Cyt c reductase, mitochondrialenzymes, were about 27 and 35 times higher in GCPs than in MCPs.On a Chi basis, the ATP content was about 9 times higher inGCPs. The amount of ATP in GCPs was decreased by respiratoryinhibitors, an energy transfer inhibitor, and uncouplers ofoxidative phosphorylation. On a volume basis, GCPs had 8- to10-fold higher respiratory activities than MCPs, but had a lowChi content and lacked the activity of NADP-glyceraldehyde-3-phosphatedehydrogenase (NADP-GAPD), the Calvin cycle enzyme. From theseresults, we concluded that oxidative phosphorylation plays amain role in ATP production in guard cells and that guard cellshave a heterotrophic feature. Salicylhydroxamic acid (SHAM)in combination with KCN or NaN₃ strongly inhibited O₂ uptake,indicating the presence of cyanide-resistant respiration inguard cells. Phenylmercuric acetate (PMA), a potent inhibitorof stomatal opening, reduced the ATP content of GCPs by about90%, whereas it had a relatively small effect on the ATP levelof MCPs. The specific effect of PMA on GCPs is discussed. (Received March 24, 1983; Accepted June 8, 1983)     

Studies on isolated starch-containing (Vicia faba) and starch-deficient (Allium cepa) guard cell protoplasts

Guard cell protoplasts from starch-containing Vicia faba and starch-deficient Allium cepa stomata were isolated, stabilized and recovered with an efficiency — in relation to the potential yield — of approx. 62% and 77%, respectively. In vitro, guard cell protoplasts (GCP) respond to abscisic acid and fusicoccin by respectively contracting and swelling, that is, decreasing or increasing in diameter by about 15% and more in comparison to the control. This in vitro response correlates with, but is more than 4 times as rapid as, the in vivo response of the stomata. Among the advantages presented by working with isolated GCPs are: greater sensitivity in response; freedom from influences of cuticular ridges, cell walls, subsidiary cells, and epidermal cells; and direct and parallel comparisons of starch-containing and starch-deficient GCP systems.Abbrecviations ABA abscisic acid - FC fusicoccin - ECP, MCP, and GCP epidermal, mesophyll, and guard cell protoplasts, respectively - PPV packed protoplast volume     

Crosstalk between blue-light- and aba-signaling pathways in stomatal guard cells

We recently established an immunohistochemical method for the detection of blue light (BL)-induced and phototropin-mediated phosphorylation of plasma-membrane H⁺-ATPase in stomatal guard cells of Arabidopsis thaliana. This technique makes it possible to detect the phosphorylation/activation status of guard-cell H⁺-ATPase in the epidermis of a single rosette leaf, without the need to prepare guard-cell protoplasts (GCPs) from a large number of plants. Moreover, it can detect guard-cell responses under more natural and stress-free conditions compared to using GCPs. Taking advantage of these properties, we examined the effect of abscisic acid (ABA) on BL-induced phosphorylation of guard-cell H⁺-ATPase by using ABA-insensitive mutants. This revealed inhibition of BL-induced phosphorylation of guard-cell H⁺-ATPase via the early ABA-signaling components PYR/PYL/RCAR-PP2Cs-SnRK2s, which are known to be early ABA-signaling components for a wide range of ABA responses in plants.      

Light-Induced De-Epoxidation of Violaxanthin in Guard Cell Protoplasts of Vicia faba

Photosynthetic pigments of Vicia guard cell protoplasts (GCPs)from abaxial epidermis were analyzed by reverse-phase HPLC.Violaxanthin decreased and zeaxanthin increased in GCPs afterlight illumination. The epoxidation state of GCPs decreasedfrom 0.82 (dark) to 0.37 (light), suggesting operation of thexanthophyll cycle in GCPs of Vicia faba. (Received March 15, 1993; Accepted May 10, 1993)     

The responses of guard and mesophyll cell photosynthesis to CO2, O2, light,and water stress in a range of species are similar

High resolution chlorophyll a fluorescence imaging was used to compare the photosynthetic efficiency of PSII electron transport (estimated by Fq'/Fm') in guard cell chloroplasts and the underlying mesophyll in intact leaves of six different species: Commelina communis, Vicia faba, Amaranthus caudatus, Polypodium vulgare, Nicotiana tabacum, and Tradescantia albifora. While photosynthetic efficiency varied between the species, the efficiencies of guard cells and mesophyll cells were always closely matched. As measurement light intensity was increased, guard cells from the lower leaf surfaces of C. communis and V. faba showed larger reductions in photosynthetic efficiency than those from the upper surfaces. In these two species, guard cell photosynthetic efficiency responded similarly to that of the mesophyll when either light intensity or CO2 concentration during either measurement or growth was changed. In all six species, reducing the O2 concentration from 21% to 2% reduced guard cell photosynthetic efficiency, even for the C4 species A. caudatus, although the mesophyll of the C4 species did not show any O2 modulation of photosynthetic efficiency. This suggests that Rubisco activity is significant in the guard cells of these six species. When C. communis plants were water-stressed, the guard cell photosynthetic efficiency declined in parallel with that of the mesophyll. It was concluded that the photosynthetic efficiency in guard cells is determined by the same factors that determine it in the mesophyll.     

Epiplastome variation of the number of chloroplasts in stomata guard cells of sugar beet (Beta vulgaris L.)

In stomata guard cells of sugar beet, variation in the number of chloroplasts was studied in successive generations: (1) hybrid generation; (2) generation yielded by uniparental apozygotic seed reproduction; (3) generation obtained after seed treatment with a colchicine solution; (4) generation obtained after seed treatment with 5-azacytidine. As compared to hybrid generation, uniparental seed reproduction increases the average number of chloroplasts in stomata guard cells (from 13.5 to 15.0) and decreases distribution variance of this trait by a factor of 3 to 4. Colchicine increases both average number of chloroplasts in stomata guard cells (from 13.5 to 18.2) and distribution variance (about twice). 5-Azacytindine reduces the number of chloroplasts in cells (from 15.0 to 12.9) but enhances distribution variance (about 1.5 times). Variation in the number of chromosomes in stomata cells is related to myxoploidy in meristem tissue, on the one hand, and to the rate of cell division, on the other. Uniparental seed reproduction is suggested to enhance the number of organelles per cell due to high myxoploidy in cell populations, which is typical of inbred plants. Colchicine blocks spindle division and sharply increases the level of myxoploidy in cell populations and the number of organelles per cell. 5-Azacytidine hypomethylates chromosome DNA, increases the rate of cell divisions, and reduces the number of organelles per cell. The described changes in the number of chloroplasts are inherited in cell lineage ("cell hereditary memory") and successive sporophyte generations.     

Involvement of intracellular Ca2+ in blue light-dependent proton pumping in guard cell protoplasts from Vicia faba

Recent studies have suggested that Ca²⁺/calmodulin (CaM) or CaM-like proteins may be involved in blue light (BL)-dependent proton pumping in guard cells. As the increase in cytosolic concentration of Ca²⁺ is required for the activation of CaM and CaM-like proteins, the origin of the Ca²⁺ was investigated by measuring BL-dependent proton pumping with various treatments using guard cell protoplasts (GCPs) from Vicia faba . BL-dependent proton pumping was affected neither by Ca²⁺ channel blockers nor by changes of Ca²⁺ concentration in the medium used for the GCPs. Addition of Ca²⁺ ionophores and an agonist to GCPs did not induce proton pumping. However, BL-dependent proton pumping was inhibited by 10 m M caffeine, which releases Ca²⁺ from the intracellular stores, and by 10 μ M 2,5-di-( tert -butyl)-1,4-benzohydroquinone (BHQ) and 10 μ M cyclopiazonic acid (CPA), inhibitors of Ca²⁺-ATPase in the sarcoplasmic and endoplasmic reticulum (ER). By contrast, the inhibitions were not observed by 10 μ M thapsigargin, an inhibitor of animal ER-type Ca²⁺-ATPase. The inhibitions by caffeine and BHQ were reversible. Light-dependent stomatal opening in the epidermis of Vicia was inhibited by caffeine, BHQ, and CPA. From these results, we conclude that the Ca²⁺ thought to be required for BL-dependent proton pumping may originate from intracellular Ca²⁺ stores, most likely from ER in guard cells, and that this origin of Ca²⁺ may generate a stimulus-specific Ca²⁺ signal for stomatal opening.     

Control of leaf cell elongation in barley. Generation rates of osmotic pressure and turgor, and growth-associated water potential gradients

In a previous study on the effects of N-supply on leaf cell elongation, the spatial distribution of relative cell elongation rates (RCER), epidermal cell turgor, osmotic pressure (OP) and water potential (Ψ) along the elongation zone of the third leaf of barley was determined (W. Fricke et al. 1997, Planta 202: 522–530). The results suggested that in plants receiving N at fixed relative addition rates (N-supply limitation of growth), cell elongation was rate-limited by the rate of solute provision, whereas in plants growing on complete nutrient solution containing excessive amounts of N (N-demand limitation), cell elongation was rate-limited by the rate of water supply or wall yielding. In the present paper, these suggestions were tested further. The generation rates of cell OP, turgor and Ψ along the elongation zone were calculated by applying the continuity equation of fluid dynamics to the previous data. To allow a more conclusive interpretation of results, anatomical data were collected and bulk solute concentrations determined. The rate of OP generation generally exceeded the rate of turgor generation. As a result, negative values of cell Ψ were created, particularly in demand-limited plants. These plants showed highest RCER along the elongation zone and a Ψ gradient of at least −0.15 MPa between water source (xylem) and expanding epidermal cells. The latter was similar to a theoretically predicted value (−0.18 MPa). Highest rates of OP generation were observed in demand-limited plants, with a maximum rate of 0.112 MPa · h⁻¹ at 16–20 mm from the leaf base. This was almost twice the rate in N-supply-limited plants and implied that the cells in the leaf elongation zone were capable of importing (or synthesising) every minute almost 1 mM of osmolytes. Potassium, Cl⁻ and NO₃ ⁻ were the main inorganic osmolytes (only determined for demand-limited plants). Their concentrations suggest that, unlike the situation in fully expanded epidermal cells, sugars are used to generate OP and turgor. Anatomical data revealed that the zone of lateral cell expansion extended distally beyond the zone of cell elongation. It is concluded that leaf cell expansion in barley relies on high rates of water and solute supply, rates that may not be sustainable during periods of sufficient N-supply (limitation by water supply: Ψ gradients) or limiting N-supply (limitation by solute provision: reduced OP-generation rates). To minimise the possibility of growth limitation by water and osmolyte provision, longitudinal and lateral cell expansion peak at different locations along the growth zone. Received: 15 October 1997 / Accepted: 12 March 1998     

Plant cell suspension culture in a bench-scale fermenter with a newly designed membrane stirrer for bubble-free aeration

In this paper, tests of an optimized membrane-stirrer geometry for bubble-free aeration of a plant cell suspension culture are described. Cell attachment and clogging of a previously described system [Piehl et al. (1988) Appl Microbiol Biotechnol 29:456–461] led to the development of a new stirrer. The volumetric oxygen transfer capacity has been measured in aqueous medium. The mass transfer coefficient, k _l a, was 3.75 h⁻¹ at 25 °C and at a stirrer speed of 34 rpm. The overall oxygen transfer capacity was investigated with a suspension culture of Aesculus hippocastanum. It was shown that the oxygen mass transfer was sufficient even at the maximum biomass of 10–12 g dry weight/l, which was obtained by using this system. Furthermore, special attention was given to medium components like C and N sources, to avoid growth limitation due to a shortage of nutrients. Received: 22 October 1996 / Revised version: 11 March 1997 / Accepted: 14 March 1997     

New approach of monitoring changes in chlorophyll a fluorescence of single guard cells and protoplasts in response to physiological stimuli

A new type of microfluorometer was applied to assess photosynthesis at the single-cell level by chlorophyll fluorescence using the saturation pulse method. A microscopy–pulse amplitude modulation (PAM) chlorophyll fluorometer was combined with a Zeiss Axiovert 25 inverted epifluorescence microscope for high-resolution measurements on single mesophyll and guard cells and the respective protoplasts. Available information includes effective quantum yield of photosystem II, relative electron transport rate and energization of the thylakoid membrane due to the transthylakoidal proton gradient. Dark–light induction curves of guard cell (GCPs) and mesophyll cell protoplasts (MCPs) displayed very similar characteristics, indicating similar functional organization of thylakoid membranes in both types of chloroplasts. Light response curves, however, revealed much earlier saturation of photosynthetic electron flow in GCPs than in MCPs. Under anaerobiosis, photosynthetic electron flow and membrane energization were severely suppressed. A similar effect was observed in guard cells when epidermal peels were incubated with the fungal toxin fusicoccin which activates the plasma membrane H⁺-ATPase and causes irreversible opening of stomata. The drop in electron transport rate was prevented by blocking ATP consumption of the H⁺ pump or by glucose addition. These results show that chlorophyll fluorescence quenching analysis allows profound insights into stomatal physiology.     

Visualization of abscisic acid-perception sites on the plasma membrane of stomatal guard cells

Abscisic acid (ABA) is a phytohormone that plays a key role as a stress signal, regulating water relations during drought conditions, by inducing stomatal closure. However, to date, no putative ABA receptor(s) has been reported at the protein sequence, gene family, or cellular localization levels. We used biotinylated ABA (bioABA) to characterize the ABA-perception sites in the stomatal guard cells of Vicia faba. Treatment with bioABA induced stomatal closure and shrinkage of guard cell protoplasts (GCPs). The ABA-perception sites were visualized by fluorescence microscopy and confocal laser scanning microscopy (CLSM), using bioABA and fluorescence-labeled avidin. Fluorescent particles were observed in patches on the surface of the GCPs. Fluorescence intensity was quantified by flow cytometry (FCM) as well as by CLSM. Binding of bioABA was inhibited by ABA in a dose-dependent manner. Pre-treatment of GCPs with proteinase K also blocked the binding of bioABA. Binding of bioABA was inhibited by RCA-7a, an ABA analog that induces stomatal closure, but not by RCA-16, which has no effect on stomatal aperture. Another ABA analog, PBI-51, inhibited ABA-induced stomatal closure. This ABA antagonist also inhibited binding of bioABA to the GCPs. These results suggest that ABA is perceived on the plasma membrane of stomatal guard cells, and that the present experimental methods constitute valuable tools for characterizing the nature of the ABA receptor(s) that perceives physiological ABA signals. These imaging studies allow us to demonstrate the spatial distribution of the ABA-perception sites. Visualization of the ABA-perception sites provides new insights into the nature of membrane-associated ABA receptor(s).     

Performance of a membrane-dialysis bioreactor with a radial-flow fixed bed for the cultivation of a hybridoma cell line

A bioreactor system for the continuous cultivation of animal cells with a high potential for scale-up is presented. This reactor system consists of radial-flow fixed-bed units coupled with a dialysis module. The dialysis membrane enables the supply of low-molecular-weight nutrients and removal of toxic metabolites, while high-molecular-weight nutrients and products (e.g. monoclonal antibodies) are retained and accumulated. This concept was investigated on the laboratory scale in a bioreactor with an integrated dialysis membrane. The efficiency of the reactor system and the reproducibility of the cell activity (hybridoma cells) under certain process conditions could be demonstrated in fermentations up to 77 days. Based on model calculations, an optimized fermentation strategy was formulated and experimentally confirmed. Compared to chemostat cultures with suspended cells, a ten-times higher mAb concentration (383 mgl⁻¹) could be obtained. The highest volumetric specific mAb production rate determined was 6.1 mg mAb (1 fixed bed)⁻¹ h⁻¹.     

Immuno-reactant to RhoA antibody co-localizes with cortical actin filaments in guard cells of open stomata inCommelina communis L.

Stomatal regulation is essential for the growth of land plants. Pairs of guard cells that delineate the stomata perceive stimuli and respond to acquire the optimum aperture. The actin cytoskeleton participates in signaling pathways of the guard cell (Kim et al., 1995; Eun and Lee, 1997; Hwang et al., 1997). To identify the upstream molecules that regulate actin dynamics in plant cells, we immunoblotted proteins extracted from leaves ofCommelina commuais L. with the RhoA antibody, and identified one band of 26KD from the epidermis. Using immunofluorescence microscopy, we examined the subcellular distribution of the immuno-reactant(s) in guard cells. When stomata were open under light, the organization of the immuno-reactant(s) resembled the radial arrangement of cortical actin filaments of guard cells. Double-labeling of the guard cells, using the RhoA and actin antibodies as primary antibodies, showed that the immuno-reactant(s) of the RhoA antibody and actin filaments co-localized in the cortex of illuminated guard cells. However, the pattern was not found in guard cells when stomata were closed under darkness or by ABA, conditions under which cortical actin proteins are disassembled in guard cells. From these observations, we can suggest the possible presence of a RhoA-like protein and its involvement in the organization of the actin cytoskeleton in guard cells.