Osmotically Induced Water Stress, Potassium Uptake, and Stomatal Aperture in Epidermal Strips of Vicia faba L.

Uptake of potassium ions, estimated microdensitometrically,and stomatal opening in isolated epidermal strips from Viciafaba L. cv. Aquadulce were both inhibited by incubation of thestrips in solutions of low solute potential. The inhibitionof potassium uptake was substantially reduced by preincubationof the strips in benzylaminopurine¹ solution. The data are discussedin terms of the induction of abscisic acid synthesis by waterstress in the epidermal strips and the antagonism to its effectsby BAP.     

Mutual Antagonism of Sulfur Dioxide and Abscisic Acid in Their Effect on Stomatal Aperture in Broad Bean (Vicia faba L.) Epidermal Strips

Abscisic acid (ABA) was found to counteract the stomatal opening in Vicia faba L. caused by SO₂. The antagonism between SO₂ and ABA was mutual, and their combined effect depended upon which compound was in the greatest concentration. Stomatal apertures were monitored in detached epidermal strips floated in the light on aqueous solutions of SO₂ (sulfurous acid) and/or ABA in 0.01 molar sodium citrate buffer (pH 5.8). Low concentrations of sulfurous acid (10⁻¹⁰ to 10⁻⁷ molar) increased stomatal aperture, but concentrations greater than 10⁻⁵ molar decreased it. A progressive decrease in aperture size occurred as ABA was increased from 10⁻¹⁰ to 10⁻⁵ molar.     

Stomatal Opening in Isolated Epidermal Strips of Vicia faba. II. Responses to KCl Concentration and the Role of Potassium Absorption

The stimulation by KCl of stomatal opening in isolated epidermal strips of Vicia faba was examined. In dark + normal air the opening response was maximal at 100 mm KCl while in light + CO₂-free air it was maximal at about 10 mm KCl. CO₂-free air was more influential than light in reducing the KCl concentration required for maximal opening. K⁺ was essential while Cl⁻ seemed to be of secondary importance in these processes.     

Circadian Stomatal Rhythms in Epidermal Peels from Vicia faba

Circadian rhythms in stomatal aperture and in stomatal conductance have been observed previously. Here we investigate circadian rhythms in apertures that persist in functionally isolated guard cells in epidermal peels of Vicia faba, and we compare these rhythms with rhythms in stomatal conductance in attached leaves. Functionally isolated guard cells kept in constant light display a rhythmic change in aperture superimposed on a continuous opening trend. The rhythm free-runs with a period of about 22 hours and is temperature compensated between 20 and 30°C. Functionally isolated guard cell pairs are therefore capable of sustaining a true circadian rhythm without interaction with mesophyll cells. Stomatal conductance in whole leaves displays a more robust rhythm, also temperature-compensated, and with a period similar to that observed for the rhythm in stomatal aperture in epidermal peels. When analyzed individually, some stomata in epidermal peels showed a robust rhythm for several days while others showed little rhythmicity or damped out rapidly. Rhythmic periods may vary between individual stomata, and this may lead to desynchronization within the population.     

Acid-Induced Stomatal Opening in Commelina communis and Vicia faba

The effects of H^$ and fusicoccin (FC) on stomatal opening inthe dark were investigated using epidermal strips of Commelinacommunis and Vicia faba cv. Ryosai Issun. Citrate-phosphatebuffer induced maximal opening of stomata at pH 3.0 when testedover the range of 2.7 to 5.0. HCl at 1 mM also induced stomatalopening without appreciable accumulation of K^$ in the guardcells. After 4 hr treatment with 10 µM FC, stomata openedwith concomitant accumulation of K^$ in the guard cells, although1–2 hr treatment caused opening without concomitant K^$increase. These results suggest that stomatal opening can be caused bysalt accumulation and/or changes of the physicochemical conditionsin the cell wall of the guard cells due to high acidity. ¹ Present address: Biological Laboratory, Faculty of Education,Nagasaki University, Nagasski 852, Japan. (Received April 30, 1982; Accepted July 17, 1982)     

Effect of Colored Light on Stomatal Opening Rates of Vicia faba L

The average opening rate of Vicia faba L. stomata was determined over an initial 20-minute light period following darkness. Nonsaturating intensities of broad band red and blue light had similar quantum effectiveness for the promotion of opening, whereas broad band green was about 40% and far red about 5% as effective. The opening rates under saturating red, green, and blue light were the same. Net photosynthesis was measured under various intensities of the same red, green, and blue light spectra. Red and blue light were equally efficient in causing photosynthesis, whereas green was 60% as effective. The light compensation points for the three colors were at higher intensities than those which saturated the opening rate response. These data suggest that only a single pigment system, probably the photosynthetic pigments, is responsible for initiating the light-induced opening response in V. faba stomata.     

Effect of Photoperiod on Stomatal Opening in Vicia faba

Stomatal apertures in darkness and subsequent average opening rates in light were measured in Vicia faba leaf discs throughout the nyctoperiods for plants grown on three light:dark cycles (8:16, 12:12, and 16:8). The time course of opening in darkness depended on the specific light:dark cycle with the maximum aperture always occurring at the time the lights normally went on. The light-induced opening rate was also maximum at the end of the nyctoperiod.     

Stomatal Opening in Isolated Epidermal Strips of Vicia faba. I. Response to Light and to CO(2)-free Air

This paper reports a consistent and large opening response to light + CO₂-free air in living stomata of isolated epidermal strips of Vicia faba. The response was compared to that of non-isolated stomata in leaf discs floating on water; stomatal apertures, guard cell solute potentials and starch contents were similar in the 2 situations. To obtain such stomatal behavior, it was necessary to float epidermal strips on dilute KCl solutions. This suggests that solute uptake is necessary for stomatal opening.

The demonstration of normal stomatal behavior in isolated epidermal strips provides a very useful system in which to investigate the mechanism of stomatal opening. It was possible to show independent responses in stomatal aperture to light and to CO₂-free air.

     

Calcium Inhibits Ion-Stimulated Stomatal Opening in Epidermal Strips of Commelina communis L.

Inoue, H. and Katoh, Y. 1987. Calcium inhibitsion-stimulatedstomatal opening in epidermal strips of Commelina communis L.—J.exp. Bot. 38: 142–149. Ca²⁺ suppressed both the ion-stimulated stomatal opening andH⁺ extrusion of pre-illuminated epidermal strips isolated fromCommelina communis L. In the absence of Ca²⁺, the rate of H⁺release was 18 nmol H⁺ cm^–2 h^–1 per epidermal stripunit area in 150 mol m^–3 KCL at pH 7?4. Half-maximum inhibitionof stomatal opening was observed with 220 mmol m^–3 ofCa²⁺. The hexavalent dye, ruthenium red, showed concentration-dependentprevention of the inhibition by Ca²⁺ of the ion-stimulated stomatalopening. The effect of ruthenium red was non-competitive, andthe K₁ for the calcium inhibition was found to be 3?6 mmol m^–3.The calcium inhibition of H⁺ extrusion was also prevented byruthenium red. These results suggest that Ca²⁺ inhibits theactivity of electrogenic H⁺ translocating ATPase of the guardcell plasma membrane and leads to the suppression of stomatalopening. Key words: Calcium, Commelina communis, ruthenium red, stomata     

Ion-Stimulated Stomatal Opening Induced by Preillumination in Epidermal Strips of Commelina communis

The effects of preillumination were investigated on ion-stimulated stomatal opening of epidermal strips isolated from Commelina communis L. leaves, which are dark-starved 24 hours or more. The rate and the extent of ion-stimulated stomatal openings were increased by preexposure of epidermal strips to light. The evidences are interpreted as indicating that the energy induced by preillumination can be conserved in guard cells for considerable time periods and then used for a delayed stomatal opening in the presence of higher concentration of potassium or sodium ions. Action spectrum showed two peaks, one in blue and one in the red light region. The ratio of the blue peak to the red peak is 1.2; which is the smallest reported value in action spectra of stomatal movements. 3-(4-chlorophenyl)-1,-1-Dimethylurea suppressed the ion-stimulated stomatal opening induced by the preillumination. We conclude that the photosynthetic electron transport system, containing photosystem II, in guard cell chloroplasts is a basic system of energy acquirement for stomatal opening.     

Energy Supply for Stomatal Opening in Epidermal Strips of Commelina benghalensis

The influence of light or darkness on stomatal opening in epidermal strips of Commelina benghalensis was evaluated in the presence or absence of O₂ and/or metabolic inhibitors. Opening was restricted in nitrogen and was promoted by NADH and acids of the tricarboxylic acid cycle (succinate and α-ketoglutarate) in CO₂-free air in light as well as in darkness. The enhancement by light of stomatal opening was prevalent under nitrogen or in the presence of the respiratory inhibitors (sodium azide and oligomycin). Respiratory inhibitors decreased the opening in light or darkness under CO₂-free air but exhibited no effect under nitrogen, whereas phosphorylation uncouplers were inhibitory in light or darkness under both CO₂-free air and nitrogen. The results suggest that oxidative phosphorylation is a basic source of energy for stomatal opening, although photophosphorylation could be an energy source.     

NADPH氧化酶参与水杨酸诱导的蚕豆气孔关闭过程

水杨酸(SA)可以浓度依赖的方式诱导蚕豆叶片的气孔关闭,1～1000μmol·L~(-1)SA所诱导的气孔关闭可以再开放,而10~(-2)mol·L~(-1)的SA导致的气孔关闭则否。质膜NADPH氧化酶抑制剂二亚苯基碘(DPI)可削弱SA作用的45%～60%。表明SA诱导的气孔关闭可能与H_2O_2的产生有关。以H_2O_2荧光探针H_2DCFDA结合显微注射技术直接检测保卫细胞内产生H_2O_2的结果显示,100μmol·L~(-1)SA可引起保卫细胞内荧光素(DCF)荧光快速增强。在DPI存在的情况下,经SA处理的保卫细胞,仅在其叶绿体部位产生H_2O_2,而质膜附近的DCF荧光增强则受到抑制。表明叶绿体可能是保卫细胞内产生H_2O_2的主要部位,质膜NADPH氧化酶也可能参与SA诱导H_2O_2的产生。     

Acid-Induced Stomatal Opening in Vicia faba L. and the Role of Guard Cell Wall Elasticity

When epidermal peels of Vicia faba L. were treated with solutions of varying pH, stomatal aperture was significantly increased at pH 4.0, 3.0, and 2.7 in darkness, but not in light. This effect was independent of the presence of KCl in the medium. Using a short-term plasmolytic method, estimates were made of the osmotic pressure (II_i) and the volumetric elastic modulus of guard cells, the aperture of which varied due to pretreatments at different pH, in darkness or light. In darkness, the lower pH pretreatments induced an increase in II_i and a decrease in volumetric elastic modulus. In comparison to the response in unbuffered solutions, 10 and 25 millimolar Mes buffer at pH 6.5 significantly reduced the degree of stomatal opening induced by light or by fusicoccin. These results indicate that acid-induced stomatal opening is, at least partially, due to an increase in guard cell wall elasticity which occurs in association with changes in II_i. It is suggested that the observed inhibitory effect of Mes buffer on stomatal opening may be due to a reduction in the degree of acidification of the guard cell wall and a consequent decrease of cell wall elasticity.     

Role of Potassium in Stomatal Opening in the Leaf of Vicia faba

With isolated epidermal strips of Vicia faba, the intensity of potassium-staining in the guard cells of stomata was calibrated against the uptake of radioactively labeled potassium. By using this calibration, the quantity of potassium that had accumulated in the guard cells, as stomata of leaves of Vicia open in the light, was estimated. Results support the hypothesis that in leaves, as well as in isolated epidermal strips, potassium and an accompanying anion comprise the major, osmotically active solutes in the guard cells of open stomata.     

H+参与茉莉酸调控蚕豆气孔运动的信号转导

以BCECF-AM为pH的荧光探针,结合激光共聚焦扫描显微技术,研究H 可能参与茉莉酸(JA)调控气孔运动信号转导途径的结果表明,0.1~100μmol·L~(-1)浓度的(-)JA可诱导蚕豆气孔关闭,在引起气孔孔径改变之前,(-)JA能引起蚕豆保卫细胞胞质的碱化;而(±)JA可诱导气孔适当开放,它未引起蚕豆保卫细胞胞质中pH的明显改变。药理学实验证明,质膜上质子泵的抑制剂矾酸钠能减弱(-)JA诱导气孔关闭的作用;而质膜上质子泵的激活剂壳梭孢菌素(fusicoccin)基本上未改变(±)JA的作用趋势。(-)JA和(±)JA刺激保卫细胞胞质Ca2 变化则表现出不同趋势。说明不同异构体形式的JA在调节气孔运动中的作用和信号转导途径有所不同。     

在非光合条件下外源蔗糖能促进蚕豆的气孔开放

蚕豆植株经暗饥饿处理40h后,取其下表皮,再用超声波“原位分离”下表皮上的保卫细胞对,然后在无菌、非光合条件下,用外源蔗糖处理蚕豆下表皮上的保卫细胞对,考察其对气孔开放的效应。结果发现,在1d的无菌培养过程中,蔗糖显著促进了气孔的开放。100mmol/L的蔗糖在10mmol/L的MES-NaOH/KOH(pH6.1)缓冲液中,开度分别增加2.0/2.6μm;在1μmol/L的气孔开放促进剂fusicoccin(FC)的存在下,100mmol/L的蔗糖在MES-NaOH/KOH(pH6.1)缓冲液中分别增加开度5.0/5.5μm。不同浓度（5～200mmol/L）的蔗糖处理结果表明气孔开度的增加与蔗糖浓度呈一定的正相关,浓度为100mmol/L的蔗糖处理表现出最大促进作用。同时还初步观察到,蔗糖可维持保卫细胞存活率和叶绿体的完整性。     

Statistical Distribution of Stomatal Apertures of Vicia faba and Hordeum vulgare and the Spannungsphase of Stomatal Opening

Plants of Vicia faba and Hordeum vulgare were grown in growthboxes with 7 mW cm^–2 PAR, 14 h day/10 h night, at 22/20°C. Stomata of attached leaves were measured under controlledconditions by means of an optical microscope and the distributionfunctions of the widths of pores were established. For Viciafaba they appeared to be symmetrical bell-shaped functions.In the process of stomatal opening or closure the shape of thedistribution remained constant, its maximum sliding left andright along the aperture axis. This result has been interpretedto mean that increments or decrements of apertures were equalfor all stomata independent of their individual apertures. Theconclusion has been drawn that the ‘driving force’is evenly distributed, equal for all stomata, and varies withinwider limits than is possible for stomatal apertures. Stomatalopening is limited by the closed state from below and by ananatomically possible maximum aperture from above.     

蚕豆水孔蛋白可能参与微丝骨架对气孔运动的调节

水孔蛋白的抑制剂HgCl2可明显抑制壳梭孢菌素（FC）和微丝骨架的解聚剂细胞松弛素D（CD）对蚕豆保卫细胞原生质体膨胀的诱导作用,而对微丝骨架的稳定剂鬼笔环肽（phalloidin）的抑制作用影响不明显。这表明水孔蛋白可能介导了FC和微丝骨架对气孔运动的调节。     

Involvement of Nitric Oxide in the Mechanism for Stomatal Opening in Vicia faba Leaves

Nitrite, as well as the nitric oxide (NO) donor S-nitroso-N-acethylpenisilamine (SNAP), was found to increase the aperture of stoma on Vicia faba leaf peels. The results demonstrated here suggest that the nitrite-dependent NO production pathway would be involved in the signal transduction for stomatal movements.     

Actin Filaments Modulate Both Stomatal Opening and Inward K+-Channel Activities in Guard Cells of Vicia faba L

Actin antagonists have previously been shown to alter responses of Commelina communis stomata to physiological stimuli, implicating actin filaments in the control of guard cell volume changes (M. Kim, P.K. Hepler, S.-O. Eun, K.S. Ha, Y. Lee [1995] Plant Physiol 109: 1077-1084). Since K+ channels in the guard cell play an important role in stomatal movements, we examined the possible regulation of K+-channel activities by the state of actin polymerization. Agents affecting actin polymerization altered light-induced stomatal opening and inward K+-channel activities measured by patch clamping in Vicia faba. Cytochalasin D, which induces depolymerization of actin filaments, promoted light-induced stomatal opening and potentiated the inward K+ current in guard cell protoplasts. Phalloidin, a stabilizer of filamentous actin, inhibited both light-induced stomatal opening and inward K+ current. Inward K+-channel activities in outside-out membrane patches showed responses to these agents that support results at the whole-cell current level, suggesting that cytochalasin D facilitates and phalloidin inhibits K+ influx in intact guard cells, thus resulting in enhancement and inhibition of stomatal opening, respectively. To our knowledge, this is the first report that provides evidence that actin filaments may regulate an important physiological process by modulating the activities of ion channels in plant cells.