The Role of the Epidermal Cells in the Stomatal Movements

The water deficit of the leaves, the osmotic values of the stomatal cells and epidermal cells at incipiment plasmolysis, as well as the width of the stomatal apparatus and pore opening, were measured every hour from 6-17 o'clock under natural environmental conditions. During the noon hours, the intensity of light in clear weather ranged from 40,000-55,000 lux in the open position, and from 15,000-20,000 lux in the shade. The temperature was usually 15–20°C. The experimental object was Vicia Faba growing in a field, both plants freely rooted and plants in pots buried in the soil. The experiments resulted in the following observations and conclusions: 1. When leaves are exposed to strong light, the osmotic value at incipient plasmolysis changes not only in the guard cells, but also in the epidermal cells. If the epidermal cells' osmotic value rises, water is sucked from the guard cells and their uptake of water by suction is decreased, which promotes closure and counteracts opening, respectively. If the value falls, the effect is the reverse. The guard cells react passively to these epidermal changes. The passive stomatal movement eliciteed in this way has therefore been denoted as “osmopassive”, in contrast to the long known passive movement caused by a change in turgor of the epidermal cells, and which has therefore been denoted as “turgorpasslve”. The osmopassive component of stomatal closure has an earlier and more rapid onset than the hydroactive closing reaction, which consists of a decrease in the guard cells' osmotic value. Stomatat closure often starts with the osmopassive rapid process, and is completed and stabilized by the hydroactive process. It has not been possible to determine whether the osmopassive closing reaction is identical with the rapid reaction previously described, and interpreted as of adenoid nature, and tlius belonging to the active group. 2. The osmotic potential of the guard cells - i.e., the difference between the osmotic value of guard cells and epidermal cells at incipient plasmolysis - is, therefore, formed not only by a cbange in the osmotic value of the former cells, but also by a cbange in that of the latter. 3. Although the pore width runs largely parallel to the osmotic value of the guard cells, there is greater agreement between pore width and osmotic potential. When the water deficit of the leaf exceeds a certain threshold value, potential and stomatal width start to decrease. Closure is completed when the fall in potential approaches the zero value. If the water deficit subsequently continues to increase, the potential becomes negative and the stomata remain closed. 4. The stomatal movements are regulated by physiological processes which form two kinds of equilibrium between increase and decrease of the osmotic potential of the guard cells, i.e. the osmopassive increase - osmopassive decrease and the photoactive increase - hydroactive decrease. These equilibria complement each other in rate and stability. The osmopassive processes start rapidly and as soon as the deficit cbanges; hydroactive closure and sometimes also photoactive opening, are, on the contrary, time-consuming. When the water deficit is suboptimal, turgorpassive opening and closing are superadded, but only in those cases in which the osmotic potential of the guard cetls is positive.     

Studies of the mechanism of action of fusicoccin,the fungal toxin that induces wilting,and its interaction with abscisic acid

Summary Effects of fusicoccin alone and together with abscisic acid were observed on the stomatal complex of Commelina communis. The experimental material consisted of isolated epidermal strips incubated in a medium containing the ions required for stomatal opening. Fusicoccin stimulated opening and this was accompanied by potassium entry into the guard cells, and hydrolysis of the starch in their chloroplasts. Abscisic acid alone inhibited potassium entry and starch hydrolysis, but these effects could be almost entirely overcome by fusicoccin.Attempts were made to measure the solute potential of the guard cells under the various treatments. Abscisic acid clearly increased their solute potential, but no absolute measurements could be made in the presence of fusicoccin owing to a failure of plasmolysis even with mannitol solutions of solute potential as low as —35 bars. Experiments using isotopically labelled mannitol indicated a massive uptake into the epidermis in the presence of fusicoccin.The mechanism of stimulation of stomatal opening by fusicoccin probably depends in part on a stimulation of the normal processes associated with opening in the guard cells, but may also involve release of pressure due to destruction of the surrounding cells. The effectiveness of this toxin under natural conditions may depend on its ability to counteract effects of abscisic acid, the stress hormone that induces stomatal closure.     

An analysis of the mechanics of guard cell motion

This paper presents a mechanical analysis of the cellular deformations which occur during the opening and closing of stomata. The aperture of the stomatal pore is shown to be a result of opposing pressures of the guard and adjacent epidermal cells. The analysis indicates that the epidermal cells have a mechanical advantage over the guard cells. With no mechanical advantage, an equal reduction in the turgor pressure of both guard and epidermal cells would have a neglible effect upon stomatal aperture. However, due to the mechanical advantage of the surrounding cells, the stomatal aperture increases with equal reductions in turgor, until the adjacent epidermal cells become flaccid. The minimum diffusion resistance of the pore occurs at this point. Further reductions in guard cell turgor lead to closure of the pore. The analysis further demonstrates how the shape, size, wall thickness and material properties of the guard cell walls influence their behavior.     

蚕豆下表皮细胞外钙调素的存在及其对气孔运动的调节

细胞外钙调素可能作为多肽第一信使,调节细胞增殖,花粉萌发,特定基因表达等生理过程,气孔能灵敏地对外界刺激作出反应,快速开闭,本文用免疫电镜和免疫荧光显微镜技术证明保卫细胞及其它表皮细胞胞外都存在钙调素;外源纯化钙调素能促进气孔关闭,抑制气孔开放,最适浓度为10^-8mol/L;不能透过质膜的大分子钙调素拮抗剂W—-agarose和钙调素抗血清都能抑制气孔关闭,促进开放,说明保卫细胞的内源胞外钙调素确实能促进气孔关闭,抑制开放。而且只能在细胞外起作用,推测在自然情况下,保卫细胞内源胞外钙调素可能作为胞外第一信使和其它信号分子一起调节气孔的开关运动,而且可能在环境刺激与细胞响应之间起重要作用。     

蚕豆下表皮细胞外钙调素的存在及其对气孔运动的调节

细胞外钙调素可能作为多肽第一信使,调节细胞增殖、花粉萌发、特定基因表达等生理过程.气孔能灵敏地对外界刺激作出反应,快速开闭.本文用免疫电镜和免疫荧光显微镜技术证明保卫细胞及其它表皮细胞胞外都存在钙调素.外源纯化钙调素能促进气孔关闭、抑制气孔开放,最适浓度为10-8mol/L;不能透过质膜的大分子钙调素拮抗剂W7-agarose和钙调素抗血清都能抑制气孔关闭、促进开放,说明保卫细胞的内源胞外钙调素确实能促进气孔关闭、抑制开放,而且只能在细胞外起作用.推测在自然情况下,保卫细胞内源胞外钙调素可能作为胞外第一信使和其它信号分子一起调节气孔的开关运动,而且可能在环境刺激与细胞响应之间起重要作用.     

Guard Cells Extrude Protons Prior to Stomatal Opening--A Study using Fluorescence Microscopy and pH Micro-electrodes

A method for the demonstration of pH changes in the apoplastis described. The fluorescent pH indicator pnmulin was usedto follow pH changes in the epidermis of leaves of Commelinacommunis during stomatal movements. Previously darkened leavesexposed to light showed quenching of fluorescence in the apoplastsurrounding theuard cells up to 20 min before the stomata opened.This indicated that proton efflux by the guard cells precededstomatal opening. This result was substantiated by apoplasticpH measurements using pH micro-electrodes. Acidification ofthe apoplast spread outwards from the guard cells to the surroundingsubsidiary and epidermal cells. This phenomenon persisted forsome time after subsequent stomatal closure, supporting thehypothesis that closure is brought about by a process otherthan the cessation of proton pumping. Key words: Commelina communis, stomata, proton pumping     

Stomatal movement and potassium transport in epidermal strips of Zea mays: The effect of CO2

Summary The correlation between stomatal action and potassium movement in the epidermis of Zea mays was examined in isolated epidermal strips floated on distilled water. Stomatal opening in the isolated epidermis is reversible in response to alternate periods of light or darkness, and is always correlated with a shift in the potassium content of the guard cells. K accumulates in guard cells during stomatal opening, and moves from the guard cells into the subsidiary cells during rapid stomatal closure. When epidermal strips are illuminated in normal air, as against CO₂-free air, the stomata do not open and there is a virtually complete depletion of K from the stomatal apparatus. In darkness CO₂-containing air inhibits stomatal opening and K accumulation in guard cells, but does not lead to a depletion of K from the stomata as observed in the light.     

Jasmonates Induce Intracellular Alkalinization and Closure ofPaphiopedilumGuard Cells

Jasmonates (jasmonic acid or methyl jasmonate) promote stomatalclosure inPaphiopedilum Supersuk(RHS, 1973) andP. tonsum(Rchb.f)Stein. Studies on guard cells loaded with pH dependent fluorescentdyes show that jasmonates cause intracellular alkalinizationof up to 0.5 pH units within 5 to 15 min. Jasmonate-inducedalkalinization always preceded stomatal closure and where alkalinizationwas not detected no closure occurred. Propionic acid inhibitedjasmonate-induced stomatal closure, suggesting that jasmonate-inducedintracellular alkalinization is involved in guard cell movements. BCECF; confocal microscopy; cytosolic pH; guard cells; jasmonic acid; methyl jasmonate; Paphiopedilum Supersuk(R. H. S.); Snarf-1; stomatal movements     

Potassium Chloride as Stomatal Osmoticum in Allium cepa L., a Species Devoid of Starch in Guard Cells

K⁺ and Cl⁻ contents of guard cells and of ordinary epidermal cells were determined in epidermal samples of Allium cepa L. by electron probe microanalysis; malate contents of the same samples were determined by enzymic oxidation. KCl was, in general, the major osmoticum in guard cells, irrespective of whether stomata had opened on leaves or in epidermal strips floating on solutions. The solute requirement varied between 50 and 110 femtomoles KCl per micrometer increase in aperture per pair of guard cells. Stomata did not open on solutions of K iminodiacetate, presumably because its anion could not be taken up. Stomata opened if KCl or KBr was provided. Taken together, the results indicate that the absence of starch from guard cells deprived them of the ability to produce malate in amounts of osmotic consequence and that the presence of absorbable Cl⁻ (or Br⁻) was necessary for stomatal opening.     

Stomatal deregulation in Plasmopara viticola-infected grapevine leaves

In grapevine, the penetration and sporulation of Plasmopara viticola occur via stomata, suggesting functional relationships between guard cells and the pathogen. This assumption was supported by our first observation that grapevine (Vitis vinifera cv. Marselan) cuttings infected by P. viticola wilted more rapidly than healthy ones when submitted to water starvation. Here, complementary approaches measuring stomatal conductance and infrared thermographic and microscopic observations were used to investigate stomatal opening/closure in response to infection. In infected leaves, stomata remained open in darkness and during water stress, leading to increased transpiration. This deregulation was restricted to the colonized area, was not systemic and occurred before the appearance of symptoms. Cytological observations indicated that stomatal lock-open was not related to mechanical forces resulting from the presence of the pathogen in the substomatal cavity. In contrast to healthy leaves, stomatal closure in excised infected leaves could not be induced by a water deficit or abscisic acid (ABA) treatment. However, ABA induced stomatal closure in epidermal peels from infected leaves, indicating that guard cells remained functional. These data indicate that the oomycete deregulates guard cell functioning, causing significant water losses. This effect could be attributed to a nonsystemic compound, produced by the oomycete or by the infected plant, which inhibits stomatal closure or induces stomatal opening; or a reduction of the back-pressure exerted by surrounding epidermal cells. Both hypotheses are under investigation.     

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.     

A constraint–relaxation–recovery mechanism for stomatal dynamics

Models of guard cell dynamics, built on the OnGuard platform, have provided quantitative insights into stomatal function, demonstrating substantial predictive power. However, the kinetics of stomatal opening predicted by OnGuard models were threefold to fivefold slower than observed in vivo. No manipulations of parameters within physiological ranges yielded model kinetics substantially closer to these data, thus highlighting a missing component in model construction. One well‐documented process influencing stomata is the constraining effect of the surrounding epidermal cells on guard cell volume and stomatal aperture. Here, we introduce a mechanism to describe this effect in OnGuard2 constructed around solute release and a decline in turgor of the surrounding cells and its subsequent recovery during stomatal opening. The results show that this constraint–relaxation–recovery mechanism in OnGuard2 yields dynamics that are consistent with experimental observations in wild‐type Arabidopsis, and it predicts the altered opening kinetics of ost2 H⁺‐ATPase and slac1 Cl^? channel mutants. Thus, incorporating solute flux of the surrounding cells implicitly through their constraint on guard cell expansion provides a satisfactory representation of stomatal kinetics, and it predicts a substantial and dynamic role for solute flux across the apoplastic space between the guard cells and surrounding cells in accelerating stomatal kinetics.     

Expression and manipulation of phosphoenolpyruvate carboxykinase 1 identifies a role for malate metabolism in stomatal closure

Malate, along with potassium and chloride ions, is an important solute for maintaining turgor pressure during stomatal opening. Although malate is exported from guard cells during stomatal closure, there is controversy as to whether malate is also metabolised. We provide evidence that phosphoenolpyruvate carboxykinase (PEPCK), an enzyme involved in malate metabolism and gluconeogenesis, is necessary for full stomatal closure in the dark. Analysis of the Arabidopsis PCK1 gene promoter indicated that this PEPCK isoform is specifically expressed in guard cells and trichomes of the leaf. Spatially distinct promoter elements were found to be required for post-germinative, vascular expression and guard cell/trichome expression of PCK1. We show that pck1 mutant plants have reduced drought tolerance, and show increased stomatal conductance and wider stomatal apertures compared with the wild type. During light-dark transients the PEPCK mutant plants show both increased overall stomatal conductance and less responsiveness of the stomata to darkness than the wild type, indicating that stomata get 'jammed' in the open position. These results show that malate metabolism is important during dark-induced stomatal closure and that PEPCK is involved in this process.     

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

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. Immunolocalization 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. Both stomatal closing induced by abscisic acid and opening under light were inhibited. Treatment of guard cells with cytochalasin D abolished the radial pattern of microfilaments; generated sparse, poorly oriented arrays; and caused partial opening of dark-closed stomata. These results suggest that microfilaments participate in stomatal aperture regulation.     

Potassium Loss from Stomatal Guard Cells at Low Water Potentials

The potassium content of guard cells and the resistance to viscousflow of air through the leaf were determined in sunflower (Helianthusannuus) subjected to low leaf water potentials under illuminatedconditions. In intact plants desiccated slowly by withholdingwater from the soil, large losses in guard cell K occurred asleaf water potentials decreased. Leaf viscous resistance increased,indicating stomatal closure. Similar results were obtained whendetached leaf segments were desiccated rapidly. Upon rehydrationof leaves, no stomatal opening was observed initially, despiteleaf water potentials at predesiccated levels. After severalhours, however, re-entry of K occurred and stomata became fullyopen. Turgid leaf segments floated on an ABA solution showedlosses of guard cell K and closure of stomata as rapidly andcompletely as those brought about by desiccation. It is concludedthat stomatal closure at low water potentials under illuminatedconditions is not controlled solely by water loss from the tissuebut involves the loss of osmoticum from the guard cells as well.This in turn decreases the turgor difference between the guardcells and the surrounding cells, and closing occurs.     

Humidity Responses of Stomata and the Potassium Content of Guard Cells

Humidity responses of stomata and changes in the potassium contentof their guard cells were investigated in intact plants anddetached epidermal strips of Valerianella locusta (L.) Betcke.Potassium content was determined by Macallum‘s stain.It was found that changes in stomatal aperture caused by decreasingor increasing humidity were followed only after a delay by changesin the potassium content of the guard cells. By comparison,if stomatal movements occurred in response to changes in illuminationthe relative potassium content of the guard cells correlatedcontinuously with the changes in stomatal aperture. Since thepotassium content of the guard cells changed only after mostof the stomatal movements in response to changes in humiditywere completed changes in potassium content and humidity responsesof stomata can be described as following a hysteresis curve.     

Die Abhängigkeit des osmotischen Potentials der Stomatazellen vom Wasserzustand der Pflanze

Summary The osmotic value (incipient plasmolysis) of the epidermal cells ofVicia Faba rises with a water deficit, if it is of several days' duration, and sometimes leads to transient wilting. The stomatal cells are an exception, because their osmotic value undergoes little change. Consequently, the osmotic potential of the stomatal cells is strongly negative in relation to that of the epidermal cells. This potential decreases and finally disappears after the plant has been watered, since the osmotic value of the epidermal cells falls; it reaches that of the guard cells after 12–14 hours.Owing to the negative osmotic potential of the guard cells, stomatal opening is prevented as long as the deficit lasts, as well as during the time required for restoring the deficit. Even if it has been restored, the impediment to opening persists for a certain time, because of the after-effect exerted by the water deficit on hydroactive closure.The expenses of the investigation were defrayed by a grant from the Science Research Council of Sweden.Valuable help in carrying out the investigation has been given by Fil. kand. Gösta Stenbeck.     

The ABC transporter AtABCB14 is a malate importer and modulates stomatal response to CO2

Carbon dioxide uptake and water vapour release in plants occur through stomata, which are formed by guard cells. These cells respond to light intensity, CO2 and water availability, and plant hormones. The predicted increase in the atmospheric concentration of CO2 is expected to have a profound effect on our ecosystem. However, many aspects of CO2-dependent stomatal movements are still not understood. Here we show that the ABC transporter AtABCB14 modulates stomatal closure on transition to elevated CO2. Stomatal closure induced by high CO2 levels was accelerated in plants lacking AtABCB14. Apoplastic malate has been suggested to be one of the factors mediating the stomatal response to CO2 (Refs 4,5) and indeed, exogenously applied malate induced a similar AtABCB14-dependent response as high CO2 levels. In isolated epidermal strips that contained only guard cells, malate-dependent stomatal closure was faster in plants lacking the AtABCB14 and slower in AtABCB14-overexpressing plants, than in wild-type plants, indicating that AtABCB14 catalyses the transport of malate from the apoplast into guard cells. Indeed, when AtABCB14 was heterologously expressed in Escherichia coli and HeLa cells, increases in malate transport activity were observed. We therefore suggest that AtABCB14 modulates stomatal movement by transporting malate from the apoplast into guard cells, thereby increasing their osmotic pressure.     

Anion-Channel Blockers Inhibit S-Type Anion Channels and Abscisic Acid Responses in Guard Cells

The effects of anion-channel blockers on light-mediated stomatal opening, on the potassium dependence of stomatal opening, on stomatal responses to abscisic acid (ABA), and on current through slow anion channels in the plasma membrane of guard cells were investigated. The anion-channel blockers anthracene-9-carboxylic acid (9-AC) and niflumic acid blocked current through slow anion channels of Vicia faba L. guard cells. Both 9-AC and niflumic acid reversed ABA inhibition of stomatal opening in V. faba L. and Commelina communis L. The anion-channel blocker probenecid also abolished ABA inhibition of stomatal opening in both species. Additional tests of 9-AC effects on stomatal aperture in Commelina revealed that application of this anion-channel blocker allowed wide stomatal opening under low (1 mM) KCI conditions and increased the rate of stomatal opening under both low and high (100 mM) KCI conditions. These results indicate that anion channels can function as a negative regulator of stomatal opening, presumably by allowing anion efflux and depolarization, which prohibits ion up-take in guard cells. Furthermore, 9-AC prevented ABA induction of stomatal closure. A model in which ABA activation of anion channels contributes a rate-limiting mechanism during ABA-induced stomatal closure and inhibition of stomatal opening is discussed.