Sensory transduction and electrical signaling in guard cells

Guard cells are a valuable model system for the study of photoreception, ion transport, and osmoregulation in plant cells. Changes in stomatal apertures occur when sensing mechanisms within the guard cells transduce environmental stimull into the ion fluxes and biosynthesis of organic solutes that regulate turgor. The electrical events mediating sensory transduction in guard cells can be characterized with a variety of electrophysiological recording techniques. Recent experiments applying the patch clamp method to guard cell protoplasts have demonstrated activation of electrogenic pumps by blue and red light as well as the presence of potassium channels in guard cell plasmalemma. Light activation of electrogenic proton pumping and the ensuing gating of voltage-dependent ion channels appear to be components of sensory transduction of the stomatal response to light. Mechanisms underlying stomatal control by environmental signals can be understood by studying electrical events associated with ion transport.     

蚕豆气孔运动中水通道和离子通道的作用和地位的探讨

 用不同浓度HgCl2、LaCl3和TEACl (Tetraethylammonium chloride)处理蚕豆(Vicia faba)叶片下表皮条,发现HgCl2能显著抑制气孔开闭,Ca2+通道阻塞剂LaCl3或K+通道阻塞剂TEACl处理也都有一定程度的抑制。三者的作用效果HgCl2>>LaCl3>TEACl。用HgCl2+LaCl3、HgCl2+TEACl或HgCl2+LaCl3+TEACl处理,则气孔开闭运动几乎完全被抑制。表明：蚕豆气孔运动中,保卫细胞胀缩主要是水通道直接参与保卫细胞与叶肉细胞间水流的调节引起的,离子通道起间接次要作用,二者共同引起保卫细胞体积变化而导致气孔开闭。     

Rapid hydropassive opening and subsequent active stomatal closure follow heat-induced electrical signals in Mimosa pudica

In Mimosa pudica L., heat stimulation triggers leaflet folding in local, neighbouring and distant leaves. Stomatal movements were observed microscopically during this folding reaction and electrical potentials, chlorophyll fluorescence, and leaf CO(2)/H(2)O-gas exchange were measured simultaneously. Upon heat stimulation of a neighbouring pinna, epidermal cells depolarized and the stomata began a rapid and pronounced transient opening response, leading to an approximately 2-fold increase of stomatal aperture within 60 s. At the same time, net CO(2) exchange showed a pronounced transient decrease, which was followed by a similar drop in photochemical quantum yield at photosystem (PS) II. Subsequently, CO(2)-gas exchange and photochemical quantum yield recovered and stomata closed partly or completely. The transient and fast stomatal opening response is interpreted as a hydropassive stomatal movement caused by a sudden loss of epidermal turgor. Thus, epidermal cells appear to respond in a similar manner to heat-induced signals as the pulvinar extensor cells. The subsequent closing of the stomata confirms earlier reports that stomatal movements can be induced by electrical signals. The substantial delay (several minutes) of guard cell turgor loss compared with the immediate response of the extensor and epidermal cells suggests a different, less direct mechanism for transmission of the propagating signal to the guard cells.     

The effect of blue light on stomatal oscillations and leaf turgor pressure in banana leaves

Stomatal oscillations are cyclic opening and closing of stomata, presumed to initiate from hydraulic mismatch between leaf water supply and transpiration rate. To test this assumption, mismatches between water supply and transpiration were induced using manipulations of vapour pressure deficit (VPD) and light spectrum in banana (Musa acuminata). Simultaneous measurements of gas exchange with changes in leaf turgor pressure were used to describe the hydraulic mismatches. An increase of VPD above a certain threshold caused stomatal oscillations with variable amplitudes. Oscillations in leaf turgor pressure were synchronized with stomatal oscillations and balanced only when transpiration equaled water supply. Surprisingly, changing the light spectrum from red and blue to red alone at constant VPD also induced stomatal oscillations – while the addition of blue (10%) to red light only ended oscillations. Blue light is known to induce stomatal opening and thus should increase the hydraulic mismatch, reduce the VPD threshold for oscillations and increase the oscillation amplitude. Unexpectedly, blue light reduced oscillation amplitude, increased VPD threshold and reduced turgor pressure loss. These results suggest that additionally, to the known effect of blue light on the hydroactive opening response of stomata, it can also effect stomatal movement by increased xylem–epidermis water supply.     

Stomatal dynamics are limited by leaf hydraulics in ferns and conifers: results from simultaneous measurements of liquid and vapour fluxes in leaves

Stomatal responsiveness to vapour pressure deficit (VPD) results in continuous regulation of daytime gas‐exchange directly influencing leaf water status and carbon gain. Current models can reasonably predict steady‐state stomatal conductance (g_s) to changes in VPD but the g_s dynamics between steady‐states are poorly known. Here, we used a diverse sample of conifers and ferns to show that leaf hydraulic architecture, in particular leaf capacitance, has a major role in determining the g_s response time to perturbations in VPD. By using simultaneous measurements of liquid and vapour fluxes into and out of leaves, the in situ fluctuations in leaf water balance were calculated and appeared to be closely tracked by changes in g_s thus supporting a passive model of stomatal control. Indeed, good agreement was found between observed and predicted g_s when using a hydropassive model based on hydraulic traits. We contend that a simple passive hydraulic control of stomata in response to changes in leaf water status provides for efficient stomatal responses to VPD in ferns and conifers, leading to closure rates as fast or faster than those seen in most angiosperms.     

Stomatal oscillations at small apertures: indications for a fundamental insufficiency of stomatal feedback-control inherent in the stomatal turgor mechanism.

Continuous measurements of stomatal aperture simultaneously with gas exchange during periods of stomatal oscillations are reported for the first time. Measurements were performed in the field on attached leaves of undisturbed Sambucus nigra L. plants which were subjected to step-wise increases of PPFD. Oscillations only occurred when stomatal apertures were small under high water vapour mole fraction difference between leaf and atmosphere (DeltaW). They consisted of periodically repeated opening movements transiently leading to very small apertures. Measurements of the area of the stomatal complex in parallel to the determination of aperture were used to record volume changes of guard cells even if stomata were closed. Stomatal opening upon a light stimulus required an antecedent guard cell swelling before a slit occurred. After opening of the slit the guard cells again began to shrink which, with some delay, led to complete closure. Opening and closing were rhythmically repeated. The time-lag until initial opening was different for each individual stoma. This led to counteracting movements of closely adjacent stomata. The tendency to oscillate at small apertures is interpreted as being a failure of smoothly damped feedback regulation at the point of stomatal opening: Volume changes are ineffective for transpiration if stomata are still closed; however, at the point of initial opening transpiration rate rises steeply. This discontinuity together with the rather long time constants inherent in the stomatal turgor mechanism makes oscillatory overshooting responses likely if at high DeltaW the 'nominal value' of gas exchange demands a small aperture.     

Evidence of Hydropassive Movement in Stomatal Oscillations of Glycyrrhiza inflata under Desert Conditions

Stomatal conductance was found to change from steady-state to a state of oscillations during daytime when vapour pressure deficit (VPD) increased to a value of 1 kPa in Glycyrrhiza inflata Batalin grown under the conditions of arid desert in north-west China. The injected metabolic inhibitors (NaN 3 or carbonyl cyanide-m-chlorophenyl-hydrazone (CCCP)) slightly reduced the stomatal conductance but did not significantly decrease the intensity of stomatal oscillations (amplitude/average). The oscillation intensity was found to be significantly correlated with VPD and root resistance, but not with the respiration rate. There might exist a minimum threshold of VPD (0.8 kPa) and root resistance (1/4 relative value) that induced stomatal oscillations. These results suggested that stomatal oscillations induced by atmospheric drought stress and root resistance were mainly a type of hydropassive movement.     

荒漠条件下甘草气孔振荡的水被动证据

生长在中国西北干旱荒漠的甘草（Glycyrrhiza inflata Batalin),当白天大气水蒸汽压差（ＶＰＤ）高于１kPa时,其气孔导度随时间的变化趋势为从稳态转为振荡态。通过茎木质部注射代谢抑制剂（ＮaN3或羰基氰化物－间－氯苯腙（ＣＣＣＰ）使气孔导度有些微降低,但是并不能显改变气孔振荡强度（振幅／平均值）。气孔振荡强度与ＶＰＤ和根阻力显相关,但与呼吸速度无明显相关,在荒漠条件下,当ＶＰＤ大于０．８kPa和至少存在１／４全根阻力的条件下才能出现气孔振荡。结果说明荒漠干旱条件诱发的甘草气孔振荡可能主要是一种水被动过程 。     

Stomatal action directly feeds back on leaf turgor: new insights into the regulation of the plant water status from non‐invasive pressure probe measurements

Uptake of CO₂ by the leaf is associated with loss of water. Control of stomatal aperture by volume changes of guard cell pairs optimizes the efficiency of water use. Under water stress, the protein kinase OPEN STOMATA 1 (OST1) activates the guard‐cell anion release channel SLOW ANION CHANNEL‐ASSOCIATED 1 (SLAC1), and thereby triggers stomatal closure. Plants with mutated OST1 and SLAC1 are defective in guard‐cell turgor regulation. To study the effect of stomatal movement on leaf turgor using intact leaves of Arabidopsis, we used a new pressure probe to monitor transpiration and turgor pressure simultaneously and non‐invasively. This probe permits routine easy access to parameters related to water status and stomatal conductance under physiological conditions using the model plant Arabidopsis thaliana. Long‐term leaf turgor pressure recordings over several weeks showed a drop in turgor during the day and recovery at night. Thus pressure changes directly correlated with the degree of plant transpiration. Leaf turgor of wild‐type plants responded to CO₂, light, humidity, ozone and abscisic acid (ABA) in a guard cell‐specific manner. Pressure probe measurements of mutants lacking OST1 and SLAC1 function indicated impairment in stomatal responses to light and humidity. In contrast to wild‐type plants, leaves from well‐watered ost1 plants exposed to a dry atmosphere wilted after light‐induced stomatal opening. Experiments with open stomata mutants indicated that the hydraulic conductance of leaf stomata is higher than that of the root–shoot continuum. Thus leaf turgor appears to rely to a large extent on the anion channel activity of autonomously regulated stomatal guard cells.     

The control of specificity in guard cell signal transduction

Stomatal guard cells have proven to be an attractive system for dissecting the mechanisms of stimulus-response coupling in plants. In this review we focus on the intracellular signal transduction pathways by which extracellular signals bring about closure and opening of the stomatal pore. It is proposed that guard cell signal transduction pathways may be organized into functional arrays or signalling cassettes that contain elements common to a number of converging signalling pathways. The purpose of these signalling cassettes may be to funnel extracellular signals down onto the ion transporters that control the fluxes of ions that underlie stomatal movements. Evidence is emerging that specificity in guard cell signalling may be, in part, encoded in complex spatio-temporal patterns of increases in the concentration of cytosolic-free calcium ([Ca²⁺]_cyt). It is suggested that oscillations in [Ca²⁺]_cyt may generate calcium signatures that encode information concerning the stimulus type and strength. New evidence is presented that suggests that these calcium signatures may integrate information when many stimuli are present.     

PHO1 expression in guard cells mediates the stomatal response to abscisic acid in Arabidopsis

Stomatal opening and closing are driven by ion fluxes that cause changes in guard cell turgor and volume. This process is, in turn, regulated by environmental and hormonal signals, including light and the phytohormone abscisic acid (ABA). Here, we present genetic evidence that expression of PHO1 in guard cells of Arabidopsis thaliana is required for full stomatal responses to ABA. PHO1 is involved in the export of phosphate into the root xylem vessels and, as a result, the pho1 mutant is characterized by low shoot phosphate levels. In leaves, PHO1 was found expressed in guard cells and up‐regulated following treatment with ABA. The pho1 mutant was unaffected in production of reactive oxygen species following ABA treatment, and in stomatal movements in response to light cues, high extracellular calcium, auxin, and fusicoccin. However, stomatal movements in response to ABA treatment were severely impaired, both in terms of induction of closure and inhibition of opening. Micro‐grafting a pho1 shoot scion onto wild‐type rootstock resulted in plants with normal shoot growth and phosphate content, but failed to restore normal stomatal response to ABA treatment. PHO1 knockdown using RNA interference specifically in guard cells of wild‐type plants caused a reduced stomatal response to ABA. In agreement, specific expression of PHO1 in guard cells of pho1 plants complemented the mutant guard cell phenotype and re‐established ABA sensitivity, although full functional complementation was dependent on shoot phosphate sufficiency. Together, these data reveal an important role for phosphate and the action of PHO1 in the stomatal response to ABA.     

Stomatal response to humidity in sugarcane and soybean: effect of vapour pressure difference on the kinetics of the blue light response

Abstract. The effect of atmospheric humidity on the kinetics of stomatal responses was quantified in gas exchange experiments using sugarcane ( Saccharum spp. hybrid) and soybean ( Glycine max ). Pulses of blue light were used to elicit pulses of stomatal conductance that were mediated by the specific blue light response of guard cells. Kinetic parameters of the conductance response were more closely related to leaf-air vapour pressure difference (VPD) than to relative humidity or transpiration. Increasing VPD significantly accelerated stomatal opening in both sugarcane and soybean, despite an approximately five-fold faster response in sugarcane. In contrast, the kinetics of stomatal recovery (closure) following the pulse were similar in the two species. Acceleration of opening by high VPD was observed even under conditions where soybean exhibited a feedforward response of decreasing transpiration (E) with increasing evaporative demand (VPD). This result suggests that epidermal, rather than bulk leaf, water status mediates the VPD effect on stomatal kinetics. The data are consistent with the hypothesis that increased cpidermal water loss at high VPD decreases the backpressure exerted by neighbouring cells on guard cells. allowing more rapid stomatal opening per unit of guard cell metabolic response to blue light.     

The magnitude of the stomatal response to blue light : modulation by atmospheric humidity

The effect of leaf-air vapor pressure difference (VPD) on the magnitude of the stomatal response to blue light was investigated in soybean (Glycine max) by administering blue light pulses (22 seconds by 120 micromoles per square meter per second) at different levels of VPD and temperature. At 20 °C and 25 °C, the magnitude of the integrated conductance response decreased with increasing VPD (0.4 to 2.6 kiloPascals), due to an earlier onset of stomatal closure that terminated the pulse response. In contrast, at 30 °C this magnitude increased with rising VPD (0.9 to 3.5 kiloPascals), due to an increasing maximum excursion of the conductance response despite the accelerated onset of stomatal closure. When the feedforward response of stomata to humidity caused steady state transpiration to decrease with increasing VPD, the magnitude of the pulse-induced conductance response correlated with VPD rather than with transpiration. This suggests that water relations or metabolite movements within epidermal rather than bulk leaf tissue interacted with guard cell photobiological properties in regulating the magnitude of the blue light response. VPD modulation of pulse magnitude could reduce water loss during stomatal responses to transient illumination in natural light environments.     

Stomatal responses to light and humidity in sugarcane: prediction of daily time courses and identification of potential selection criteria

Abstract Stomatal sensitivities to light and VPD have potential as quantitative selection criteria in programs designed to enhance water-use efficiency of sugarcane and other crops. These responses were characterized using gas exchange techniques and then simulated by a mathematical relationship describing conductance as a function of photon fluence rates and VPD values. The same form of relationship simulated stomatal responses of well-watered greenhouse- and field-grown plants. A comparison between simulated and measured conductance values showed a close correlation, indicating that light and VPD responses of stomata are dominant input signals modulating stomatal conductance in sugarcane. Observed conductance of Hawaiian sugarcane in a commerical production area appeared larger than required to support prevailing rates of carbon assimilation, since predicted intercellular CO₂ was greater than required to saturate its C₄ photosynthesis. Manipulation of the relationship describing stomatal conductance allowed us to simulate the responses of plants with hypothetically altered stomatal sensitivities to VPD or to light, using micrometeorological data collected in the field. Further simulation indicated that selection for clones with altered stomatal sensitivity to either light or VPD could improve the water-use efficiency of sugarcane without inhibiting current high levels of productivity.     

The chemiosmotic model of stomatal opening revisited

Stomata are light‐activated biological valves in the otherwise gas‐impermeable epidermis of aerial organs of higher plants. Stomata often regulate rates of photosynthesis and transpiration in ways that optimize whole‐plant carbon gain against water loss. Each stoma is flanked by a pair of opposing guard cells. Stomatal opening occurs by light‐activated increases in the turgor pressure of guard cells, which causes them to change shape so that the stomatal pore between them widens. These increases in turgor pressure oppose increases in cellular osmotic pressure that result from uptake of K⁺. K⁺ uptake occurs by a chemiosmotic mechanism in response to light‐activated extrusion of H⁺ outward across the plasma membrane of the guard cell. The initial changes in cellular membrane potential lead to the opening of inward‐rectifying K⁺ channels, after which K⁺ is taken up along its electrochemical gradient. Changes in membrane potential resulting from K⁺ uptake may be balanced by accumulation of Cl^?ions by guard cells and/or by synthesis of malic acid within each cell. Malic acid also acts to buffer increases in cytosolic pH caused by H⁺ extrusion. This review describes how the application of patch‐clamp technology to guard cell protoplasts has enabled investigators to elucidate the mechanisms by which H⁺ is extruded from guard cells, the types of ion channels present in the guard cell plasma membrane, how those ion channels are regulated, and the signal transduction processes that trigger stomatal opening and closing.     

A Cationic Channel in the Guard Cell Tonoplast of Allium cepa

Stomatal movements depend on an osmoregulation process in which swelling or shrinking of the guard cells opens or closes the stomatal pore. Ions and water fluxes are an essential aspect of guard cell osmoregulation. Thus far, studies of these fluxes have focused on the guard cell plasma membrane. Guard cells, however, are a multi-compartment system that includes a prominent vacuole, which has a primary role in turgor regulation. This study reports on a detailed characterization of an ion channel at the guard cell tonoplast of Allium cepa (onion). We used patch-clamp methodology with isolated tonoplast patches to study conduction and gating at the single channel level. A voltage-dependent outward-rectifying cationic channel (210 picosiemens) was the dominant conductance. In symmetrical solutions the channel displayed an ohmic behavior in its current-voltage relationship. It also showed a very large rectification in the open probability. The channel was predominantly cationic and its sequence of ionic selectivity was weak (Na+ > K+ > Rb+ > Cs+). The channel conductance was not affected by intravacuolar pH. Analysis of membrane patches with multiple channels showed that the probability of a channel to open was independent of the opening of the other channels present in the patch and that there was a conservation of the open probability for different channels. Ensemble records generated using a pulse protocol showed slow activation and deactivation kinetics. A first-latency analysis of single-channel records in response to protocols with different prepulse duration indicated that this channel has more than one closed state.     

Roles of ion channels and transporters in guard cell signal transduction

Stomatal complexes consist of pairs of guard cells and the pore they enclose. Reversible changes in guard cell volume alter the aperture of the pore and provide the major regulatory mechanism for control of gas exchange between the plant and the environment. Stomatal movement is facilitated by the activity of ion channels and ion transporters found in the plasma membrane and vacuolar membrane of guard cells. Progress in recent years has elucidated the molecular identities of many guard cell transport proteins, and described their modulation by various cellular signal transduction components during stomatal opening and closure prompted by environmental and endogenous stimuli.     

Light perception in guard cells

Abstract. Guard cells perceive light via two photoreceptor systems: a blue-light-dependent photosystem and the guard cell chloroplast. Chloroplasts stimulate stomatal opening by transducing photosynthetic active radiation into proton pumping at the guard cell plasma membrane. In addition, guard cell chloroplasts fix CO₂ photosynthetically. Sugar from guard cell photosynthesis can contribute to the osmotic build-up required for opening. The blue-light-dependent photosystem activates proton pumping at the guard cell plasma membrane and stimulates starch hydrolysis. Available information on the photobiological properties of guard cells makes it possible to describe stomatal function in terms of the cellular components regulating stomatal movements. The blue light response is involved in stomatal opening in the early morning and stomatal responses to sunflecks. The guard cell chloroplast is likely to be involved in stomatal adaptations to sun, shade and to temperature. Interactions between these photosystems, a third photoreceptor in guard cells, phytochrome, and other mechanisms transducing stomatal responses such as VPD and carbon dioxide, provide the cellular basis for stomatal regulation.     

Stomatal responses to light in the facultative Crassulacean acid metabolism species, Portulacaria afra

Guard cell responses to light are mediated by guard cell chlorophyll and by a specific blue light photoreceptor. Gas exchange and epidermal peel techniques were employed to investigate these responses in the facultative Crassulacean acid metabolism (CAM) species, Portulacaria afra (L.) Jacq. In P. afra individuals performing C₃ metabolism, red light stimulated an increase in leaf conductance in intact leaves and stomatal opening in isolated epidermal peels, indicating the presence in guard cells of the chlorophyll-mediated response to light. Under a background of continuous red illumination, conductance exhibited transient increases following pulses of blue but not red light, indicating that the specific stomatal response to blue light was also operative. In contrast, in CAM individuals, conductance in gas exchange experiments and stomatal opening in epidermal peel experiments were not stimulated by red light. In CAM plants, conductance did not increase following blue light pulses administered over a range of temperatures, vapor pressure differences (VPD), ambient CO₂ concentrations and background red light intensities. These results indicate that P. afra does possess typical guard cell responses to light when performing C₃ metabolism. The metabolic pathways mediating these responses are either lost or inhibited when CAM is induced.     

Light-Induced Stomatal Opening Is Affected by the Guard Cell Protein Kinase APK1b

Guard cells allow land plants to survive under restricted or fluctuating water availability. They control the exchange of gases between the external environment and the interior of the plant by regulating the aperture of stomatal pores in response to environmental stimuli such as light intensity, and are important regulators of plant productivity. Their turgor driven movements are under the control of a signalling network that is not yet fully characterised. A reporter gene fusion confirmed that the Arabidopsis APK1b protein kinase gene is predominantly expressed in guard cells. Infrared gas analysis and stomatal aperture measurements indicated that plants lacking APK1b are impaired in their ability to open their stomata on exposure to light, but retain the ability to adjust their stomatal apertures in response to darkness, abscisic acid or lack of carbon dioxide. Stomatal opening was not specifically impaired in response to either red or blue light as both of these stimuli caused some increase in stomatal conductance. Consistent with the reduction in maximum stomatal conductance, the relative water content of plants lacking APK1b was significantly increased under both well-watered and drought conditions. We conclude that APK1b is required for full stomatal opening in the light but is not required for stomatal closure.