Cytokinin and auxin inhibit abscisic acid-induced stomatal closure by enhancing ethylene production in Arabidopsis

Cytokinins and auxins are major phytohormones involved in various aspects of plant growth and development. These phytohormones are also known to antagonize the effects of abscisic acid (ABA) on stomatal movement, and to affect ethylene biosynthesis. As ethylene has an antagonistic effect on ABA-induced stomatal closure, the possibility that the antagonistic effects of these phytohormones on ABA were mediated through ethylene biosynthesis was investigated. Both the cytokinin, 6-benzyladenine (BA), and the auxin, 1-naphthaleneacetic acid (NAA), antagonized ABA-induced stomatal closure in a manner similar to that following application of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). However, these effects were negated when ethylene signalling, perception, or biosynthesis were blocked. As stomatal aperture is regulated by changes in guard cell volume, ABA application was found to reduce the volume of the guard cell protoplasts (GCP). It was found that BA, NAA, or ACC application compensated perfectly for the reduction in GCP volume by ABA application in WT plants. The above observations suggest that cytokinins and auxins inhibit ABA-induced stomatal closure through the modulation of ethylene biosynthesis, and that ethylene inhibits the ABA-induced reduction of osmotic pressure in the guard cells.     

The stomata frontline of plant interaction with the environment-perspectives from hormone regulation

Plants have evolved elaborate mechanisms to perceive and integrate signals from various environmental conditions.On leaf surface,stomata formed by pairs of guard cells mediate gas exchange,water transp...     

Oligogalacturonic Acid and Chitosan Reduce Stomatal Aperture by Inducing the Evolution of Reactive Oxygen Species from Guard Cells of Tomato and Commelina communis

Stomatal opening provides access to inner leaf tissues for many plant pathogens, so narrowing stomatal apertures may be advantageous for plant defense. We investigated how guard cells respond to elicitors that can be generated from cell walls of plants or pathogens during pathogen infection. The effect of oligogalacturonic acid (OGA), a degradation product of the plant cell wall, and chitosan (beta-1,4-linked glucosamine), a component of the fungal cell wall, on stomatal movements were examined in leaf epidermis of tomato (Lycopersicon esculentum L.) and Commelina communis L. These elicitors reduced the size of the stomatal aperture. OGA not only inhibited light-induced stomatal opening, but also accelerated stomatal closing in both species; chitosan inhibited light-induced stomatal opening in tomato epidermis. The effects of OGA and chitosan were suppressed when EGTA, catalase, or ascorbic acid was present in the medium, suggesting that Ca(2+) and H(2)O(2) mediate the elicitor-induced decrease of stomatal apertures. We show that the H(2)O(2) that is involved in this process is produced by guard cells in response to elicitors. Our results suggest that guard cells infected by pathogens may close their stomata via a pathway involving H(2)O(2) production, thus interfering with the continuous invasion of pathogens through the stomatal pores.     

Stomatal responses to ABA and IAA in isolated epidermal strips ofVicia faba L.

Epidermal strips from well-watered faba-bean plants were subjected to a range of abscisic acid (ABA) and indolyl-3-acetic acid (IAA) concentrations (10^-5 to 1 mM) in the presence or absence of CO₂ in light or dark. ABA had inhibitory effect on abaxial stomatal apertures in all the concentrations studied and retained them closed even after addition of KCl (SO and 100 mM) to the incubation medium. It also influenced stomatal responses to CO₂. In the presence of CO₂ apertures were greater than in its absence in light as well as in darkness. This relationship remained unchanged also after addition of KCl. The action of ABA inhibited accumulation of potassium in the guard cells. IAA stimulated stomatal opening and its effect was quite opposite to ABA; in the presence of CO₂ the apertures were smaller than in its absence. IAA, however, was able to inhibit the closing effect of darkness, CO₂, and ABA, and stimulated potassium accumulation in the guard cells. Simultaneous action of ABA+IAA manifested effects of both substances.     

The Arabidopsis small G protein ROP2 is activated by light in guard cells and inhibits light-induced stomatal opening

ROP small G proteins function as molecular switches in diverse signaling processes. Here, we investigated signals that activate ROP2 in guard cells. In guard cells of Vicia faba expressing Arabidopsis thaliana constitutively active (CA) ROP2 fused to red fluorescent protein (RFP-CA-ROP2), fluorescence localized exclusively at the plasma membrane, whereas a dominant negative version of RFP-ROP2 (DN-ROP2) localized in the cytoplasm. In guard cells expressing green fluorescent protein-ROP2, the relative fluorescence intensity at the plasma membrane increased upon illumination, suggesting that light activates ROP2. Unlike previously reported light-activated factors, light-activated ROP2 inhibits rather than accelerates light-induced stomatal opening; stomata bordered by guard cells transformed with CA-rop2 opened less than controls upon light irradiation. When introduced into guard cells together with CA-ROP2, At RhoGDI1, which encodes a guanine nucleotide dissociation inhibitor, inhibited plasma membrane localization of CA-ROP2 and abolished the inhibitory effect of CA-ROP2 on light-induced stomatal opening, supporting the negative effect of active ROP2 on stomatal opening. Mutant rop2 Arabidopsis guard cells showed phenotypes similar to those of transformed V. faba guard cells; CA-rop2 stomata opened more slowly and to a lesser extent, and DN-rop2 stomata opened faster than wild-type stomata in response to light. Moreover, in rop2 knockout plants, stomata opened faster and to a greater extent than wild-type stomata in response to light. Thus, ROP2 is a light-activated negative factor that attenuates the extent of light-induced changes in stomatal aperture. The inhibition of light-induced stomatal opening by light-activated ROP2 suggests the existence of feedback regulatory mechanisms through which stomatal apertures may be finely controlled.     

Effect of brassinolide, alone and in concert with abscisic acid, on control of stomatal aperture and potassium currents of Vicia faba guard cell protoplasts

The essential role of brassinosteroids (BRs) in normal plant growth, development and physiology has been established by the analysis of biosynthesis and signal transduction mutants. Some of the BR-related mutants also display altered sensitivity to the phytohormone abscisic acid (ABA) suggesting that BRs normally counteract the effects of ABA on root growth, seed germination, and possibly stomatal movement. In this study, the effect of a specific BR, brassinolide (BL), on guard cell function of Vicia faba was examined alone and in conjunction with ABA. Unlike other described plant responses, BL did not oppose the effect of ABA in regulation of stomatal movement. On the contrary, BL modulated stomatal aperture by promoting stomatal closure and inhibiting stomatal opening, functions of this hormone that were previously undescribed. This study also demonstrated a role for plant steroidal hormones in ion channel regulation: BL inhibited inwardly rectifying K⁺ currents of V. faba guard cell protoplasts in a manner similar to ABA. In both stomatal movement assays and whole-cell patch clamp experiments, the effects of BL and ABA applied together were not additive, suggesting that these two hormones may function in interacting pathways to regulate stomatal apertures and guard cell physiology.     

The stomatal response to CO2 is linked to changes in guard cell zeaxanthin*

The mechanisms mediating CO₂ sensing and light–CO₂ interactions in guard cells are unknown. In growth chamber-grown Vicia faba leaves kept under constant light (500 μ mol m^–2 s^–1) and temperature, guard cell zeaxanthin content tracked ambient [CO₂] and stomatal apertures. Increases in [CO₂] from 400 to 1200 cm³ m^–3 decreased zeaxanthin content from 180 to 80 mmol mol^–1 Chl and decreased stomatal apertures by 7·0 μ m. Changes in zeaxanthin and aperture were reversed when [CO₂] was lowered. Guard cell zeaxanthin content was linearly correlated with stomatal apertures. In the dark, the CO₂-induced changes in stomatal aperture were much smaller, and guard cell zeaxanthin content did not change with chamber [CO₂]. Guard cell zeaxanthin also tracked [CO₂] and stomatal aperture in illuminated stomata from epidermal peels. Dithiothreitol (DTT), an inhibitor of zeaxanthin formation, eliminated CO₂-induced zeaxanthin changes in guard cells from illuminated epidermal peels and reduced the stomatal CO₂ response to the level observed in the dark. These data suggest that CO₂-dependent changes in the zeaxanthin content of guard cells could modulate CO₂-dependent changes of stomatal apertures in the light while a zeaxanthin-independent CO₂ sensing mechanism would modulate the CO₂ response in the dark.     

Role of Ca and EGTA on Stomatal Movements in Commelina communis L

Ca²⁺ (0.1-1.0 millimolar) accelerated dark-induced stomatal closure and reduced stomatal apertures in the light in epidermal peels of Commelina communis L. In contrast, ethyleneglycol-bis-(β-aminoethyl ether) N,N′tetraacetic acid (EGTA) (2 millimolar), a Ca²⁺ chelator, prevented closure in the dark and accelerated opening in the light. EGTA did not promote significant opening in the dark. It is therefore concluded that EGTA does not increase ion uptake into guard cells, but rather prevents ion efflux. Addition of EGTA to incubating solutions with 10 millimolar KCl resulted in steady state apertures of 15.6 micrometers, whereas in the absence of EGTA similar apertures required 55 millimolar KCl and 150 millimolar KCl was needed in the presence of 1 millimolar CaCl₂. The results demonstrate the importance of Ca²⁺ in the regulation of stomatal closure and point to a role of Ca²⁺ in the regulation of K⁺ efflux from stomatal guard cells.     

Possible involvement of phospholipase A2 in light signal transduction of guard cells of Commelina communis

Polyunsaturated fatty acids induce stomatal opening (Y. Lee, H. Lee, R. C. Crain, A. Lee and S. J. Korn. 1994. Cell Signal. 6: 181–186), but it is not known whether they function as second messengers in guard cells exposed to signals that open stomata. To test the hypothesis that phospholipase A₂ (PLA₂), which produces fatty acids and lysophospholipids, is involved in light signal transduction in guard cells, we treated epidermal peels of Commelina communis L. with PLA₂ inhibitors and followed the changes in stomatal apertures in response to light. Stomatal opening by white, blue, or red light was inhibited by 2–3 different PLA₂ inhibitors in concentration ranges that have been reported to inhibit PLA₂ activity. However, the PLA₂ inhibitors could not block stomatal opening induced by a polyunsaturated fatty acid. These results suggest that PLA₂ functions as a signal transducer for both blue and red light in guard cells.     

Membrane trafficking in guard cells during stomatal movement: Application of an image processing technique

Pairs of guard cells form small pores called stoma in the epidermis, and the reversible swelling and shrinking of these guard cells regulate the stomatal apertures. The well-documented changes in guard cell volume have been associated with their vacuolar structures. To investigate the contribution of the guard cell vacuoles to stomatal movement, the dynamics of these vacuolar structures were recently monitored during stomatal movement in vacuolar-membrane visualized Arabidopsis plants. Calculation of the vacuolar volume and surface area after reconstruction of three-dimensional images revealed a decrease in the vacuolar volume but an increase in the vacuolar surface area upon stomatal closure. These results implied the possible acceleration of membrane trafficking to the vacuole upon stomatal closure and membrane recycling from the vacuole to the plasma membrane upon stomatal opening. To clarify and quantify membrane trafficking during stomatal movement, we describe in this addendum our development of an improved image processing system.Key words: stomata, guard cells, vacuole, membrane traffic, image processing     

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.     

Two Arabidopsis guard cell-preferential MAPK genes,MPK9 and MPK12, function in biotic stress response

Abscisic acid (ABA) plays a major role in plant development and adaptation to severe environmental conditions. ABA evokes cellular events to regulate stomatal apertures and thus contributes to the plant’s ability to respond to abiotic stresses. Reactive oxygen species (ROS) are produced in response to ABA and mediate ABA-induced stomatal closure. We have shown that two MAP kinases, MPK9 and MPK12, are highly and preferentially expressed in guard cells and function as positive regulators of ROS-mediated ABA signaling in guard cells. Cell biological and electrophysiological analyses demonstrated that MPK9 and MPK12 act downstream of ROS and cytosolic Ca²⁺ and upstream of anion channels in the guard cell ABA signaling cascade. Plant pathogens use stomata as the primary gateway to enter into their hosts, and previous studies have indicated crosstalk between ABA and defense signaling. Here we show that mpk9-1/12-1 double mutants are highly susceptible to Pseudomonas syringae DC3000 compared to WT plants. These results suggest that the regulation of stomatal apertures by MPK9 and MPK12 contributes to the first line of defense against pathogens.     

Guard cell zeaxanthin tracks photosynthetically active radiation and stomatal apertures in Vicia faba leaves*

Zeaxanthin, antheraxanthin and violaxanthin concentrations in guard cells from sonicated abaxial epidermal peels of Vicia faba were measured from dawn to dusk, and compared with concentrations in mesophyll tissue of the same leaves. Measured changes in guard cell zeaxanthin and violaxanthin concentrations indicate that guard cells operate the xanthophyll cycle throughout the day. Mesophyll tissue had no detectable zeaxanthin at dawn, whereas guard cells had 30–50 mmol mol^?1 chlorophyll a+b. On a chlorophyll basis, maximal zeaxanthin levels were 3–4 fold higher in guard cells than in mesophyll cells. Zeaxanthin concentrations tracked levels of photosynthetically active radiation (PAR) in both mesophyll and guard cells. In the mesophyll, most of the zeaxanthin changes occurred in mid-morning and mid-afternoon. In guard cells, zeaxanthin concentrations changed nearly linearly with PAR in the early morning and late afternoon, and closely tracked PAR levels throughout the day. Guard cell zeaxanthin concentrations were also closely correlated with stomatal apertures. The close relationship between zeaxanthin concentrations and PAR levels in guard cells indicates that zeaxanthin is well suited to function as a molecular photosensor in stomatal movements.     

Intra-vacuolar reserves of membranes during stomatal closure: the possible role of guard cell vacuoles estimated by 3-D reconstruction

Stomatal apertures are regulated by morphological changes in guard cells which have been associated with guard cell vacuolar structures. To investigate the contribution of guard cell vacuoles to stomatal movement, we examined the dynamics of vacuolar membrane structures in guard cells and evaluated the changes in vacuolar volumes and surface areas during stomatal movement. Using a transgenic Arabidopsis line expressing green fluorescent protein (GFP)-AtVAM3, we have found that the guard cell vacuolar structures became complicated during stomatal closure with the appearance of numerous intra-vacuolar membrane structures. A three-dimensional (3-D) reconstruction using our originally developed software, REANT (reconstructor and analyzer of 3-D structure), and photobleaching analysis revealed the continuity of the vacuolar structures, even when they appeared to be compartmented in confocal images of closed stomata. Furthermore, calculations of the surface area by REANT revealed an increase in vacuolar surface area during stomatal closure but a decrease in the surface area of the guard cells. Movement of a vital staining dye, FM4-64, to the vacuolar membrane was accelerated during ABA-induced stomatal closure in Vicia faba. These results suggest that the guard cell vacuoles store some portion of the excess membrane materials produced during stomatal closure as intra-vacuolar structures.     

Guard Cell Pressures and Wall Properties during Stomatal Opening

Pressures required to produce stomatal apertures of differentwidths have been measured in guard cells of T. virginiana andC. communis. These pressures are lower than those that coulddevelop in guard cells at full turgor on account of their osmoticproperties. During the Spannungsphase the metabolism of guardcells seems to contribute towards a reduction of the elasticmodulus of their walls, whereas during the motorphase the volumetricmodulus of the cell as a whole, apparently increases gentlyat first and then more markedly as maximum apertures are approached.     

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.     

The Effects of Temperature and ABA on Stomata of Zea mays L.

Epidermal fragments were removed from maize leaves by tearingparallel to the veins. These were incubated at a number of differenttemperatures in several concentrations of ABA. The sensitivityof stomata to temperature was dependent upon the technique usedto incubate epidermis. Generally, the widest apertures wererecorded at around 25°C. In all experiments, stomata incubatedat low (10°C) temperatures on 5.6 x 10^–4 M ABA showedwider apertures than those incubated on distilled water. ThisABA-stimulated stomatal opening was accompanied by an increasein the intensity of potassium staining in the guard cells. At25 °C, epidermis incubated on several concentrations ofABA showed some stomatal closure, decreased potassium stainingin the guard cells and increased potassium staining in the subsidiarycells.     

Phosphatidylinositol 3- and 4-phosphate are required for normal stomatal movements

Phosphatidylinositol (PI) metabolism plays a central role in signaling pathways in both animals and higher plants. Stomatal guard cells have been reported to contain PI 3-phosphate (PI3P) and PI 4-phosphate (PI4P), the products of PI 3-kinase (PI3K) and PI 4-kinase (PI4K) activities. In this study, we tested the roles of PI3P and PI4P in stomatal movements. Both wortmannin (WM) and LY294002 inhibited PI3K and PI4K activities in guard cells and promoted stomatal opening induced by white light or the circadian clock. WM and LY294002 also inhibited stomatal closing induced by abscisic acid (ABA). Furthermore, overexpression in guard cells of GFP:EBD (green fluorescent protein:endosome binding domain of human EEA1) or GFP:FAPP1PH (PI-four-P adaptor protein-1 pleckstrin homology domain), which bind to PI3P and PI4P, respectively, increased stomatal apertures under darkness and white light and partially inhibited stomatal closing induced by ABA. The reduction in ABA-induced stomatal closing with reduced levels of PI monophosphate seemed to be attributable, at least in part, to impaired Ca(2+) signaling, because WM and LY294002 inhibited ABA-induced cytosolic Ca(2+) increases in guard cells. These results suggest that PI3P and PI4P play an important role in the modulation of stomatal closing and that reductions in the levels of functional PI3P and PI4P enhance stomatal opening.     

Catalases negatively regulate methyl jasmonate signaling in guard cells

Methyl jasmonate (MeJA)-induced stomatal closure is accompanied by the accumulation of hydrogen peroxide (H?O?) in guard cells. In this study, we investigated the roles of catalases (CATs) in MeJA-induced stomatal closure using cat mutants cat2, cat3-1 and cat1 cat3, and the CAT inhibitor, 3-aminotriazole (AT). When assessed with 2',7'-dichlorodihydrofluorescein, the reduction of catalase activity by means of mutations and the inhibitor accumulated higher basal levels of H?O? in guard cells whereas they did not affect stomatal aperture in the absence of MeJA. In contrast, the cat mutations and the treatment with AT potentiated MeJA-induced stomatal closure and MeJA-induced H?O? production. These results indicate that CATs negatively regulate H?O? accumulation in guard cells and suggest that inducible H?O? production rather than constitutive elevation modulates stomatal apertures in Arabidopsis.     

Phospholipase C is required for the control of stomatal aperture by ABA

The calcium-releasing second messenger inositol 1,4,5-trisphosphate is involved in the regulation of stomatal aperture by ABA. In other signalling pathways, inositol 1,4,5-trisphosphate is generated by the action of phospholipase C. We have studied the importance of phospholipase C in guard cell ABA-signalling pathways. Immunolocalisation of a calcium-activated phospholipase C confirmed the presence of phospholipase C in tobacco guard cells. Transgenic tobacco plants with considerably reduced levels of phospholipase C in their guard cells were only partially able to regulate their stomatal apertures in response to ABA. These results suggest that phospholipase C is involved in the amplification of the calcium signal responsible for reductions in stomatal aperture in response to ABA. As full ABA-induced inhibition of stomatal opening was not observed, our results support a role for the action of other calcium-releasing second messengers in the guard cell ABA-signalling pathway. It is not known whether these different calcium-releasing second messengers act in the same or parallel ABA-signalling pathways.