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.     

Microtubule dynamics are involved in stomatal movement ofVicia faba L.

Summary To obtain a full picture of microtubule (MT) behavior during the opening and closure of guard cells we have microinjected living guard cells ofVicia faba with fluorescent tubulin, examined fine detail by freeze shattering fixed cells, and used drug treatments to confirm aspects of MT dynamics. Cortical MTs in fully opened guard cells are transversely oriented from the ventral wall to the dorsal wall. When the stomatal aperture was decreased by darkness, these MTs became twisted and patched and broken down into diffuse fragments when stomata were closed. When the closed stomata were opened in response to light, the MTs in guard cells changed from the diffused, transitional pattern back to one in which MTs are transversely oriented from stomatal pore to dorsal wall. This observation indicates a linkage between these MT changes and stomatal movement. To confirm this, we used the MT-stabilizing agent taxol and the MT-depolymerizing herbicide oryzalin and observed their effects on the stomatal aperture and MT dynamics. Both drugs suppressed light-induced stomatal opening and dark-induced closure. MTs are known to be necessary for maintaining the static kidney shape of guard cells; the present data now show that the dynamic properties of polymeric tubulin accompany changes in shape with stomatal movement and may be functionally involved in stomatal movement.     

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.

     

Overexpression of the Arabidopsis ��-expansin gene AtEXPA1 accelerates stomatal opening by decreasing the volumetric elastic modulus

Guard cell walls of stomata are highly specialized in plants. Previous research focused on the structure and anatomy of guard cell walls, but little is known about guard cell regulation during stomata movement. In this work, we investigate the possible biological role of the Arabidopsis expansin gene AtEXPA1 in stomatal opening. The AtEXPA1 promoter drove the expression of the GUS reporter gene specifically in guard cells. Light-induced stomatal opening was accelerated in 35S::AtEXPA1 lines, whereas the anti-AtEXPA1 antibody decelerated light-induced stomatal opening. The inhibition of the anti-AtEXPA1 antibody on stomatal opening was largely dependent on the environmental pH. The volumetric elastic modulus (ε) was measured as an indicator of changes in the cell wall. The ε value of guard cells in 35S::AtEXPA1 lines was smaller than in the wild types. The putative role of AtEXPA1 as controller of stomatal opening rate and its regulation are discussed.     

PECTATE LYASE LIKE12 patterns the guard cell wall to coordinate turgor pressure and wall mechanics for proper stomatal function in Arabidopsis

Plant cell deformations are driven by cell pressurization and mechanical constraints imposed by the nanoscale architecture of the cell wall, but how these factors are controlled at the genetic and molecular levels to achieve different types of cell deformation is unclear. Here, we used stomatal guard cells to investigate the influences of wall mechanics and turgor pressure on cell deformation and demonstrate that the expression of the pectin-modifying gene PECTATE LYASE LIKE12 (PLL12) is required for normal stomatal dynamics in Arabidopsis thaliana. Using nanoindentation and finite element modeling to simultaneously measure wall modulus and turgor pressure, we found that both values undergo dynamic changes during induced stomatal opening and closure. PLL12 is required for guard cells to maintain normal wall modulus and turgor pressure during stomatal responses to light and to tune the levels of calcium crosslinked pectin in guard cell walls. Guard cell-specific knockdown of PLL12 caused defects in stomatal responses and reduced leaf growth, which were associated with lower cell proliferation but normal cell expansion. Together, these results force us to revise our view of how wall-modifying genes modulate wall mechanics and cell pressurization to accomplish the dynamic cellular deformations that underlie stomatal function and tissue growth in plants.     

The function of guard cells does not require an intact array of cortical microtubules

The development of stomatal guard cells is known to require cortical microtubules; however, it is not known if microtubules are also required by mature guard cells for stomatal function. To study the role of microtubules in guard cell function, epidermal peels of Vicia faba were subjected to conditions known to open or close stomata in the presence or absence of microtubule inhibitors. To verify the action of the inhibitors, microtubules in appropriately treated epidermal peels were localized by cryofixation followed by freeze substitution and embedding in butyl-methyl methacrylate. Mature guard cells had a radial array of microtubules, focused toward the thick cell wall of the pore, and the appearance of this array was the same for stomata remaining closed in darkness or induced to open by light. Treatment of epidermal peels with 1 mM colchicine for 1 h depolymerized nearly all cortical microtubules. Measurements of stomatal aperture showed that neither 1 mM colchicine nor 20 M taxol affected any of the responses tested: remaining closed in the dark, opening in response to light or fusicoccin, and closing in response to calcium and darkness. We conclude that intact microtubule arrays are not invariably required for guard cell function.     

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.     

Effect of Potassium Levels on the Stomatal Behavior of the Hemi-Parasite Striga hermonthica

The hemi-parasite Striga hermonthica, exhibits an anomalous pattern of stomatal response, stomata remaining open in darkness and when subjected to water stress. This suggests irregularity in stomatal response due to malfunction of the stomatal mechanism. To test this suggestion guard cells were isolated from the effects of surrounding cells, by incubating epidermal strips at low pH. These stomata responded rapidly to low CO₂ concentrations, darkness, and ABA. Thus, a paradox exists between stomatal behavior observed in whole leaves and that in isolated guard cells. However, when incubated in the presence of high potassium concentrations (>200 millimolar KCl) stomatal responses in epidermal strips resembled those found in whole leaves, with enhanced opening and reduced closing responses. It is suggested that the anomalous behavior of stomata in Striga and other leafy hemiparasites can be explained by the modulatory effects of high potassium concentrations which accumulate in the leaves as a consequence of high transpiration rates and the lack of a retranslocation system.     

Changes in K+, Cl− and P contents in stomata of Zea mays leaves exposed to different light and CO2 levels

Maize plants (Zea mays L. hybrid INRA 508) were placed under controlled conditions of light and CO₂ partial pressure. The K⁺, Cl^? and P contents were then determined by X-ray microanalysis in the bulbous end of guard cells and in the center of subsidiary cells. The results were interpreted in connection with the stomatal conductance at the time of sampling. In normal air, the K⁺ and Cl^? contents in guard cells only rose from a light threshold of about 300 μmol m^?2 s^?1 at which stomata were already largely open. At 600 μmol m^?2 s^?1, the K⁺ and Cl^? levels in guard cells attained values that were 3- and 8-fold greater, respectively, than the values observed in darkness. The K⁺ and Cl^? contents in the subsidiary cells remained quite constant irrespective of the light conditions. CO₂-free air in darkness induced a significant K⁺ influx towards guard and subsidiary cells. Under light and in CO₂-free air, the K⁺ and Cl^? contents dramatically increased in the guard cells, but slightly decreased in the subsidiary cells. Thus, when subjected to strong light in CO₂-free air, the K⁺ and Cl^? contents in the subsidiary cells were approximately equal to those measured in normal air conditions. In the guard cells, stomatal opening was associated with a marked shift of the Cl^?/K⁺ ratio – from 0.3 for closed stomata to ca 1 for fully open stomata. This could imply a slow change in the nature of the principal counterion accompanying K⁺ during stomatal opening. The content of P in guard cells appeared, in contrast to that of K⁺ and Cl^?, to be practically independent of stomatal aperture.     

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.     

Roles of phosphoinositides in regulation of stomatal movements

Guard cells sense various environmental and internal stimuli and, in response, modulate the stomatal aperture to a size optimal for growth and adaptation. Among the many factors involved in the fine regulation of stomata, we have focused our studies on the role of phosphoinositides. Our recent study published in the Plant Journal (52:803–16) provides evidence for an important role for phosphatidylinositol 4,5-bis-phosphate (PtdIns(4,5)P₂) in inducing stomatal opening. Light induces translocation of a PtdIns(4,5)P₂-binding protein from the cytosol to the plasma membrane and treatments that increase the intracellular PtdIns(4,5)P₂ level induce stomatal opening in the absence of light irradiation. Inhibition of anion channel activity, a negative regulator for stomatal opening, was suggested as a mechanism of PtdIns(4,5)P₂-induced stomatal opening. We also reported that phosphatidylinositol 3-phosphate (PtdIns(3)P) and phosphatidylinositol 4-phosphate (PtdIns(4)P) regulate actin dynamics in guard cells. The effects of the phosphoinositides were specific, and were not induced by other lipids with similar structures. The roles of different interacting partners are likely to be important for these lipids to produce specific changes in guard cell activity.Key words: PtdIns(4,5)P₂; PtdIns(4)P; Ins(1,4,5)P₃; anion channel; PIP kinase; phospholipase C; stomatal opening; guard cells     

Malate Dehydrogenases in Guard Cells of Pisum sativum

Guard cell protoplasts of Pisum sativum show considerable NADP-dependent malate dehydrogenase (MDH) activity in darkness which can be enhanced severalfold by illumination or treatment with dithiothreitol (DTT). The question arose whether guard cells possess an NADP-MDH different from that present in the chloroplasts of the mesophyll (which is inactive in darkness or in the absence of DTT). MDH activities were determined in extracts of isolated protoplasts from mesophyll and epidermis, and in mechanically prepared epidermal pieces (with guard cells as the only living cells and no interference from proteases originating from the cell wall digesting enzymes). Guard cells possessed NAD-dependent MDHs of high activity and incomplete exclusion of NADP as a coenzyme. This NADP-dependent activity of the NAD-MDH(s) could not be stimulated by DTT or, inferentially, by light. The DTT- (and light-) dependent NADP-MDH represented 0.05% of the total protein of the guard cells and had a specific activity of 0.1 unit per milligram protein; both values are in the same range as the corresponding ones of the mesophyll cells. Agreement was also found in the extent of light activation, in subunit molecular weight, immunological cross-reactions, and in the behavior on an ion exchange column. The activity of the chloroplastic NADP-MDH in guard cells barely suffices to meet the malate requirement for stomatal opening in the light. It is therefore likely that NAD-MDHs residing in other compartments of the guard cells supplement the activity of the chloroplastic NADP-MDH particularly during stomatal opening in darkness.     

Metabolomics of red‐light‐induced stomatal opening in Arabidopsis thaliana: Coupling with abscisic acid and jasmonic acid metabolism

Environmental stimuli‐triggered stomatal movement is a key physiological process that regulates CO₂ uptake and water loss in plants. Stomata are defined by pairs of guard cells that perceive and transduce external signals, leading to cellular volume changes and consequent stomatal aperture change. Within the visible light spectrum, red light induces stomatal opening in intact leaves. However, there has been debate regarding the extent to which red‐light‐induced stomatal opening arises from direct guard cell sensing of red light versus indirect responses as a result of red light influences on mesophyll photosynthesis. Here we identify conditions that result in red‐light‐stimulated stomatal opening in isolated epidermal peels and enlargement of protoplasts, firmly establishing a direct guard cell response to red light. We then employ metabolomics workflows utilizing gas chromatography mass spectrometry and liquid chromatography mass spectrometry for metabolite profiling and identification of Arabidopsis guard cell metabolic signatures in response to red light in the absence of the mesophyll. We quantified 223 metabolites in Arabidopsis guard cells, with 104 found to be red light responsive. These red‐light‐modulated metabolites participate in the tricarboxylic acid cycle, carbon balance, phytohormone biosynthesis and redox homeostasis. We next analyzed selected Arabidopsis mutants, and discovered that stomatal opening response to red light is correlated with a decrease in guard cell abscisic acid content and an increase in jasmonic acid content. The red‐light‐modulated guard cell metabolome reported here provides fundamental information concerning autonomous red light signaling pathways in guard cells.     

Guard Cell Chloroplasts Are Essential for Blue Light-Dependent Stomatal Opening in Arabidopsis

Blue light (BL) induces stomatal opening through the activation of H⁺-ATPases with subsequent ion accumulation in guard cells. In most plant species, red light (RL) enhances BL-dependent stomatal opening. This RL effect is attributable to the chloroplasts of guard cell, the only cells in the epidermis possessing this organelle. To clarify the role of chloroplasts in stomatal regulation, we investigated the effects of RL on BL-dependent stomatal opening in isolated epidermis, guard cell protoplasts, and intact leaves of Arabidopsis thaliana. In isolated epidermal tissues and intact leaves, weak BL superimposed on RL enhanced stomatal opening while BL alone was less effective. In guard cell protoplasts, RL enhanced BL-dependent H⁺-pumping and DCMU, a photosynthetic electron transport inhibitor, eliminated this effect. RL enhanced phosphorylation levels of the H⁺-ATPase in response to BL, but this RL effect was not suppressed by DCMU. Furthermore, DCMU inhibited both RL-induced and BL-dependent stomatal opening in intact leaves. The photosynthetic rate in leaves correlated positively with BL-dependent stomatal opening in the presence of DCMU. We conclude that guard cell chloroplasts provide ATP and/or reducing equivalents that fuel BL-dependent stomatal opening, and that they indirectly monitor photosynthetic CO₂ fixation in mesophyll chloroplasts by absorbing PAR in the epidermis.     

Sugar Concentrations in Guard Cells of Vicia faba Illuminated with Red or Blue Light : Analysis by High Performance Liquid Chromatography

Concentrations of soluble sugars in guard cells in detached, sonicated epidermis from Vicia faba leaves were analyzed quantitatively by high performance liquid chromatography to determine the extent to which sugars could contribute to changes in the osmotic potentials of guard cells during stomatal opening. Stomata were illuminated over a period of 4 hours with saturating levels of red or blue light, or a combination of red and blue light. When stomata were irradiated for 3 hours with red light (50 micromoles per square meter per second) in a solution of 5 millimolar KCl and 0.1 millimolar CaCl₂, stomatal apertures increased a net maximum of 6.7 micrometers and the concentration of total soluble sugar was 289 femtomoles per guard cell (70% sucrose, 30% fructose). In an identical solution, 2.5 hours of irradiation with 25 micromoles per square meter per second of blue light caused a maximum net increase of 7.1 micrometers in stomatal aperture and the total soluble sugar concentration was 550 femtomoles per guard cell (91% sucrose, 9% fructose). Illumination with blue light at 25 micromoles per square meter per second in a solution lacking KCl caused a maximum net increase in stomatal aperture of 3.5 micrometers and the sugar concentration was 382 femtomoles per guard cell (82% sucrose, 18% fructose). In dual beam experiments, stomata irradiated with 50 micromoles per square meter per second of red light opened steadily with a concomitant increase in sugar production. Addition of 25 micromoles per square meter per second of blue light caused a further net gain of 3.7 micrometers in stomatal aperture and, after 2 hours, sugar concentrations had increased by an additional 138 femtomoles per guard cell. Experiments with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) were performed with epidermis illuminated with 50 micromoles per square meter per second of red light or with 25 micromoles per square meter per second of blue light in solutions containing or lacking KCl. DCMU completely inhibited sugar production under red light, had no effect on guard cell sugar production under blue light when KCl was present, and inhibited sugar production by about 50% when guard cells were illuminated with blue light in solutions lacking KCl. We conclude that soluble sugars can contribute significantly to the osmoregulation of guard cells in detached leaf epidermis of V. faba. These results are consistent with the operation of two different sugar-producing pathways in guard cells: a photosynthetic carbon reduction pathway and a pathway of blue light-induced starch degradation.     

Responses of Stomata to IAA and Fusicoccin at the Opposite Phases of an Entrained Rhythm

The stomata of Commelma communis showed reduced opening responsesto light and low CO₂ concentrations during the night phase oftheir entrained circadian rhythm. Increased supplies of potassiumions, and treatments with indol-3-ylacetic acid and fusicoccin,failed to promote opening during the night phase to a levelequivalent to that in the day phase. The inability of fusiccocinto overcome the suppression of opening during the night phasecontrasts with its ability to counteract the closure inducedby agents such as CO₂, darkness and abscisic acid. It is concludedthat there are at least two basic mechanisms by which the turgorof guard cells can be regulated, one which is susceptible tooverriding control by fusicoccin and another which is unaffectedby fusicoccin. Several previous studies had shown a positive correlation betweenmalate in the epidermis (mainly located in guard cells) andstomatal opening. In the present experiments the aperture/malatecorrelation was broken in epidermis treated with fusicoccinduring the night phase of the rhythm. The amount of malate presentexceeded that associated with the same stomatal aperture inthe day phase. Possible explanations are (1) that fusicoccinstimulates similar proton fluxes out of the guard cells duringboth phases of the rhythm, but an unknown factor imposes a restrictionon stomatal opening during the night phase; (2) that there arelower proton fluxes in the night phase (limited, for example,by a reduced supply of ATP) but chloride availability or transportis reduced to an even greater extent so that a larger productionof malate in the guard cells is required. Key words: Stomata, IAA, Fusicoccin, Rhythms     

Role of vacuolar invertase in regulating Arabidopsis stomatal opening

Guard cells sense and integrate environmental signals to modulate stomatal aperture in response to diverse conditions. In this study, the effect of vacuolar invertase on Arabidopsis stomatal opening was investigated. The technology of enzyme activity detection in situ was used to show that the vacuolar invertase activity was much higher in guard cells than in other epidermal cells. The stomatal aperture of T-DNA insertion mutant in At1g12240 (inv-7) was significantly lower than that in wild-type plants. Increased stomatal aperture was observed in the transgenic Arabidopsis overexpressing cotton vacuolar invertase gene. These results indicated that Arabidopsis stomatal aperture was correlated with vacuolar invertase, and that vacuolar invertase may play an important role in regulating Arabidopsis stomatal opening.     

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.     

Metabolic energy for stomatal opening. Roles of photophosphorylation and oxidative phosphorylation

The supply of energy for stomatal opening was investigated with epidermal peels of Commelina communis L. and Vicia faba L., under white, blue and red irradiation or in darkness. Fluencerate response curves of stomatal opening under blue and red light were consistent with the operation of two photosystems, one dependent on photosynthetic active radiation (PAR) and the other on blue light, in the guard cells. The PAR-dependent system was 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-sensitive and KCN-resistant and showed a relatively high threshold irradiance for its activation; its activity was most prominent at moderate to high irradiances. The blue-light-dependent photosystem was KCN-sensitive, was active at low irradiances, and interacted with the PAR-dependent photosystem at high blue irradiances. Stomatal opening in darkness, caused by CO₂-free air, fusicoccin or high KCl concentrations, was KCN-sensitive and DCMU-resistant. These data indicate that stomatal opening in darkness depends on oxidative phosphorylation for the supply of high-energy equivalents driving proton extrusion. Light-dependent stomatal opening appears to require photophosphorylation from guard-cell chloroplasts and the activation of the blue-light photosystem which could rely either on oxidative phosphorylation or a specific, membrane-bound electron-transport carrier.Abbreviations DCMU 3(3,4-dichlorophenyl)-1-1-dimethylurea - FC fusicoccin - KCN potassium cyanide - PAR photosynthetic active radiation - WL white light