Guard cells of Commelina communis L. do not respond metabolically to osmotic stress in isolated epidermis: Implications for stomatal responses to drought and humidity

We investigated the hypothesis that stomatal aperture is regulated by epidermal water status. Detached epidermal peels of Commelina communis L. or leaf disks with epidermis attached were incubated in graded solutions of mannitol (0–1.2 M) containing KCl. In isolated epidermis, guard-cell solute content of open stomata did not decrease in response to desiccation. Guard cells of closed stomata accumulated solutes to the same extent in all levels of mannitol tested. There was no evidence of stress-induced hydroactive closure nor of inhibition of hydroactive opening, even when guard cells of closed stomata were initially plasmolyzed. Hydropassive, osmometer-like, changes in stomatal aperture in the isolated epidermis were induced by addition or removal of mannitol, but these did not involve changes in guard-cell solute content. In leaf disks, stomata exhibited clear hydroactive stomatal responses. Steady-state guard-cell solute content of initially open and initially closed stomata decreased substantially with increasing mannitol. Stomata were completely closed above approx. 0.4 M mannitol, near the turgor-loss point for the bulk leaf tissue. Stomata of Commelina did not exhibit direct hydroactive responses to environmental or epidermal water status. Stomatal responses to water deficit and low humidity may be indirect, mediated by abscisic acid or other signal metabolite(s) from the mesophyll.Abbreviations ABA abscisic acid - EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Mes 2-(N-morpholino)ethanesulfonic acid     

Changes in apoplastic pH and membrane potential in leaves in relation to stomatal responses to CO2, malate, abscisic acid or interruption of water supply

Low CO2 concentrations open CO2-sensitive stomata whereas elevated CO2 levels close them. This CO2 response is maintained in the dark. To elucidate mechanisms underlying the dark CO2 response we introduced pH- and potential-sensitive dyes into the apoplast of leaves. After mounting excised leaves in a gas-exchange chamber, changes in extracellular proton concentration and transmembrane potential differences as well as transpiration and respiration were simultaneously monitored. Upon an increase in CO2 concentration transient changes in apoplastic pH (occasionally brief acidification, but always followed by alkalinization) and in membrane potential (brief hyperpolarization followed by depolarization) accompanied stomatal closure. Alkalinization and depolarization were also observed when leaves were challenged with abscisic acid or when water flow was interrupted. During stomatal opening in response to CO2-free air the apoplastic pH increased while the membrane potential initially depolarized before it transiently hyperpolarized. To examine whether changes in apoplastic malate concentrations represent a closing signal for stomata, malate was fed into the transpiration stream. Although malate caused apoplastic alkalinization and membrane depolarization reminiscent of the effects observed with CO2 and abscisic acid, this dicarboxylate closed the stomata only partially and less effectively than CO2. Apoplastic alkalinization was also observed and stomata closed partially when KCl was fed to the leaves. Respiration increased on feeding of malate or KCl, or while abscisic acid closed the stomate. From these results we conclude that CO2 signals modulate the activity of plasma-membrane ion channels and of plasmalemma H+-ATPases during changes in stomatal aperture. Responses to potassium malate and KCl are not restricted to guard cells and neighbouring cells.     

Stomatal responses to water stress and to abscisic Acid in phosphorus-deficient cotton plants

Cotton (Gossypium hirsutum L.) plants were grown in sand culture on nutrient solution containing adequate or growth-limiting levels of P. When water was withheld from the pots, stomata of the most recently expanded leaf closed at leaf water potentials of approximately −16 and −12 bars in the normal and P-deficient plants, respectively. Pressure-volume curves showed that the stomata of P-deficient plants closed when there was still significant turgor in the leaf mesophyll. Leaves of P-deficient plants accumulated more abscisic acid (ABA) in response to water stress, but the difference was evident only at low water potentials, after initiation of stomatal closure. In leaves excised from unstressed plants, P deficiency greatly increased stomatal response to ABA applied through the transpiration stream. Kinetin blocked most of this increase in apparent sensitivity to ABA. The effect of P nutrition on stomatal behavior may be related to alterations of the balance between ABA and cytokinins.     

Effect of the Mesophyll on Stomatal Opening in Commelina communis

The effect of a number of factors on the opening of stomatain the intact leaf and in the isolated leaf epidermis of Commelinacommunishas been investigated. Stomata in the intact leaf opened widein the light and closed rapidly on transfer to the dark. Theywere also sensitive to CO₂. In contrast, stomata in isolatedepidermis floated on an incubation solution containing 100 molm^–3KCl responded neither to light nor CO₂. They opened as widelyas those in the intact leaf when treated with fusicoccin. Stomata in isolated epidermis opened almost as wide as thosein the intact leaf when they were incubated with isolatedmesophyllcells in the light. The solution in which the mesophyll cellswere incubated was separated by centrifugation. Themedium fromcells previously incubated in the light caused the stomata inisolated epidermis to open but that from cells kept inthe darkhad no effect. A similar effect was observed when isolated chloroplastswere incubated with the isolated epidermis.However, the supernatantfrom the chloroplast suspension had no significant effect onstomatal opening. These results indicate that the mesophyll plays an importantrole in stomatal opening in the light. The mesophyll appearstoproduce in the light, but not in the dark, a soluble compoundwhich moves to the guard cells to bring about stomatal opening.Theexperiments with isolated chloroplasts suggest that this substanceis a product of photosynthesis. Key words: Commelina communis, stomata, light, mesophyll     

Evidence for the Uptake of Large Anions through Stomatal Pores

Experiments were conducted with leek (Allium porrum L.) leaves to investigate whether aqueous solutions are able to penetrate stomata. Epidermal strips were used for the determination of transport rates. Stomata were opened by fusicoccin or closed by darkness or abscisic acid. A droplet containing the anionic fluorescent dye, uranine, was placed on the physiologically outer side of the epidermis and allowed to dry. With open stomata 30 times more uranine penetrated through the epidermal strips than with closed stomata (comparison of medians). In another experiment droplets of uranine solution were placed on leaf segments and epidermal strips were removed after drying of the droplets. Penetration of uranine through stomata was detectable under the microscope both with epidermal strips from the transport experiments and with strips obtained after application on leaf segments. As maximum uptake rates occurred during the drying process, it is concluded that penetration took place via water films. These results show that the physical restrictions preventing stomatal penetration of static droplets are not decisive for drying droplets and that stomatal uptake of dissolved ionic substances occurs under natural conditions, i.e. without surfactants or applied pressure.     

Stomatal opening by fusicoccin is accompanied by depolymerization of actin filaments in guard cells

Actin in guard cells is assembled in a radial pattern when stomata are induced to open under light, but the filaments are disassembled when stomata are closed under darkness or by abscisic acid (S.-O. Eun and Y. Lee, 1997, Plant Physiol. 115: 1491–1498). To test if signals that open stomata commonly generate the polymerized form of actin in guard cells, leaves of Commelina communis L. were treated with a potent stomatal opening agent, fusicoccin, and the actin organization examined by immunolocalization techniques. When stomata were induced to open by fusicoccin, hardly any of the filamentous form of actin was detected; instead, the actin resembled that present in guard cells that had been treated with an antagonist to actin filaments, cytochalasin D, and showed a sharp contrast to the long filaments developed in illuminated guard cells. Furthermore, treatment of illuminated leaves with fusicoccin disintegrated actin filaments that had already been formed in the guard cells. Preincubation of leaves with phalloidin, which interferes with fusicoccin-induced actin depolymerization, delayed fusicoccin-induced opening during the early phase. These observations suggest that the prevention of actin filament formation and/or depolymerization of actin filaments may accelerate the stomatal opening process in response to fusicoccin. Received: 1 October 1999 / Accepted: 29 November 1999     

Age-related Changes in Stomatal Response to Cytokinins and Abscisic Acid

Kinetin and zeatin(100 mmol m^–3)reversald the ABA-mediated(100mmol ^m-2)closure of stomata of young maize leaves but did notaffect stomatal apertures of these leaves when applied alone.As leaves aged, kinetin or zeatin alone promoted increased stomatalapertures, while abscisic acid (ABA) applied alone had a reducedeffect on stomata. Even with older leaves, cytokinins reversadthe effect of ABA on stomata. Maize, stomata, abscisic acid, kineusc, zeatin, Zea mays     

Malate metabolism in isolated epidermis of Commelina communis L. in relation to stomatal functioning

Epidermal strips with closed stomata were exposed to malic acid labelled with ¹⁴C either uniformly or in 4-C only. During incubation with [U-¹⁴C]malate, radioactivity appeared in products of the tricarboxylic-acid cycle and in transamination products within 10 min, in sugars after 2 h. Hardly any radioactivity was found in sugars if [4-¹⁴C]malate had been offered. This difference in the degree of labelling of sugars indicates that gluconeogenesis can occur in epidermal tissue, involving the decarboxylation of malate. Epidermis incubated with labelled malate was hydrolyzed after extraction with aqueous ethanol. The hydrolysate contained glucose as the only radioactive product, indicating that starch had been formed from malate. Microautoradiograms were black above stomatal complexes, showing that the latter were sites of starch formation. In order to follow the fate of malate during stomatal closure, malate was labelled in guard cells by exposing epidermes with open stomata to ¹⁴CO₂ and then initiating stomatal closure. Of the radioactive fixation products of CO₂ only malate was released into the water on which the epidermal samples floated; the epidermal strips retained some of the malate and all of its metabolites. In the case of rapid stomatal closure initiated by abscisic acid and completed within 5 min, 63% of the radioactivity was in the malate released, 22% in the malate retained, the remainder in aspartate, glutamate, and citrate. We conclude that during stomatal closing guard cells can dispose of malate by release, gluconeogenesis, and consumption in the tricarboxylic-acid cycle.Abbreviations ABA abscisic acid - NAD nicotinamide adenine dinucleotide - NADP nicotinamide adenine dinucleotide phosphate - PEP phosphoenolpyruvate     

Stomatal Responses to Water Deficits and Abscisic Acid in Leaves of Sunflower Plants (Helianthus annuus L.) Grown under Different Conditions

The decrease in diffusive conductance of a leaf exposed to waterstress or to exogenous abscisic acid (ABA) was smaller in leavesof sunflower plants (Helianthus annuus L. cv. NK285) that hadbeen grown in a phytotron in humid air than in leaves of sunflowersgrown outdoors. Stomata of the phytotron-grown plants were slowerto close after detachment of a leaf than those of the outdoorplants. When stomata closed rapidly, as they did in detachedleaves and after treatment with ABA, the extent of closure wasvaried over the leaf's surface, in particular in the case ofphytotron-grown plants, and the extent of the heterogeneitywas greater in the phytotrongrown plants than in the outdoorplants. When stomata closed gradually, for example, under conditionsof limited moisture in the soil, closure occurred uniformlyover leaves of plants of both types. The smaller decrease indiffusive conductance of leaves from phytotron-grown plantsafter treatment with ABA resulted from the presence of patcheson the surface in which stomata remained open. The smaller decreaseof diffusive conductance in the phytotron-grown plants underconditions of limited moisture in the soil resulted from theuniformly lower responsiveness of stomata on a leaf to the decreasein water potential. When estimates are made of the intercellularconcentration of CO₂ (Ci) from gas-exchange measurements, heterogeneityin stomatal closure should be monitored when stomata close rapidly,in particular in plants grown in humid air, because heterogeneousstomatal closure can lead to overestimates of Ci. (Received April 18, 1994; Accepted May 25, 1995)     

Electrical potentials in stomatal complexes

Guard cells of several species, but predominantly Commelina communis, were impaled by micropipette electrodes and potential differences measured that occurred between cell compartments and the flowing bathing medium. The wall developed a Donnan potential that was between −60 and −70 millivolt in 30 millimolar KCl at pH 7. The density of the fixed charges ranged from 0.3 to 0.5 molar; its dependence on pH was almost identical with the titration curve of authentic polygalacturonic acid. The vacuolar potential of guard cells of Commelina communis L., Zea mays L., Nicotiana glauca Graham, Allium cepa L., and Vicia faba L. was between −40 and −50 millivolt in 30 millimolar KCl when stomata were open and about −30 millivolt when stomata were closed. The vacuolar potential of guard cells of C. communis was almost linearly related to stomatal aperture and responded to changes in the ionic strength in the bathing medium in a Nernstian manner. No specificity for any alkali ion (except Li⁺), ammonium, or choline appeared. Lithium caused hyperpolarization. Calcium in concentrations between 1 and 100 millimolar in the medium led to stomatal closure, also caused hyperpolarization, and triggered transient oscillations in the intracellular potential. Gradients in the electrical potential existed across stomatal complexes with open pores. When stomata closed, these gradients almost disappeared or slightly reverted; all epidermal cells were then at potentials near −30 millivolt in 30 millimolar KCl.     

Transcriptome Analysis of Plant Hormone-Related Tomato (Solanum lycopersicum) Genes in a Sunlight-Type Plant Factory

In plant factories, measurements of plant conditions are necessary at an early stage of growth to predict harvest times of high value-added crops. Moreover, harvest qualities depend largely on environmental stresses that elicit plant hormone responses. However, the complexities of plant hormone networks have not been characterized under nonstress conditions. In the present study, we determined temporal expression profiles of all genes and then focused on plant hormone pathways using RNA-Seq analyses of gene expression in tomato leaves every 2 h for 48 h. In these experiments, temporally expressed genes were found in the hormone synthesis pathways for salicylic acid, abscisic acid, ethylene, and jasmonic acid. The timing of CAB expression 1 (TOC1) and abscisic acid insensitive 1 (ABA1) and open stomata 1 (OST1) control gating stomata. In this study, compare with tomato and Arabidopsis thaliana, expression patterns of TOC1 have similarity. In contrast, expression patterns of tomato ABI1 and OST1 had expression peak at different time. These findings suggest that the regulation of gating stomata does not depend predominantly on TOC1 and significantly reflects the extracellular environment. The present data provide new insights into relationships between temporally expressed plant hormone-related genes and clock genes under normal sunlight conditions.     

Isotachophoretic Analysis of Ions in Guard Cells of Vicia faba

Isotachophoretic analysis of ions was performed on guard cells of Vicia faba cv. Ryosai Issun with either open or closed stomata. In guard cells of open stomata, K⁺ and malic acid concentrations were 5–7 and 5–10 times higher, respectively, than in guard cells of closed stomata. The content of citric acid (plus isocitric acid) also increased during stomatal opening, but the increment was smaller than that of malic acid. Sodium ions, phosphoric and glyceric acids were present in low concentrations but did not increase during the opening. Other cations and anions could not be measured because of low concentrations. Malic acid provided 68–79% of the counter anions for the potassium taken up by guard cells during stomatal opening.     

The Effect of Flowering on Stomatal Response to Water Stress in Pearl Millet (Pennisetum americanum [L.] Leeke)

Stomata on upper leaves of drought-stressed pearl millet (Pennisetumamericanum [L.] Leeke) crops were more open in flowering (F)than in pre-flowering (PF) plants. This was not due to differencesin leaf water potential (). Stomata of PF plants closed when fell to about –1.7 MPa, while on F plants stomata closedonly when approached –2.3 MPa. Osmotic adjustment did not account for these differences asrelations between turgor potential (_P) and were similar inF and PF plants. While stomata of PF plants closed as W becamezero, in F plants stomata remained open even after bulk leafturgor was lost. Differences between F and PF plants were not explained by differencesin age of leaves sampled. However, leaves of water-stressedPF plants had higher levels of abscisic acid (ABA) than leavesof F plants, despite similarities in water status. From theseresults and from relationships between g_L and stage of panicledevelopment, it is concluded that the tendency of stomata toremain open despite water stress and loss of bulk leaf _P isrelated to the presence of an emerged panicle. Hypotheses whichaccount for this effect are discussed. Key words: Pennisetum americanum [L.] Leeke, Pearl millet, Flowering, Stomata, Water stress, Abscisic acid     

Stomatal responses of Argenteum — a mutant of Pisum sativum L. with readily detachable leaf epidermis

Epidermis is easily detached from both adaxial and abaxial surfaces of leaf four of the Argenteum mutant of Pisum sativum L. The isolated epidermis has stomata with large, easily-measured pores. Hairs and glands are absent. The density of stomata is high and contamination by mesophyll cells is low. In the light and in CO₂-free air, stomata in isolated adaxial epidermis of Argenteum mutant opened maximally after 4 h incubation at 25°C. The response of stomata to light was dependent on the concentration of KCl in the incubation medium and was maximal at 50 mol m^-3 KCl. Stomata did not respond to exogenous kinetin, but apertures were reduced by incubation of epidermis on solutions containing between 10^-5 and 10^-1 mol m^-3 abscisic acid (ABA). The responses of stomata of Argenteum mutant to light, exogenous KCl, ABA and kinetin were comparable with those described previously for stomata in isolated epidermis of Commelina communis. A method for preparing viable protoplasts of guard cells from isolated epidermis of Argenteum mutant is described. The response of guard cell protoplasts to light, exogenous KCl, ABA and kinetin were similar to those of stomata in isolated epidermis except that the increase in volume of the protoplasts in response to light was maximal at a lower concentration of KCl (10 mol m^-3) and that protoplasts responded more rapidly to light than stomata in isolated epidermis. The protoplasts did not respond to exogenous kinetin, but when incubated for 1 h in the light and in CO₂-free air on a solution containing 10^-3 mol m^-3 ABA, they decreased in volume by 30%. The advantages of using epidermis from Argenteum mutant for experiments on stomatal movements are discussed.Abbreviations ABA abscisic acid - MES 2-(N-morpholino)ethanesulfonic acid     

Water Relations of Cotton Plants under Nitrogen Deficiency: III. STOMATAL CONDUCTANCE, PHOTOSYNTHESIS, AND ABSCISIC ACID ACCUMULATION DURING DROUGHT

Nitrogen nutrition exerted a strong effect on stomatal sensitivity to water stress in cotton. In well-watered plants grown with 0.31 millimolar N in the nutrient solution, stomata closed at a water potential of -9 bars even though the wilting point was below -15 bars. For each doubling of nutrient N level, the water potential for stomatal closure decreased by about 2 bars. Elevated intercellular CO₂ concentrations caused only slight stomatal closure regardless of N nutrition. Exogenous abscisic acid (ABA) greatly increased stomatal sensitivity to elevated CO₂ concentrations.     

Stomatal responses of Vicia faba L. to indole acetic acid and abscisic acid

Evidence is presented that stomata in isolated epidermal peelsof Vicia faba L. open in darkness in response to the externalpresence of indole acetic acid (IAA) in the incubation medium.The effect of IAA is found to be overcome completely in thepresence of either TRIS or MES buffers. In the absence of buffer,V. faba stomata are shown to be influenced by IAA in a concentration-dependenttrend which reached a maximum at an [IAA] of 10^–3 molm^–3. Further investigations reveal that stomata in thisspecies can be shown to respond to the presence of IAA and anotherphytohormone, abscisic acid (ABA). IAA and ABA are demonstratedto be antagonistic in their effects provided the incubationconditions are suitable. The data are discussed in relationto stomatal responses of other species in different treatmentconditions. Recommendations are made with respect to standardizationof incubation media during epidermal peel experiments. Key words: Vicia faba, stomata, indole acetic acid, abscisic acid, buffers     

Some new observations and interpretations on the vital staining of leaf epidermal tissue by neutral red

Summary The lower leaf epidermis from 5 plant species was stained with neutral red at 2 pH's (7.1 and 5.6) in the light and dark when the stomata were open or closed. At pH 5.6 no globule (= droplet) formation was observed in the guard cells whether the stomata were open or closed and cell walls possessed a high affinity for the stain. At pH 7.1 globules appeared in guard cells of open stomata, but not closed stomata, within 15 minutes. Anaerobic conditions prevented this globule formation. InZea mays, globules also appeared in subsidiary cells when the stomata were closed and in certain epidermal cells. Where globule formation did not occur increased diffuse staining of certain epidermal cells was considered to be the indication of cell integrity. In old leaf material very large numbers of dark blue globules appeared in epidermal cells ofCommelina diffusa, C. communis andSenecio odoris and this was associated with cell senescence.The staining characteristics were discussed in terms of cellular K⁺, Cl^–, tannin and flavonoid content and vacuolar pH.     

Visualizing Changes in Cytosolic-Free Ca2+ during the Response of Stomatal Guard Cells to Abscisic Acid

In this paper, we report the results of a detailed investigation into abscisic acid (ABA)[mdash]stimulated elevations of guard cell cytosolic-free Ca2+ ([Ca2+]cyt). Fluorescence ratio photometry and ratio imaging techniques were used to investigate this phenomenon. Guard cells of open and closed (opened to 10 to 12 [mu]m before treatment with ABA) stomata were microinjected with the fluorescent Ca2+ indicator Indo-1. Resting [Ca2+]cyt ranged from 50 to 350 nM. ABA (100 nM) stimulated an increase in [Ca2+]cyt in 68 and 81% of guard cells microinjected in the open and closed configuration, respectively. All stomata were observed to close in response to ABA. Increases ranged from 100 to 750 nM above the resting concentration and were arbitrarily grouped into five "classes." ABA-stimulated increases in [Ca2+]cyt were not uniformly distributed across the cytosol of guard cells. Rapid transient increases in [Ca2+]cyt were also observed in the guard cells of stomata microinjected in the closed configuration. We concluded that the ABA-induced turgor loss in guard cells is a Ca2+-dependent process.     

Abscisic Acid and stomatal regulation

The closure of stomata by abscisic acid was examined in several species of plants through measurements of CO₂ and H₂O exchange by the leaf. The onset of closure was very rapid, beginning at 3 minutes from the time of abscisic acid application to the cut base of the leaf of corn, or at 8 or 9 minutes for bean, Rumex and sugarbeet; rose leaves were relatively slow at 32 minutes. The timing and the concentration of abscisic acid needed to cause closure were related to the amounts of endogenous abscisic acid in the leaf. Closure was obtained in bean leaves with 8.9 picomoles/cm². (+)-Abscisic acid had approximately twice the activity of the racemic material. The methyl ester of abscisic acid was inactive, and trans-abscisic acid was likewise inactive. The effects of stress on levels of endogenous abscisic acid, and the ability of very small amounts of abscisic acid to cause rapid closure suggests that stomatal control is a regulatory function of this hormone.