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.     

Calcium Inhibits Ion-Stimulated Stomatal Opening in Epidermal Strips of Commelina communis L.

Inoue, H. and Katoh, Y. 1987. Calcium inhibitsion-stimulatedstomatal opening in epidermal strips of Commelina communis L.—J.exp. Bot. 38: 142–149. Ca²⁺ suppressed both the ion-stimulated stomatal opening andH⁺ extrusion of pre-illuminated epidermal strips isolated fromCommelina communis L. In the absence of Ca²⁺, the rate of H⁺release was 18 nmol H⁺ cm^–2 h^–1 per epidermal stripunit area in 150 mol m^–3 KCL at pH 7?4. Half-maximum inhibitionof stomatal opening was observed with 220 mmol m^–3 ofCa²⁺. The hexavalent dye, ruthenium red, showed concentration-dependentprevention of the inhibition by Ca²⁺ of the ion-stimulated stomatalopening. The effect of ruthenium red was non-competitive, andthe K₁ for the calcium inhibition was found to be 3?6 mmol m^–3.The calcium inhibition of H⁺ extrusion was also prevented byruthenium red. These results suggest that Ca²⁺ inhibits theactivity of electrogenic H⁺ translocating ATPase of the guardcell plasma membrane and leads to the suppression of stomatalopening. Key words: Calcium, Commelina communis, ruthenium red, stomata     

Stomatal Responses to Applied ABA and CO2 in Epidermis Detached from Well-Watered and Water-Stressed Plants of Commelina communis L.

Epidermal strips from either well-watered or water-stressedplants of Commelina communis L. were subjected to a range ofABA concentrations (10^–6–10^–3 mol m^–3)in the presence (330 parts 10^–6 in air) or virtual absence(3 parts 10–6 in air) of CO₂. The stomatal response toCO₂ was greater in epidermis from water-stressed plants, althoughthere was a distinct CO₂ response in epidermis from well-wateredplants. Additions of ABA via the incubation medium had littleeffect on the relative CO₂ response. Stomata responded to ABAboth in the presence and virtual absence of CO₂, but the relativeresponse to ABA was greatest in the high CO₂ treatment. Whenwell-watered plants were sprayed with a 10^–1 mol m^–3ABA solution 1 d prior to use, the stomatal response of detachedepidermis to both CO₂ and ABA was very similar to that of epidermisdetached from water-stressed leaves. It is hypothesized thata prolonged exposure to ABA is necessary before there is anymodification of the CO₂ response of stomata.     

Abaxial and Adaxial Stomatal Responses to Light of Different Wavelengths and to Phenylacetic Acid on Isolated Epidermes of Commelina communis L.

Abaxial and adaxial stomatal responses to light of differentwavelengths and to phenylacetic acid (PAA), a molecule knownto form complexes with irradiated flavins, were examined onisolated epidermes of Commelina communis L. Blue light was superiorto red and green in promoting opening. Potassium accumulationand malate production were common to both abaxial and adaxialstomatal cells, but the photosensitivity was markedly higherin the former than in the latter. PAA suppressed opening andpotassium accumulation in guard cells, but hardly affected thelevel of epidermal malate; CO₂-free air failed to reverse thesesuppressions. The PAA-effect was more substantial in blue lightthan in red, green or darkness; thus, a flavin photoreceptoris indicated. Because of the overall effect of PAA under allconditions it is suggested that, in addition to its interactionwith blue light reception, PAA also has a more general effecton guard cells.     

Stomatal opening in isolated epidermis of Commelina benghalensis L. heterophasic response to KCl concentration

The pattern of stomatal opening in epidermal strips detached from leaves of Commelina benghalensis was examined. Two different phases could be distinguished in the stomatal response to KCl, one at low concentrations of KCl (up to 60 mM) and the other at high KCl concentrations (above 100 mM). The stomatal opening at low KCl concentrations was stimulated remarkably by light or fusicoccin and was suppressed by abscisic acid. At higher KCl concentrations, the stimulation by light or FC as well as the inhibition by ABA was limited. Both phases of stomatal response to KCl were sensitive to carbonyl cyanide-m-chlorophenyl hydrazone. The results suggest that illumination or FC favours selectively stomatal opening only at low KCl concentrations. The ionic participation in the stomatal opening is similar to the heterophasic uptake of ions by plant cells/roots.Abbreviations FC fusicoccin - ABA abscisic acid - CCCP carbonyl cyanide-m-chlorophenyl hydrazone     

Light Saturation of Stomatal Opening on the Adaxial and Abaxial Epidermis of Commelina communis

The responses of adaxial and abaxial stomata to light were examinedon detached epidermis of Commelina communis. Stomata on theabaxial epidermis were considerably more sensitive to lightthan those on the adaxial epidermis, supporting the view thatthe differences in photosensitivity are inherent rather thanthe result of differences in the microenvironment within theleaf. The sensitivity of adaxial and abaxial stomata to bluelight was also examined. The quantum flux received by a pairof guard cells appears to be sufficient to support a directeffect of blue light on ion transport into the guard cells,but there is no evidence to suggest that blue light is essentialfor stomatal opening.     

Effects of Light/Dark on Cation Fluxes in Guard Cells of Commelina communis L.

The effects of light/dark on cation fluxes in isolated guardcells of Commelina communis L. have been studied, using ⁸⁶RbCland ²²NaCl. Transfer to the dark has no effect on ⁸⁶Rb influx,but produces a marked transient stimulation of ⁸⁶Rb efflux,similar to that seen previously on adding ABA. The ⁸⁶Rb effluxfalls on return to light only during the period of stimulatedflux; after the transient, return to light has no effect onefflux. The ability to produce this transient stimulation ontransfer to the dark is recovered in a subsequent light period.In general, in Na-loaded cells, the stimulated efflux is notseen. and the cells do not close in the dark. The results arenot consistent with a simple permeability or potential change,but suggest a specific ion excretion activated by the transferto the dark. Key words: Commelina communis L., Light/dark effects, Cation flux, Guard cells     

Guard Cell Metabolism in Epidermis of Commelina communis L. During Stomatal Opening and Closing

The rates of CO² incorporation into the epidermis of C. communiswere linear and were similar during the completion of opening(2 h) and closing (1 h) movements of stomata. The kinetics of¹⁴C turnover between metabolites and the rates of ‘leakage’of metabolites were determined for opening and closing movements.When stomata were opening there was a slow turnover of ¹⁴C frommalate chiefly into sugars. Upon stomatal closure ¹⁴C was initiallymainly in sugars, malate, and sugar phosphates. Thereafter,there was a slight loss of label from sugar phosphates witha corresponding increase in malate. Starch became labelled duringopening and closing movements. Rates of incorporation of CO₂found in the ‘leakage’ fraction were greatest whenstomata were opening. Of the labelled compounds Most‘from the tissue, malate was the most highly labelled whetherstomata were opening or closing. Although interpretation of the turnover patterns is difficultwithout knowledge of pool sizes for the metabolites it is suggestedthat a pool of sugars exists within the guard cells, which havefairly direct and reversible access to carbon from starch andmalate. The implications of loss of malate from guard cellsduring stomatal opening and closing are discussed.     

Redox Processes in the Blue Light Response of Guard Cell Protoplasts of Commelina communis L

Guard cell protoplasts from Commelina communis L. illuminated with red light responded to a blue light pulse by an H⁺ extrusion which lasted for about 10 minutes. This proton extrusion was accompanied by an O₂ uptake with a 4H⁺ to O₂ ratio. The response to blue light was nil in darkness without a preillumination period of red light and increased with the duration of the red light illumination until about 40 minutes. However, acidification in response to a pulse of blue light was obtained in darkness when external NADH (1 millimolar) was added to the incubation medium, suggesting that redox equivalents necessary for the expression of the response to blue light in darkness may be supplied via red light. In accordance with this hypothesis, the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (10 micromolar) decreased the acidification in response to blue light more efficiently when it was added before red light illumination than before the blue light pulse. In the presence of hexacyanoferrate, the acidification in response to a blue light pulse was partly inhibited (53% of control), suggesting a competition for reducing power between ferricyanide reduction and the response to blue light.     

Effects of Light/Dark on Anion Fluxes in Isolated Guard Cells of Commelina communis L.

The effects of light/dark on anion fluxes in isolated guardcells of Commelina communis L. have been studied, using ⁸²Brand ³⁶Cl. Transfer of open guard cells from light to dark hasno effect on the ⁸²Br influx, but produces a marked transientstimulation of ⁸²Br or ³⁶Cl efflux, similar to the effect ofsuch transfer on the ⁸⁶Rb fluxes, and to the effects on both⁸⁶Rb and ⁸²Br fluxes of adding ABA. On return of guard cellsto light, after the transient, there is a further reductionin Cl/Br efflux. It is argued that control of a specific processof ion extrusion is important in regulating the ability of guardcells to stay open. In three out of four batches of steady-statetissue labelled with ⁸²Br, the plasmalemma fluxes were highenough, relative to the tonoplast fluxes, for the efflux kineticsto be separable into two exponential components, allowing estimationof bromide contents in cytoplasm and vacuole (Q_c and Q_v), andfluxes at plasmalemma and tonoplast. With opening in light,Q_c increased by 3.9 ± 0.4 pmol mm^–2 µm^–1and Qy by 5.2 ± 0.6 pmol mm^–2 µm^–1(change in content per mm² of epidermis perµm change inaperture). Using rough estimates for the volumes of cytoplasmand vacuole these figures suggest that at 6.1 µm in thedark the concentrations were about 63 mol m^–3 in the cytoplasmand 35 mol m^–3 in the vacuole, rising to about 185 molm^–3 in the cytoplasm and 125 mol m^–3 in the vacuole,at 16.7 µm aperture in light. Neither increase can providean adequate increase in salt concentration to account for theosmotic change required, and some solute other than potassiumsalt must also be involved. In one experiment with 82Br andin the only experiment with 36Cl the plasmalemma flux was lower,and not high enough relative to the tonoplast flux to allowseparation of two phases in the efflux curves, and calculationof cytoplasmic and vacuolar contents and fluxes. The effectsof transfer from light to dark were, nevertheless, similar inboth types of tissue. Key words: Commelina communis L., Light/dark effects, Anion fluxes, Guard cells     

Stomatal Movement and Sucrose Uptake by Guard Cell Protoplasts of Commelina benghalensis L.

Sucrose concentration in guard cells of epidermal strips ofCommelina benghalensis increased with stomatal opening. Sucroseuptake patterns were investigated using guard cell protoplastsof C. benghalensis. Sucrose (0.5 mM) uptake into these protoplastswas sensitive to pH, with an optimum at pH 6. Uptake of sucroseinto guard cell protoplasts was inhibited by 2,4-dinitrophenol(DNP), diethylstilbestrol (DES) and (ptrifluoromethoxy)carbonylcyanide phenylhydrozone (FCCP), while DCMU and o-phenanthrolinehad no effect on the uptake of sucrose. Fusicoccin (FC) stimulatedsucrose influx. The influence of pH and the effect of the metabolicinhibitors on the sucrose uptake into the guard cell protoplastsare consistent with an energy dependent membrane-function. (Received July 7, 1986; Accepted September 26, 1986)     

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.

     

Effects of ABA in 'Isolated' Guard Cells of Commelina communis L.

The effects of 2 x 10^–3 M ABA on ion fluxes in isolatedguard cells of Commelina communis L. have been studied, using⁸⁶RbCl and K⁸²Br, in epidermal strips in which all cells otherthan guard cells have been killed by treatment at low pH. Theeffect of ABA on influx is small, if present, and the majoreffect is a marked transient stimulation of the efflux of both⁸⁶Rb and ⁸²Br at the plasmalemma; there is also an increasein the flux of ⁸²Br from vacuole to cytoplasm. The stimulationis transient, and the cells do not simply become more leaky.The results are not consistent with previous speculations onthe mechanism by which ABA reduces aperture.     

Stomatal movement in response to long distance-communicated signals initiated by heat shock in partial roots of Commelina communis L.

The systematic or long-distance signal transmission plays crucial roles in animal lives. Compared with animals, however, much less is known about the roles of long-distance signal communication in plant lives. Using the model plant Commelina communis L., we have probed the root to shoot communication mediated by heat-shock signals. The results showed that a heat shock of 5 min at 40℃ in partial roots, i.e. half or even 1/4 root system, could lead to a significant decrease in stomatal conductance. The regulation capability depends on both heat shock temperature and the amount of root system, i.e. with higher temperature and more roots stressed, the leaf conductance would decrease more significantly. Interestingly, the stomatal regulation by heat shock signal is in a manner of oscillation: when stomata conductance decreased to the lowest level within about 30 min, it would increase rapidly and sometimes even exceed the initial level, and after several cycles the stomata conductance would be finally stabilized at a lower level. Feeding xylem sap collected from heat-shocked plants could lead to a decrease in stomata conductance, suggesting that the heat shock-initiated signal is basically a positive signal. Further studies showed that heat shock was not able to affect ABA content in xylem sap, and also, not able to lead to a decrease in leaf water status, which suggested that the stomatal regulation was neither mediated by ABA nor by a hydraulic signal. Heat shock could lead to an increase in xylem sap H₂O₂ content, and moreover, the removal of H₂O₂ by catalase could partially recover the stomatal inhibition by xylem sap collected from heat-shocked plants, suggesting that H₂O₂ might be able to act as one of the root signals to control the stomatal movement. Due to the fact that heat-shock and drought are usually two concomitant stresses, the stomatal regulation by heat-shock signal should be of significance for plant response to stresses. The observation for the stomatal regulation in an oscillation manner by presently identified new signals should contribute to further understanding of the mystery for the pant systematic signaling in response to stresses.     

Stomatal Opening Is Induced in Epidermal Peels of Commelina communis L. by GTP Analogs or Pertussis Toxin

Pretreatment with pertussis toxin or microinjection of guanosine- 5[prime]-(3-thiotriphosphate) (GTP-[gamma]-S) into guard cells in peeled epidermis of Commelina communis L. promoted stomatal opening under subsaturating white light. Guanosine-5[prime]-(2-thiodiphosphate) (GDP-[beta]-S) and adenosine-5[prime]-(3-thiotriphosphate) (ATP-[gamma]-S) did not change stomatal aperture under identical conditions. These results indicate that G proteins may be involved in the regulation of stomatal opening.     

Stomatal movement in response to long distance-communicated signals initiated by heat shock in partial roots of Commelina communis L.

It is well known that animals are able to positively respond to environmental stimuli, thereby avoiding or reducing the possible injures or impacts on them, and the base for the rapid and positive responses is long- distance signal transmission mediated b…     

Calcium Fluxes across the Plasma Membrane of Commelina communis L. Assayed in a Cell-Free System

The inside-out fraction of plasma membrane-rich vesicles prepared from leaves of Commelina communis L. by aqueous twophase partitioning was loaded with ⁴⁵Ca²⁺ through the action of the plasma membrane Ca²⁺-ATPase. While the Ca²⁺-loaded vesicles were tightly sealed, trifluoperazine (TFP) (effective concentration giving 50% of maximum effect [EC₅₀] = 70 micromolar) and W-7 (EC₅₀ = 100 micromolar), but to a much lesser extent, W-5 (EC₅₀ = 500 micromolar) led to a rapid efflux of ⁴⁵Ca²⁺ from the vesicles. This efflux could be blocked efficiently with low (<1 millimolar) concentrations of La³⁺, but it remained unaffected by the addition of calmodulin (CM). Further experiments with vesicles incubated in ⁴⁵Ca²⁺ in the absence of ATP, as well as experiments performed with control liposomes and nonloaded as well as Ca²⁺-loaded plasma membrane vesicles using the indicator dye arsenazo III showed, that TFP and W-7 and, again to a lesser extent, W-5 mobilized a pool of membrane-bound Ca²⁺ from the vesicles. No indications for a detergent effect of TFP and W-7 were obtained. The EC₅₀-values of these compounds for mobilizing membrane-associated Ca²⁺ (TFP = 100 micromolar, W-7 = 100 micromolar, W-5 = 500 micromolar) or for the triggering of Ca²⁺ release from Ca²⁺-loaded vesicles (see above) were very similar, suggesting a common basis of antagonist action on both processes. Our results suggest the presence of a Ca²⁺ channel in the plasma membrane of C. communis. The channel is obtained in a Ca²⁺-inactivated state after preparation and Ca²⁺-loading of the vesicles. The inactivation is removed by TFP or W-7, presumably due to the Ca²⁺-mobilizing effect of these compounds. The activated Ca²⁺ channel is La³⁺ sensitive and, in the cell, would allow for passage of Ca²⁺ into the cell. The possibility that TFP or W-7 act independent of CM, or through CM tightly associated with the plasma membrane, is discussed. The system described allows a cell free analysis of Ca²⁺ influx, displaying channel properties, in a higher plant.     

Response of Photosynthesis, Stomatal Conductance and Water Use Efficiency to Elevated CO2 and Nutrient Supply in Acclimated Seedlings of Phaseolus vulgaris L.

Plants of Phaseolus vulgaris L were grown from seed in open-topgrowth chambers at present day (350 µmol mol^–1)and double the present day (700 µmol mol^–1) atmosphericCO₂ concentration with either low (L, without additional nutrientsolution) or relatively high (H, with additional nutrient solution)nutrient supply Measurements of assimilation rate, stomatalconductance and water use efficiency were started 17 d aftersowing on each fully expanded, primary leaf of three plantsper treatment Measurements were made in external CO₂ concentrations(C₂) of 200, 350, 450, 550 and 700 µmol mol^–1 andrelated to both C_a and to C₁, the mean intercellular space CO₂concentration Fully adjusted, steady state measurements weremade after approx 2 h equilibration at each CO₂ concentration The rate of CO₂ assimilation by leaves increased and stomatalconductance decreased similarly over the range of C_a or C₁ inall four CO₂ and nutrient supply treatments but both assimilationrate and stomatal conductance were higher in the high nutrientsupply treatment than in the low nutrient treatment The relationbetween assimilation rate or stomatal conductance and C₁ wasnot significantly different amongst plants grown in present-dayor elevated CO₂ concentration in either nutrient supply treatment,i e there was no evidence of down regulation of photosynthesisor stomatal response Increase in CO₂ concentration from 350to 700 µmol mol^–1 doubled water use efficiency ofindividual leaves in the high nutrient supply treatment andtripled water use efficiency in the low nutrient supply treatment The results support the hypothesis that acclimation phenomenaresult from unbalanced growth that occurs after the seed reservesare exhausted, when the supply of resources becomes growth limiting CO₂ enrichment, Phaseolus vulgaris L., net CO₂ assimilation rate, stomatal conductance, water use efficiency     

The Effects of Fusicoccin on Anion Fluxes in Isolated Guard Cells of Commelina communis L.

Clint, G. M. 1987. The effects of fusicoccin on anion fluxesin isolated guard cells of Commelina communis L.—J. exp.BoL 38: 863–876. The effects of 3?10^–2 mol m^–3 fusicoccin (FC) onbromide fluxes and contents in isolated guard cells of Commelinacommunis L. have been studied using K⁸²Br at pH 3?9 and pH 6?7.At pH 3?9 FC caused a reduction in both the influx and the effluxof ⁸²Br, whereas at pH 6?7 FC had no effect on the influx butcaused a transient increase in the efflux of ⁸²Br. There wasno obvious change in bromide content with FC treatment at eitherpH. The behaviour of the anion fluxes in response to FC suggeststhat FC does not act solely via a hyperpolarization at the plasmalemma.A redistribution of bromide between the intracellular compartmentssuggests that anion flux from the cytoplasm to the vacuole maybe stimulated by FC at pH 3?9. The failure of guard cells toincrease their anion content on treatment with FC despite anincrease in stomatal aperture and in cation content suggeststhat in FC-induced stomatal opening excess cation is balancedby organic acid synthesis within the guard cell. Key words: Fusicoccin, guard cells, ion fluxes, Commelina communis     

Saturation Kinetics of the Velocity of Stomatal Closing in Response to CO(2)

Stomatal closing movements in response to changes from CO₂-free to CO₂-containing air were recorded in leaf sections of Zea mays using air flow porometers. The response to CO₂ was fast; the shortest lag between the application of 300 microliters CO₂ per liter of air and the beginning of a stomatal response was 3 seconds. The velocity of stomatal closing increased with CO₂ concentration and approached its maximal value between 10³ and 10⁴ microliters CO₂ per liter of air. The CO₂ concentration at which the closing velocity reached half its maximal value was approximately 200 microliters CO₂ per liter of air, both in the light and in darkness. This indicates that the mechanism of stomatal responses to CO₂ is the same in both light regimes and that the range of stomatal sensitivity to changes in CO₂ concentration coincides with the range of CO₂ concentrations known to occur in the intercellular spaces of illuminated leaves.