The Circadian Rhythm of Stomatal Opening: Evidence for the Involvement of Potassium and Chloride Fluxes

Epidermal strips were taken from plants of Commelina communisat opposite phases of an entrained circadian rhythm, at timeswhich corresponded to the middle of the day phase and the middleof the night phase. Earlier observations of much reduced openingin response to external stimuli in the night phase were confirmed. Evidence is now presented that uptake or retention of specificions by the guard cells is affected by the phase of the rhythm.The overt expression of the rhythm was reduced if the epidermiswas presented with potassium iminodiacetate instead of potassiumchloride. Iminodiacetate is a non-absorbable anion, and thisresult suggested that chloride uptake is important for the fullexpression of the rhythm. The use of an anion channel inhibitor(DIDS) gave strong support for this conclusion. Reduced uptakeof chloride would explain an earlier observation that formationof malate is greater during the night phase. When the guard cells were presented with sodium chloride insteadof potassium chloride there was still an overt expression ofthe rhythm, but this was of reduced magnitude. This suggeststhat potassium movements might contribute to the rhythm, andstudies of ionic fluxes using ⁸⁶Rb as a tracer showed greatereffluxes during the night phase. It is concluded that the circadian rhythm in stomatal openingmay be the result of a varying ability of the guard cells toaccumulate or retain both chloride and potassium ions. Key words: Circadian rhythm, stomatal opening, Commelina communis, potassium and chloride fluxes     

Differential Stimulation of PEP-carboxylation in Guard Cells and Mesophyll Cells by Ammonium or Fusicoccin

Dark CO₂-fixation in guard cells of Vicia faba was much moresensitive to ammonium than in mesophyll cells. Addition of ammonium(5.0 mol m^–3; pH₀ 7.6) caused up to a 7-fold increasein dark CO₂-fixation rates in guard cell protoplasts (GCP),whereas in leaf slices, mesophyll cells, and mesophyll protoplaststhe increase was only about 1.4-fold. In both cell or tissuetypes, total CO₂-fixation rates were higher in the light (2–12-foldhigher in GCP and 28-fold in mesophyll); these rates were onlyslightly changed by ammonium treatment. However, separationof ¹⁴C-labelled products after fixation of CO₂ in the lightby GCP revealed a large ammonium-induced shift in carbon flowfrom starch and sugars to typical products of C₄-metabolism(mainly malate and aspartate). In contrast, in mesophyll cellsamino acid and malate labelling was only moderately increasedby ammonium at the expense of sucrose. The data suggest thatin vivo ammonium might facilitate stomatal opening and/or delaystomatal closing through an increased production of organicacids. Key words: PEP-carboxylation, guard cell protoplasts, ammonium, fusicoccin     

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     

Responses of Stomata in Epidermal Strips of Vicia fabato Carbon Dioxide and Growth Hormones when Incubated on Potassium Chloride and Potassium Iminodiacetate

The effect of various K⁺ levels in combination with Cl^− or iminodiacetate (IDA^{& minus;}) on stomatal responsesin isolated epidermal strips of Vicia faba L. were examinedin order to determine the role of malate during guard cell movements.Responses of guard cells to ABA, kinetin, and varying CO₂ levelswere similar when epidermal strips were floated on KCL or KIDAat 10 mM; such responses were typical in that ABA caused closure,kinetin stimulated opening in ambient air, and apertures weregreater in CO₂-free than ambient air. Maximal stomatal openingwas observed in both ambient and CO₂-free air with KCL at 100mM. The transfer of epidermal strips from 100 mM KCL to solutionsof 100 mM KCL supplemented with ABA or kinetin did not bringabout changes in stomatal aperture. KCL at 100 mM supporteda greater degree of stomatal opening than did 100 mM KIDA irrespectiveof the CO₂ content of the air. In CO₂-free air transfer of epidermalstrips from 100 mM KIDA to solutions containing 100 mM KIDAsupplemented with ABA or kinetin caused little change in stomatalaperture, whereas, in ambient air, the same treatments resultedin stomatal opening. The results are discussed in relation tothe role of malate during guard cell movements.     

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.     

Stomatal Responses to High Temperature in Darkness

The effect of a temperature increase from 25 to 35°C onstomatal opening in darkness (‘night opening’) onexcised, turgid leaves of Stachytarpheta indica was investigatedby microscopic examination of a baxial epidermis fixed in absoluteethanol. An appreciable degree of opening occurred towards theend of a 14-h night at 25°C, and this was substantiallyenhanced by the temperature increase to 35°C in the dark,which also promoted a marked increase in starch hydrolysis andaccumulation of potassium in the guard cells. The degree oftemperature-induced night opening was somewhat smaller thanthat of light-induced opening, and was higher in CO₂-free airthan in normal air. 2,4-dinitrophenol (DNP) was effective inarresting stomatal opening and suppressing starch hydrolysisand increase in stomatal potassium. The temperature-inducednight opening is related, to a great extent, to the enhancementby high temperature of starch hydrolysis and potassium accumulationin the guard cells, and the inhibitory effect of DNP on stomatalopening is attributed largely to its suppression of these twometabolic processes. The importance of oxidative phosphorylationas a possible source of energy for stomatal opening is brieflydiscussed.     

Studies on the Mechanisms of Action of the Antitranspirant Phenylmercuric Acetate, and its Penetration into the Mesophyll

Experiments have examined the effect of phenylmercuric acetate(PMA) on the guard cells of Commelina communis. In one series,PMA was supplied to the leaf surface; after different time intervalsthe epidermis was removed and the ability of the stomata toopen was determined. In the other series, different concentrationsof PMA were included in the medium used for inccubating epidermalstrips with which ion-stimulated stomatal opening was assayed.At concentrations of 10^-54 M and above the effect of PMA wassevere and the structural integrity of the guard cells was affected;they were unable to accumulate neutral red. At concentrationsarpound 10^-6 M the guard cells were less affected and PMA broughtabout a transient stimulation of stomatal opening by releasingsubsidiary-cell turgor pressure. A solution of 5 x 10^-4 M PMA applied to leaves reduced by halfthe photosynthetic ¹⁴CO₂ incorporation into C. communis mesophyll.In Zea mays it increased the CO₂ compensation point and alsothe resistance to diffusion in the gas phase (R_A, but therewas a proportionately greater increase in the apparent liquidphase resistance (R_t). This direct inhibition of mesophyll photosynthesisundermines one of the major objectives of applying anatitranspirants,and for this reason it is suggested that PMA is unsuitable forgeneral application to crops.     

ACTION OF FUSICOCCIN ON THE POTASSIUM BALANCE OF GUARD CELLS OF PHASEOLUS VULGARIS

Fusicoccin induces stomatal opening in both the light and dark. The stomatal aperture and K content of guard cells was measured to determine whether the action of fusicoccin in inducing stomatal opening is directly related to the uptake of K by the guard cells. Both detached and attached epidermis was treated with fusicoccin and the K content was determined by staining with cobalt sodium nitrite or by electron probe microanalysis. The K content of guard cells in detached epidermal strips floated on 10 μm fusicoccin in 10 mm KCl and aqueous CH₃OH (0.02%, v/v) increased in the light and dark as the stomata opened. After exposure to fusicoccin for 6 hr in the light, however, the stomata were closed and no K could be detected in the guard cells. The K content of guard cells of attached epidermis painted with fusicoccin also increased as the stomata opened, but the concentration of K in the subsidiary cells was not significantly altered by fusicoccin-stimulated opening. Moreover, painting with fusicoccin did not significantly change the Ca and P content of the guard or subsidiary cells. Stomata of epidermal strips, opened to their maximum width by fusicoccin, showed only a small and temporary closure when transferred to a solution of 10 μm abscisic acid. The use of metabolic inhibitors suggested that energy for the uptake of the K may be provided by both photophosphorylation and oxidative phosphorylation.     

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.     

The effect of Cl- upon the sensitivity of starch-containing and starch-deficient stomata and guard cell protoplasts towards potassium ions,fusicoccin and abscisic acid

Chloride ions are necessary to compensate for the positively charged potassium ions imported into guard cells of Allium cepa L. during stomatal opening. Therefore an external Cl^- supply of intact Allium plants is important. But high levels of chloride have been found to reduce the sensitivity of the starch-lacking stomata and isolated guard cell protoplasts (GCPs) from Allium to potassium ions, fusicoccin and abscisic acid. Furthermore, with high levels of chloride, malate anions disappear from the guard cells of Allium, a finding which contrasts with situation in Vicia where the stomatal sensitivity to K⁺ ions, fusicoccin and ABA is not influenced by Cl^- ions and malate levels are unaffected. It is suggested that the absence of malate as a proton yielding primer inhibits the mechanism of H⁺/K⁺ exchange in Allium.Abbreviations ABA abscisic acid - FC fusicoccin - GCPs guard cell protoplasts     

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     

Inhibition of the Stomatal Blue Light Response by Verapamil at High Concentration

Blue light-dependent proton pumping in guard cell protoplastsand light-induced stomatal opening in the epidermis were inhibitedby 1 mM verapamil, a Ca²⁺ channel blocker. Proton pumping andstomatal opening induced by fusicoccin, an activator of plasmamembrane proton pump, were not inhibited by verapamil. Theseresults suggest that verapamil inhibits blue light signalingin guard cells without inhibiting the pump. (Received January 6, 1997; Accepted March 26, 1997)     

The role of microtubules in guard cell function

Guard cells are able to sense a multitude of environmental signals and appropriately adjust the stomatal pore to regulate gas exchange in and out of the leaf. The role of the microtubule cytoskeleton during these stomatal movements has been debated. To help resolve this debate, in vivo stomatal aperture assays with different microtubule inhibitors were performed. We observed that guard cells expressing the microtubule-binding green fluorescent fusion protein (green fluorescent protein::microtubule binding domain) fail to open for all major environmental triggers of stomatal opening. Furthermore, guard cells treated with the anti-microtubule drugs, propyzamide, oryzalin, and trifluralin also failed to open under the same environmental conditions. The inhibitory conditions caused by green fluorescent protein::microtubule binding domain and these anti-microtubule drugs could be reversed using the proton pump activator, fusicoccin. Therefore, we conclude that microtubules are involved in an upstream event prior to the ionic fluxes leading to stomatal opening. In a mechanistic manner, evidence is presented to implicate a microtubule-associated protein in this putative microtubule-based signal transduction event.     

Stomatal Responses to Two Herbicidal Auxins

The effects of 1-naphthylacetic acid (NAA) and 2-naphthoxyaceticacid (NOXA) on stomatal opening on illumination of excised,turgid leaves of Stachytarpheta indica were investigated bymicroscopic examination of abaxial epidermises fixed in absoluteethanol. Both chemicals were effective in restricting, but notcompletely preventing, stomatal opening and suppressing starchhydrolysis and potassium accumulation in the guard cells. Therepressive effects were only partly reversed by CO₂-free air.It is concluded that NAA and NOXA do not greatly affect passiveopening mediated by changes in the leaf water balance, but partlysupress photoactive opening by arresting starch hydrolysis andpotassium accumulation in the guard cells and partly by disturbingthe intercellular CO₂ concentration. A possible link betweenstarch hydrolysis and potassium accumulation in the guard cellsis briefly mentioned.     

Change in Levels of Starch and Sugars in Epidermis of Commelina benghalensis during Fusicoccin Stimulated Stomatal Opening

The concentrations of malate, starch and sugars were determinedin presonicated epidermal strips of Commelina benghalensis.During opening, the starch content of epidermis decreased whilethe level of sugars or malate increased. Fusicoccin (FC) stimulatedstomatal opening and elevated the levels of malate and sugars.However, the contribution from sugars was nearly 50% of theosmotic effect of malate and it increased to more than 60% inthe presence of FC. We conclude that FC stimulates stomatalopening by enhancing not only potassium influx into guard cellsbut also hydrolysis of starch into sugars (and malate). Significantcorrelations were noticed between the width of stomatal apertureand epidermal starch (negative), malate and sugars (both positive).The negative relationship between starch and malate or sugarswithin epidermis indicated that starch hydrolysis lead to formationof sugars as well as malate. Starch—sugar interconversioncan therefore play a significant role in modulating the solutepotential of guard cells. Key words: Commelina benghalensis, Stomatal opening, Fusicoccin, Epidermal starch and sugars     

CO2 fixation in leaves of a CAM plant without lower epidermis and the effect of CO2 on their deacidification

The effects of peeling the epidermis off Bryophyllum daigremontianumleaves on CO₂ uptake in light and darkness were investigated.Light-induced CO₂ uptake in the daytime was markedly enhancedin the peeled leaves, but dark fixation of CO₂ carried out atmidnight was not. The difference in promotion of CO₂ uptakein light and darkness was due to stomatal closing in the dayand opening at night. Also, deacidification was strikingly inhibitedby CO₂ in peeled leaves. (Received February 3, 1977; )     

Presence of Chloride Reduces Malate Production in Epidermis during Stomatal Opening

When stomata of isolated epidermis of Vicia faba are allowed to open in the presence of K⁺ and iminodiacetate (an impermeant zwitterion), malate is formed in the epidermis; the increases in malate content follow a nearly linear relationship with stomatal aperture. Stomata of leaf sections of V. faba floated on water during opening also exhibit this relationship. When isolated epidermis is offered KCI, this relationship is not observed and less malate is detected at comparable stomatal apertures. The data indicate that Cl⁻, if present at concentrations ≥ 10⁻⁵ eq liter⁻¹, can partially satisfy the anion requirement of guard cells of V. faba during stomatal opening. Discrepancies between earlier reports on the relative roles Cl⁻ and malate play as counterions for K⁺ in guard cells of V. faba could now be explained as resulting from variations in the availability of Cl⁻ to guard cells.     

An Investigation into the Stomatal Behaviour of a Wilty Mutant of Pisum sativum

The water relations and stomatal behaviour of a wilty line ofpea (JI 1069) were investigated and compared with those of severalnon-wilty lines (JI 1180, JI 1194, and JI 74). The leaves ofthe wilty line were found to have a lower percent water content,water potential and diffusive resistance and the dimensionsof the stomatal cells were larger than those of the non-wiltytypes. The aperture of stomata on epidermal samples taken from plantsin the light or dark period of a diurnal rhythm was consistentlylarger for the wilty pea than for the non-wilty lines, however,their stomatal responses on detached epidermis to light, CO₂and KC1 concentration were similar. There was no differencein response to ABA of stomata on detached epidermis of wiltyor non-wilty types of pea. Key words: Pisum sativum, Wilty mutant, Water relations, Stomatal behaviour     

Abnormal Behaviour of Stomata in Barley Leaves Infected with Rhynchosporium secalis (Oudem.) J. J. Davis

Infection of barley leaves by the fungus Rhynchosporium secalisincreases the degree to which stomata open in the light. Openingis enhanced at CO₂, levels between 0 and 4000 parts/10⁶. Theability of stomata to close in the dark, as normal, is retaineduntil an advanced stage of tissue necrosis is reached. Increased stomatal opening is confined to those areas of theleaf which have been colonized by the fungus. Abnormal stomatalbehaviour results from the loss of osmotically active substancesfrom the epidermis of diseased leaves with a consequent alterationof the turgor relations between guard cells and their surroundingepidermal cells.     

Studies of the mechanism of action of fusicoccin,the fungal toxin that induces wilting,and its interaction with abscisic acid

Summary Effects of fusicoccin alone and together with abscisic acid were observed on the stomatal complex of Commelina communis. The experimental material consisted of isolated epidermal strips incubated in a medium containing the ions required for stomatal opening. Fusicoccin stimulated opening and this was accompanied by potassium entry into the guard cells, and hydrolysis of the starch in their chloroplasts. Abscisic acid alone inhibited potassium entry and starch hydrolysis, but these effects could be almost entirely overcome by fusicoccin.Attempts were made to measure the solute potential of the guard cells under the various treatments. Abscisic acid clearly increased their solute potential, but no absolute measurements could be made in the presence of fusicoccin owing to a failure of plasmolysis even with mannitol solutions of solute potential as low as —35 bars. Experiments using isotopically labelled mannitol indicated a massive uptake into the epidermis in the presence of fusicoccin.The mechanism of stimulation of stomatal opening by fusicoccin probably depends in part on a stimulation of the normal processes associated with opening in the guard cells, but may also involve release of pressure due to destruction of the surrounding cells. The effectiveness of this toxin under natural conditions may depend on its ability to counteract effects of abscisic acid, the stress hormone that induces stomatal closure.