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.     

Accumulation of malate in guard cells of Vicia faba during stomatal opening

Summary The level of malate in the epidermis from illuminated leaves of Vicia faba was greater than in that from dark-treated leaves. A difference in the malate level was still detected after the epidermis had been treated by rolling so that only the guard cells remained alive. The results suggest that malate may accumulate in guard cells on illumination. In subsequent experiments, stomatal apertures were measured, and potassium as well as malate was analysed in extracts of epidermis. In illuminated leaves, the potassium content of rolled epidermis increased from about 90 to about 335 picoequivalents mm^-2 of epidermis whele malate increased from about zero to about 71 pmoles mm^-2 and the stomata opened; in dark-treated leaves, the potassium content of rolled epidermis decreased slightly, the malate level remained about zero, and the stomata showed very slight further closure. The measured increase in potassium is likely to represent an increase in potassium concentration in the guard cells of about 0.4 Eq l^-1 with stomatal opening; the increase in malate could correspond to 0.23 Eq l^-1 (with respect to potassium) in the guard cells. Thus, malate accumulating in guard cells could balance about half of the potassium taken up by guard cells when stomata open in the light.     

PHOTOCONTROL OF STOMATAL MOVEMENTS

1. Opening in light is a feature common to the majority of functional stomata, but the current argument is against the traditional view that light is the principal environmental promoter of opening, because stomata can open in the dark in response to CO₂ removal and/or temperature increase. In this review, evidence is provided that light is more efficient and effective than other physical factors in both producing and maintaining wide opening. However, light acts on stomata both directly and indirectly, in conjunction with changes in, for example, CO₂ balance, water regime and temperature of the leaf tissue. 2. Three general categories of light effects on stomata are recognized: (a) photosynthetic effects driven by metabolic processes, induced or enhanced by light, (b) hydrophotic effects mediating through light-induced changes in epidermal turgor, and (c) photothermal effects arising from light-dependent changes in leaf temperature. 3. Photosynthetic effects involve both CO₂ depletion, and starch mobilization, malate synthesis, H⁺ extrusion, and accumulation of K⁺ and C1^- in guard cells; these processes are triggered by light of different qualities: (a) Both blue and red light are involved in photosynthetic CO₂ fixation, utilizing energy from photosynthetic light reaction(s), which provides C precursors for synthesis of stornatal starch. (b) Blue light, but not red, enhances starch mobilization, PEP carboxylase activity and respiration. Accordingly, blue light is postulated to enhance hydrolysis of stornatal starch providing C₃ precursors for malate synthesis via PEP-fixation of endogenous CO₂; the active extrusion of H⁺, derived from malate, is coupled with K⁺ influx to guard cells. Malate and C1^- are competitive anions, for K⁺, and one begins to play a progressively more important role as the other becomes limiting; in intact leaves, however, malate plays a more decisive role. These processes are driven by the energy from blue-light-enhanced respiration. (c) Both photosynthetic fixation and PEP carboxylation act as CO₂ sensors, but the exact role of CO₂ in the stornatal mechanism has yet to be determined. 4. Hydrophotic and photothermal effects facilitate guard cell expansion by releasing epidermal pressure through enhanced evaporative water loss, and are, therefore, indirect effects of light; photothermal effects may also contribute to metabolic processes outlined in paragraph 3. 5. Stomatal closure in the dark accompanies starch synthesis, malate reduction, efflux of K⁺ and C1^- from guard cells, and accumulation of CO₂ in substomatal cavities. Malate may be converted to starch via C₂ compounds. Guard cells release K⁺ and C1- into apoplastic space, from which they are removed by neighbouring cells. The entry of K⁺ into neighbouring cells is supposed to be coupled with H⁺ extrusion. These processes are dependent on respiratory energy. 6. The differential abaxial and adaxial stomatal light responses are related to inherent metabolic differences between the two epidermes, but the biochemical basis is not known.     

Guard cell photosynthesis is critical for stomatal turgor production,yet does not directly mediate CO2‐ and ABA‐induced stomatal closing

Stomata mediate gas exchange between the inter‐cellular spaces of leaves and the atmosphere. CO₂ levels in leaves (Ci) are determined by respiration, photosynthesis, stomatal conductance and atmospheric [CO₂]. [CO₂] in leaves mediates stomatal movements. The role of guard cell photosynthesis in stomatal conductance responses is a matter of debate, and genetic approaches are needed. We have generated transgenic Arabidopsis plants that are chlorophyll‐deficient in guard cells only, expressing a constitutively active chlorophyllase in a guard cell specific enhancer trap line. Our data show that more than 90% of guard cells were chlorophyll‐deficient. Interestingly, approximately 45% of stomata had an unusual, previously not‐described, morphology of thin‐shaped chlorophyll‐less stomata. Nevertheless, stomatal size, stomatal index, plant morphology, and whole‐leaf photosynthetic parameters (PSII, qP, qN, F_V′/F_M′) were comparable with wild‐type plants. Time‐resolved intact leaf gas‐exchange analyses showed a reduction in stomatal conductance and CO₂‐assimilation rates of the transgenic plants. Normalization of CO₂ responses showed that stomata of transgenic plants respond to [CO₂] shifts. Detailed stomatal aperture measurements of normal kidney‐shaped stomata, which lack chlorophyll, showed stomatal closing responses to [CO₂] elevation and abscisic acid (ABA), while thin‐shaped stomata were continuously closed. Our present findings show that stomatal movement responses to [CO₂] and ABA are functional in guard cells that lack chlorophyll. These data suggest that guard cell CO₂ and ABA signal transduction are not directly modulated by guard cell photosynthesis/electron transport. Moreover, the finding that chlorophyll‐less stomata cause a ‘deflated’ thin‐shaped phenotype, suggests that photosynthesis in guard cells is critical for energization and guard cell turgor production.     

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.     

ABNORMAL GUARD CELL DEVELOPMENT IN AN OLIVE NECROTIC MUTANT OF MAIZE

A study of a mutant variety of Zea mays (ON8147) revealed that the mutant plants, in contrast with normal maize plants, do not exhibit a light-induced increase in the rate of transpiration, and that the ontogeny of the stomatal complex is abnormal. In later stages of differentiation, the guard cells of mutant plants deteriorate, leaving the mature stomata with only the two subsidiary cells. The subsidiary cells in stomata of mutant leaves are similar to those of normal leaves with respect to their capacity to accumulate K⁺ in the dark, but they do not lose K⁺ in the light, as do subsidiary cells of stomata of nonmutant plants. It is suggested that impairment of guard cell function causes death of the mutant plant seedlings primarily by restricting CO₂ entry into the leaf.     

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.     

Effects of turgor potentials of epidermal cells neighbouring guard cells on stomatal opening in detached leaf epidermis and intact leaflets of Vicia faba L. (faba bean)

Leaflets of Vicia faba L. (faba bean) were used to determine whether the mechanical forces resulting from the turgor potentials (Φ_p) of the larger epidermal cells neighbouring guard cells play a significant role in regulating stomatal aperture. When Φ_p, of epidermis and Φ_p of bulk leaflet tissue were compared at midday, Φ_p of epidermis were only 15–25% those of bulk leaflet tissue at all but the most negative leaflet water potentials (Φ). When plants were bagged to increase Φ by reducing vapour pressure differences between leaflets and air, Φ_p of bulk leaflet tissue increased to predawn values, but Φ_p, of epidermis increased to only = 20% of predawn values and stomata opened to their widest apertures. Stomatal apertures were positively correlated with Φ_p of bulk leaflet tissue but they were not correlated with Φ_p of epidermis. Reductions in epidermal Φ_p, began predawn, before stomata were open, and reached minimum values at midday, when stomata were open. We conclude that, in Vicia faba, (1) reduction of Φ_p of epidermal cells begins predawn, reducing the counterforce to stomatal opening that would exist if full epidermal turgor were maintained throughout the day, and (2) changes in Φ_p, of leaf epidermal cells do not play a significant role in regulating stomatal aperture.     

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     

Inhibition of dark‐induced stomatal closure by fusicoccin involves a removal of hydrogen peroxide in guard cells of Vicia faba

Fusicoccin (FC) treatment prevents dark‐induced stomatal closure, the mechanism of which is still obscure. By using pharmacological approaches and laser‐scanning confocal microscopy, the relationship between FC inhibition of dark‐induced stomatal closure and the hydrogen peroxide (H₂O₂) levels in guard cells in broad bean was studied. Like ascorbic acid (ASA), a scavenger of H₂O₂ and diphenylene iodonium (DPI), an inhibitor of H₂O₂‐generating enzyme NADPH oxidase, FC was found to inhibit stomatal closure and reduce H₂O₂ levels in guard cells in darkness, indicating that FC‐caused inhibition of dark‐induced stomatal closure is related to the reduction of H₂O₂ levels in guard cells. Furthermore, like ASA, FC not only suppressed H₂O₂‐induced stomatal closure and H₂O₂ levels in guard cells treated with H₂O₂ in light, but also reopened the stomata which had been closed by darkness and reduced the level of H₂O₂ that had been generated by darkness, showing that FC causes H₂O₂ removal in guard cells. The butyric acid treatment simulated the effects of FC on the stomata treated with H₂O₂ and had been closed by dark, and on H₂O₂ levels in guard cells of stomata treated with H₂O₂ and had been closed by dark, and both FC and butyric acid reduced cytosol pH in guard cells of stomata treated with H₂O₂ and had been closed by dark, which demonstrates that cytosolic acidification mediates FC‐induced H₂O₂ removal. Taken together, our results provide evidence that FC causes cytosolic acidification, consequently induces H₂O₂ removal, and finally prevents dark‐induced stomatal closure.     

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.     

Environmental regulation of stomatal response in the <Emphasis Type="Italic">Arabidopsis</Emphasis> Cvi-0 ecotype

The Arabidopsis Cape Verde Islands (Cvi-0) ecotype is known to differ from other ecotypes with respect to environmental stress responses. We analyzed the stomatal behavior of Cvi-0 plants, in response to environmental signals. We investigated the responses of stomatal conductance and aperture to high [CO₂] in the Cvi-0 and Col-0 ecotypes. Cvi-0 showed constitutively higher stomatal conductance and more stomatal opening than Col-0. Cvi-0 stomata opened in response to light, but the response was slow. Under low humidity, stomatal opening was increased in Cvi-0 compared to Col-0. We then assessed whether low humidity affects endogenous ABA levels in Cvi-0. In response to low humidity, Cvi-0 had much higher ABA levels than Col-0. However, epidermal peels experiments showed that Cvi-0 stomata were insensitive to ABA. Measurements of organic and inorganic ions in Cvi-0 guard cell protoplasts indicated an over-accumulation of osmoregulatory anions (malate and Cl⁻). This irregular anion homeostasis in the guard cells may explain the constitutive stomatal opening phenotypes of the Cvi-0 ecotype, which lacks high [CO₂]-induced and low humidity-induced stomatal closure.     

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     

Stomatal Responses to Flooding of the Intercellular Air Spaces Suggest a Vapor-Phase Signal Between the Mesophyll and the Guard Cells

Flooding the intercellular air spaces of leaves with water was shown to cause rapid closure of stomata in Tradescantia pallida, Lactuca serriola, Helianthus annuus, and Oenothera caespitosa. The response occurred when water was injected into the intercellular spaces, vacuum infiltrated into the intercellular spaces, or forced into the intercellular spaces by pressurizing the xylem. Injecting 50 mm KCl or silicone oil into the intercellular spaces also caused stomata to close, but the response was slower than with distilled water. Epidermis-mesophyll grafts for T. pallida were created by placing the epidermis of one leaf onto the exposed mesophyll of another leaf. Stomata in these grafts opened under light but closed rapidly when water was allowed to wick between epidermis and the mesophyll. When epidermis-mesophyll grafts were constructed with a thin hydrophobic filter between the mesophyll and epidermis stomata responded normally to light and CO₂. These data, when taken together, suggest that the effect of water on stomata is caused partly by dilution of K⁺ in the guard cell and partly by the existence of a vapor-phase signal that originates in the mesophyll and causes stomata to open in the light.Stomatal responses to the environment have been studied in leaves for well over 100 years. More recently, the mechanisms for these responses have been investigated using isolated epidermes or isolated guard cell protoplasts. Despite the combination of these two approaches, the mechanisms by which stomata respond to environmental signals are not well understood. Since stomata control CO₂ uptake and water loss from leaves, the responses of stomata to environmental factors are important determinants of terrestrial productivity and water use. It is therefore critical that we understand the mechanisms by which stomata respond to the environment if we are to accurately predict the effects of future climates on productivity and water cycles (Randall et al., 1996).There are two assumptions about stomata that are implicit in much of the recent literature: (1) that stomatal responses result from sensory mechanisms that reside within the guard cells, and (2) that stomata in isolated epidermes respond similarly to those in a leaf. The exception to this generalization is the stomatal response to humidity, which has been suggested to be the result of changes in guard cell water potential (Dewar, 1995, 2002) or of signaling from other cells in the leaf to the guard cells (Buckley et al., 2003). The assumption that guard cells directly sense CO₂ and light is largely based on data from isolated epidermes that show effects of light and CO₂ on stomatal apertures. As pointed out by Mott (2009), however, stomatal responses to light and CO₂ in isolated epidermes are generally much different from those observed in leaves; e.g. responses in isolated epidermes are generally smaller than those in leaves, opening in response to light is slower, and closing in darkness is rarely observed. These observations were used to suggest that the mesophyll is somehow involved in stomatal responses to red light and CO₂. This idea is supported by several recent studies that suggest that guard cells do not respond directly to red light. In the first of these studies it was shown that guard cells in an intact leaf do not show hyperpolarization of the plasma membrane in response to red light if the red light is applied to only the guard cell (Roelfsema et al., 2002). In contrast, blue light applied only to the guard cell does cause hyperpolarization, and red light does cause hyperpolarization if applied to the guard cell and the underlying mesophyll. The second study showed that stomata in albino areas of a leaf do not respond to red light, although they contain chloroplasts and do respond to blue light (Roelfsema et al., 2006). Finally, a third study has shown that isolated epidermes are much more sensitive to light and CO₂ when placed in close contact with an exposed mesophyll from a leaf from the same or a different species (Mott et al., 2008). These epidermis-mesophyll grafts showed stomatal responses to light and CO₂ that were indistinguishable from those in an intact leaf—a sharp contrast to the behavior of stomata in isolated epidermes that are floating on buffer solutions. In that study, illumination of a single stoma in a leaf using a small-diameter fiber optic did not produce stomatal opening, but opening did occur if several stomata and the underlying mesophyll were illuminated. Furthermore, this treatment actually caused opening of adjacent, but unilluminated, stomata (Mott et al., 2008).In constructing the epidermis-mesophyll grafts in the study described above (Mott et al., 2008), it was noticed that functional grafts could be produced only if both the mesophyll and the epidermis were blotted completely dry of any free water before placing them together. Although the tissues were apparently still fully hydrated, there was very little free water present (i.e. water not contained within the walls of the leaf cells), and both the mesophyll and epidermis felt and looked dry prior to assembly. In addition, even when free water was blotted away initially, stomata did not open in grafts that ended up with visible water on the epidermis or mesophyll that was caused by condensation during the experiment. These observations suggest that the presence of free water somehow prevented the stomata in the grafts from opening. Assuming that the mechanisms operating in the grafts were similar to those in an intact leaf, this result also suggests that free water may have an effect on stomata in leaves as well. In addition, it seems possible that the effect of free water on stomata could be related to the disruption of the signal from the mesophyll that was proposed in an earlier study (Mott et al., 2008). We hypothesize that disruption of this signal could be caused by (1) dilution of some solute that is necessary for opening (such as K⁺) in the guard cell walls, (2) dilution of an apoplastic, liquid-phase opening signal from the mesophyll to the guard cells, and (3) blockage of a vapor-phase opening signal from the mesophyll to the guard cells. This study was initiated to test these three hypotheses by examining the effect of free water and other liquids on stomatal functioning.     

The trafficking protein SYP121 of Arabidopsis connects programmed stomatal closure and K⁺ channel activity with vegetative growth

The vesicle‐trafficking protein SYP121 (SYR1/PEN1) was originally identified in association with ion channel control at the plasma membrane of stomatal guard cells, although stomata of the Arabidopsis syp121 loss‐of‐function mutant close normally in ABA and high Ca²⁺. We have now uncovered a set of stomatal phenotypes in the syp121 mutant that reduce CO₂ assimilation, slow vegetative growth and increase water use efficiency in the whole plant, conditional upon high light intensities and low relative humidity. Stomatal opening and the rise in stomatal transpiration of the mutant was delayed in the light and following Ca²⁺‐evoked closure, consistent with a constitutive form of so‐called programmed stomatal closure. Delayed reopening was observed in the syp121, but not in the syp122 mutant lacking the homologous gene product; the delay was rescued by complementation with wild‐type SYP121 and was phenocopied in wild‐type plants in the presence of the vesicle‐trafficking inhibitor Brefeldin A. K⁺ channel current that normally mediates K⁺ uptake for stomatal opening was suppressed in the syp121 mutant and, following closure, its recovery was slowed compared to guard cells of wild‐type plants. Evoked stomatal closure was accompanied by internalisation of GFP‐tagged KAT1 K⁺ channels in both wild‐type and syp121 mutant guard cells, but their subsequently recycling was slowed in the mutant. Our findings indicate that SYP121 facilitates stomatal reopening and they suggest that K⁺ channel traffic and recycling to the plasma membrane underpins the stress memory phenomenon of programmed closure in stomata. Additionally, they underline the significance of vesicle traffic for whole‐plant water use and biomass production, tying SYP121 function to guard cell membrane transport and stomatal control.     

Effects of light/dark and calcium-channel drugs on fluxes of 86Rb+ in “isolated” guard cells of Vicia faba L.

Experiments on ⁸⁶Rb⁺ fluxes were used to identify the changes in ionic state induced by transferring stomata from light to darkness. Guard-cell fluxes and contents were measured on isolated epidermal strips from Vicia faba L. in which all cells other than the guard cells had been killed by ultrasonic disruption. Closure of stomata in response to darkness was achieved by a large, transient stimulation of ⁸⁶Rb⁺ efflux from the guard cells, combined with a reduction of ion transfer from cytoplasm to vacuole, but there was little significant change in influx. Removal of blue but not red light appeared to trigger the flux responses associated with darkening. Attempts to inhibit the closing response (using methoxyverapamil, nifedipine and bepridil, compounds possessing activity against the calcium channels of animal cells) were mostly unsuccessful and the significance of this result is discussed.Abbreviations and Symbols A amplitude - K rate constant - Mes 2-(N-morpholino)ethanesulfonic acid - Q ^* tracer content - s specific activity - R rate of trace loss - Q _T, Q _C, Q _V total, cytoplasmic and vacuolar contents - flux This work was supported by a Research Studentship from the Science and Engineering Research Council. I thank Professor E.A.C. MacRobbie for much helpful discussion and advice.     

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.     

Inhibition of darkness-induced stomatal closure by ethylene involves a removal of hydrogen peroxide from guard cells of <Emphasis Type="Italic">Vicia faba</Emphasis>

Ethylene regulates many aspects of plant growth and development; however, its effect on the behavior of the stomata is still largely obscure. Here, the association between ethylene inhibition of darkness-induced stomatal closure and hydrogen peroxide (H₂O₂) levels in Vicia faba guard cells was studied. Like ascorbic acid (ASA), the most important reducing substrate for H₂O₂ removal, catalase (CAT), one of H₂O₂-scavenging enzymes, and diphenylene iodonium (DPI), an inhibitor of the H₂O₂-generating enzyme NADPH-oxidase, both ethylene-releasing compound 2-chloroethylene phosphonic acid (ethephon, ETH) and 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, were found to inhibit stomatal closure by darkness and to reduce H₂O₂ levels in guard cells, indicating that ethylene-caused inhibition of darkness-induced stomatal closure involves reduction in the H₂O₂ level in guard cells. Additionally, similar to ASA and CAT, ACC/ETH not only suppressed H₂O₂-induced stomatal closure and H₂O₂ level in guard cells treated with exogenous H₂O₂ in the light, but also reopened the stomata, which had been closed by darkness, and reduced H₂O₂ level that had been generated by darkness, showing that ethylene causes H₂O₂ removal from guard cells. However, the above-mentioned effect of ACC/ETH was dissimilar from that of DPI, which not only was incapable to reduce H₂O₂ level induced by exogenous H₂O₂ but also could not abolish H₂O₂ that had been generated by darkness. Thus, we suggest that ethylene probably induces H₂O₂ removal and reduces H₂O₂ level in guard cells and finally inhibits stomatal closure induced by darkness. Furthermore, the mechanism of H₂O₂ removal caused by ethylene was also discussed.     

Light quality and osmoregulation in vicia guard cells : evidence for involvement of three metabolic pathways

Osmoregulation in opening stomata of epidermal peels from Vicia faba L. leaves was investigated under a variety of experimental conditions. The K⁺ content of stomatal guard cells and the starch content of guard cell chloroplasts were examined with cobaltinitrite and iodine-potassium iodide stains, respectively; stomatal apertures were measured microscopically. Red light (50 micromoles per square meter per second) irradiation caused a net increase of 3.1 micrometers in aperture and a decrease of −0.4 megapascals in guard cell osmotic potential over a 5 hour incubation, but histochemical observations showed no increase in guard cell K⁺ content or starch degradation in guard cell chloroplasts. At 10 micromoles per square meter per second, blue light caused a net 6.8 micrometer increase in aperture over 5 hours and there was a substantial decrease in starch content of chloroplasts but no increase in guard cell K⁺ content. At 25 micromoles per square meter per second of blue light, apertures increased faster (net gain of 5.7 micrometers after 1 hour) and starch content decreased. About 80% of guard cells had a higher K⁺ content after 1 hour of incubation but that fraction decreased to 10% after 5 hours. In the absence of KCl in the incubation medium, stomata opened slowly in response to 25 micomoles per square meter per second of blue light, without any K⁺ gain or starch loss. In dual beam experiments, stomata irradiated with 50 micomoles per square meter per second of red light for 3 hours opened without detectable starch loss or K⁺ gain; addition of 25 micomoles per square meter per second of blue light caused a further net gain of 4.4 micometers in aperture accompanied by substantial K⁺ uptake and starch loss. Comparison of K⁺ content in guard cells of opened stomata in epidermal peels with those induced to open in leaf discs showed a substantially higher K⁺ content in the intact tissue than in isolated peels. These results are not consistent with K⁺ (and its counterions) as the universal osmoticum in guard cells of open stomata under all conditions; rather, the data point to sugars arising from photosynthesis and from starch degradation as additional osmotica. Biochemical confirmation of these findings would indicate that osmoregulation during stomatal opening is the result of three key metabolic processes: ion transport, photosynthesis, and sugar metabolism.     

Stomatal opening quantitatively related to potassium transport: evidence from electron probe analysis

When stomata of Vicia faba opened (from a stomatal aperture of about 2 micrometers to one of 12 micrometers) the solute content of the guard cells increased by 4.8 × 10⁻¹² osmoles per stoma. During the same time an average of 4.0 × 10⁻¹² gram equivalents of K⁺ were transported into each pair of guard cells. This amount of K⁺, if associated with dibasic anions, is sufficient to produce the changes in guard cell volume and osmotic pressure associated with stomatal opening. Analysis of Cl, P, and S showed that these elements were not transported in significant amounts during stomatal opening. This finding suggests that the anions balancing K⁺ were predominantly organic. K⁺ was specifically required because no other elements, likely to be present as cations, were found to accumulate in appreciable quantities in guard cells of open stomata.