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.

     

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

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

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.     

Tentoxin Suppresses Stomatal Opening by Inhibiting Photophosphorylation

Tentoxin and, to a lesser extent, dihydrotentoxin (both at 10mmol m^–3) reduce stomatal opening in epidermal stripsof Commelina communis in the light but not in darkness. Thiseffect was significantly greater in normal air than in CO₂-freeair. Fusicoccin overcame the tentoxin effect. However, tentoxindid not inhibit stomatal opening in the light in epidermal stripsof Paphiopedilum harrisianum, a species which lacks guard cellchloroplasts. It is concluded that tentoxin exerts its actionon stomata not by an ionophorous effect in the plasmalemma ofguard cells but by the inhibition of photophosphorylation intheir chloroplasts. The effects of DCMU and tentoxin on guardcells are discussed in terms of their effects on chloroplastsand the extent to which energy is supplied from this organelleduring stomatal opening in the light. The results indicate thatneither photophosphorylation nor non-cyclic electron transportin guard cell chloroplasts are essential for stomatal opening. Key words: Commelina, epidermal strips, Paphiopedilum, photophosphorylation, stomata, tentoxin     

Is the signal from the mesophyll to the guard cells a vapour‐phase ion?

Previous studies have suggested that the red light and CO₂ responses of stomata are caused by a signal from the mesophyll to the guard cells. Experiments were conducted to test the idea that this signal is a vapour‐phase ion. Stomata in isolated epidermes of Tradescantia pallida were found to respond to air ions created by an electrode that was positioned under the epidermes. Anthocyanins in the epidermes of this species were observed to change colour in response to these air ions, and this change in colour was attributed to changes in pH. A similar change in lower epidermal colour was observed in intact leaves upon illumination and with changes in CO₂ concentration. Based on the change in epidermal colour, the pH of the epidermis was estimated to be approximately 7.0 in darkness and 6.5 in the light. Stomata in isolated epidermes responded to pH when suspended over (but not in contact with) solutions of different pH. We speculate that stomatal responses to CO₂ and light are caused by vapour‐phase ions, possibly hydronium ions that change the pH of the epidermis.     

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

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

Energy Supply for Stomatal Opening in Epidermal Strips of Commelina benghalensis

The influence of light or darkness on stomatal opening in epidermal strips of Commelina benghalensis was evaluated in the presence or absence of O₂ and/or metabolic inhibitors. Opening was restricted in nitrogen and was promoted by NADH and acids of the tricarboxylic acid cycle (succinate and α-ketoglutarate) in CO₂-free air in light as well as in darkness. The enhancement by light of stomatal opening was prevalent under nitrogen or in the presence of the respiratory inhibitors (sodium azide and oligomycin). Respiratory inhibitors decreased the opening in light or darkness under CO₂-free air but exhibited no effect under nitrogen, whereas phosphorylation uncouplers were inhibitory in light or darkness under both CO₂-free air and nitrogen. The results suggest that oxidative phosphorylation is a basic source of energy for stomatal opening, although photophosphorylation could be an energy source.     

Stomatal responses to ABA and IAA in isolated epidermal strips ofVicia faba L.

Epidermal strips from well-watered faba-bean plants were subjected to a range of abscisic acid (ABA) and indolyl-3-acetic acid (IAA) concentrations (10^-5 to 1 mM) in the presence or absence of CO₂ in light or dark. ABA had inhibitory effect on abaxial stomatal apertures in all the concentrations studied and retained them closed even after addition of KCl (SO and 100 mM) to the incubation medium. It also influenced stomatal responses to CO₂. In the presence of CO₂ apertures were greater than in its absence in light as well as in darkness. This relationship remained unchanged also after addition of KCl. The action of ABA inhibited accumulation of potassium in the guard cells. IAA stimulated stomatal opening and its effect was quite opposite to ABA; in the presence of CO₂ the apertures were smaller than in its absence. IAA, however, was able to inhibit the closing effect of darkness, CO₂, and ABA, and stimulated potassium accumulation in the guard cells. Simultaneous action of ABA+IAA manifested effects of both substances.     

Action Spectra for Guard Cell Rb Uptake and Stomatal Opening in Vivia faba

Abaxial epidermal strips, containing guard cells as the only viable cells, were prepared from leaves of Vicia faba following a period in darkness, and floated, under CO₂-free air, on 2 mm RbCl + 0.1 mm CaCl₂ labeled with ⁸⁶Rb⁺. Under white light (high pressure mercury vapor lamp), stomatal opening in these strips approached its maximum at less than 0.02 calorie per square centimeter per minute. Under light of different wavelengths, 20 nanometers apart, and at a low quantum flux density of 7 × 10¹⁴ quanta per square centimeter per second, Rb⁺ uptake and stomatal opening were activated only in the blue and long ultraviolet regions, with a peak at 420 to 460 nanometers. The action spectrum suggests that the underlying process is not photosynthesis. At higher quantum flux density (38 × 10¹⁴ quanta per square centimeter per second), uptake and opening also responded to red (600-680 nanometers) and somewhat to green light, with a minimum at 540 to 560 nanometers, indicating a possible involvement of the photosynthetic process. This light-induced opening appeared not to be mediated by a lowering of CO₂ concentration, since CO₂-free air was used in all treatments and controls. Stomatal opening paralleled Rb⁺ uptake in all cases. This constitutes further evidence for the potassium transport hypothesis of stomatal movement.     

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.     

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.     

Role of Potassium in Stomatal Opening in the Leaf of Vicia faba

With isolated epidermal strips of Vicia faba, the intensity of potassium-staining in the guard cells of stomata was calibrated against the uptake of radioactively labeled potassium. By using this calibration, the quantity of potassium that had accumulated in the guard cells, as stomata of leaves of Vicia open in the light, was estimated. Results support the hypothesis that in leaves, as well as in isolated epidermal strips, potassium and an accompanying anion comprise the major, osmotically active solutes in the guard cells of open stomata.     

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

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

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     

Light and stomatal metabolism : I. Possible involvement of light modulation of enzymes in stomatal movement

New evidence is provided regarding the direct effect of light on stomatal opening in the epidermis of the pea (Pisum sativum L. var Little Marvel) leaf. Light modulates the activity of a number of key enzymes involved in stomatal metabolism. When isolated epidermal strips are illuminated, phosphoenolpyruvate carboxylase, NADP-malate dehydrogenase, and NADP-isocitrate dehydrogenase are activated; and aspartate aminotransferase is inactivated. Sulfhydryl compounds, dithiothreitol and glutathione, enhance stomatal opening in epidermal strips both in light or darkness while the sulfhydryl reagent N-ethylmaleimide inhibits, indicating the possible involvement of sulfhydryl groups in stomatal movements. Further, light treatment increases measureable thiol levels in the epidermis about 3-fold. These results suggest that light modulation of enzymes in the epidermis may play a significant role in the mechanism of stomatal movement.     

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.     

Specific requirement of potassium for light-activated opening of stomata in epidermal strips

The effect of various ions on stomatal opening was studied in isolated epidermal strips of Vicia faba L. Stomata in strips floating on 10 mm KCl and in CO₂-free air opened in light, closed in subsequent darkness, then opened fully again when illuminated. A light-activated highly specific effect of K^- (and Rb⁺) on opening was found. When strips were floated on high concentrations (50 or 100 meq/liter) of Li⁺, Na⁺ or Cs⁺, stomata opened but light had very little effect on the concentrations required for opening. With K⁺, the opening produced in the dark was the same as with the other alkali ions. Light, however, lowered more than 100-fold the concentration of K⁺ required for maximal opening. Thus only the effect of K⁺ (and Rb⁺) was greatly accentuated by light. NH₄⁺ and Mg²⁺ did not produce opening.     

Stomatal responses in isolated epidermis of the crassulacean acid metabolism plant Kalanchoe daigremontiana Hamet et Perr.

The optimal conditions for opening of stomata in detached epidermis of the Crassulacean Acid Metabolism (CAM) plant Kalanchoe daigremontiana were determined. Stomatal opening in CO₂–free air was unaffected by light so subsequently all epidermal strips were incubated in the dark and in CO₂–free air. Apertures were maximal after 3 h incubation and were significantly greater at 15° C than 25° C. Thus stomata in isolated epidermis of this species can respond directly to temperature. Stomatal opening was greatest when the incubating buffer contained 17.6 mol m^–3 K⁺, but decreased linearly with increasing K⁺ concentrations between 17.6 and 300 mol m^–3; the decrease in aperture was shown to be associated with increasing osmotic potentials of the solutions. Reasons for this behaviour, which differs from that of many C₃ and C₄ species, are discussed. Stomatal apertures declined linearly upon incubation of epidermis on buffer solutions containing between 10^–11 and 10^–5 mol m^–3 abscisic acid (ABA). Hence stomata on isolated epidermis of K. daigremontiana respond to lower concentrations of ABA than those of any species reported previously.     

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

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

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