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 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.     

Different stomatal responses of maize leaves after blue or red illumination under anoxia

Abstract In normal air, illumination with a low level of blue or red light (40 μmol m^?2 s^?1) did not induce stomatal opening in maize plantlets. In CO₂-free air, 40 μmol m^?2 s^?1 of blue or red light promoted an enhancement in stomatal opening. At the same quantum flux, blue light was more efficient than red light and stomatal closure occurred more rapidly with a significantly shorter lag phase after blue light. Anoxia inhibited light-dependent stomatal opening, even under 320 μmol m^?2 s^?1 illumination. However, after 60 min of illumination with 40 μmol m^?2 s^?1 of blue light in anoxia, transient stomatal opening was observed when the plant was returned to darkness and normal air. This transient stomatal opening was weaker after pretreatment with red light. We conclude that a blue-light-dependent process induced under anoxia leads to stomatal opening provided oxygen is present. Possible mechanisms associated with blue-light-effect and the nature of the oxygen-consuming processes are discussed.     

Environmental effects on circadian rhythms in photosynthesis and stomatal opening

Persistent circadian rhythms in photosynthesis and stomatal opening occurred in bean (Phaseolus vulgaris L.) plants transferred from a natural photoperiod to a variety of constant conditions. Photosynthesis, measured as carbon assimilation, and stomatal opening, as conductance to water vapor, oscillated with a freerunning period close to 24 h under constant moderate light, as well as under light-limiting and CO₂-limiting conditions. The rhythms damped under constant conditions conducive to high photosynthetic rates, as did rates of carbon assimilation and stomatal conductance, and this damping correlated with the accumulation of carbohydrate. No rhythm in respiration occurred in plants transferred to constant darkness, and the rhythm in stomatal opening damped rapidly in constant darkness. Damping of rhythms also occurred in leaflets exposed to constant light and CO₂-free air, demonstrating that active photosynthesis and not simply light was necessary for sustained expression of these rhythms. This is CIWDPB Publication No. 1142 This research was supported by National Science Foundation grant BSR 8717422 (C.B.F.) and a U.S. Department of Agriculture training grant to Stanford University (T.L.H.).     

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.

     

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.     

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.     

Effects of Metabolic Inhibitors on the Rates of CO(2) Evolution in Light and in Darkness by Detached Spruce Twigs, Wheat, and Soybean Leaves

Detached spruce twigs, wheat and soybean leaves were infiltrated with various metabolic inhibitors, placed in a closed system in CO₂-free air and the amounts of CO₂ evolved in either light or darkness were determined with an infra-red CO₂ analyzer. In light, metabolic inhibitors always greatly suppressed evolution of CO₂, the magnitude of suppression varying between 50 to 80% of that without an inhibitor. This depressing effect became less pronounced with increasing oxygen. In darkness, metabolic inhibitors sometimes suppressed and sometimes stimulated CO₂ evolution. These observations have been taken as further support for a conclusion made earlier, that evolution of CO₂ in light and darkness is not the same process.     

Stomatal Opening in Isolated Epidermal Strips of Vicia faba. II. Responses to KCl Concentration and the Role of Potassium Absorption

The stimulation by KCl of stomatal opening in isolated epidermal strips of Vicia faba was examined. In dark + normal air the opening response was maximal at 100 mm KCl while in light + CO₂-free air it was maximal at about 10 mm KCl. CO₂-free air was more influential than light in reducing the KCl concentration required for maximal opening. K⁺ was essential while Cl⁻ seemed to be of secondary importance in these processes.     

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.     

The role of the epidermis in the generation of the circadian rhythm of carbon dioxide fixation in leaves of Bryophyllum fedtschenkoi

The role of the epidermis in the generation of the endogenous circadian rhythm of CO₂ exchange in leaves of Bryophyllum fedtschenkoi has been examined. At 25° C the rhythm of CO₂ output exhibited by whole leaves kept in continuous darkness and an initially CO₂-free air stream also occurs in isolated pieces of mesophyll. The sensitivity to light of the rhythms in whole leaves and in isolated mesophyll appears to be identical. At 15° C, however, no rhythm is observed in isolated mesophyll tissue, despite there being a conspicuous rhythm in intact leaves. The rhythm of net CO₂ assimilation in whole leaves kept in continuous light and a stream of normal air at either 25° C or at 15° C is abolished by removal of the epidermis, although at 15° C and under the higher of the two light levels used, there is an indication that rhythmicity may begin to reappear after the third day of the experiment. Thus, only under certain environmental conditions is the rhythm of CO₂ exchange in Bryophyllum leaves independent of the epidermis. The results indicate that the rhythm of carbon dioxide fixation in continuous darkness and CO₂-free air is generated primarily in the mesophyll cells, whereas the rhythm in continuous light and normal air is generated in the stomatal guard cells or in an interaction of these cells with the mesophyll cells.Abbreviation PEPCase phosphoenolpyruvate carboxylase     

Effect of ouabain on stomatal movements and transpiration rate of Secale cereale

Young rye seedlings (Secale cereale cv. Petkus) were grown for 12 or 18 din aerated nutrient solution. CO2-free air in light or in darkness induced stomatal opening. When light and CO2-free air were applied together, maximum stomatal opening was observed. Na+ channel inhibitor [3H]ouabain was taken up from nutrient medium and translocated into the leaves via the xylem sap. The presence of 10-5 M ouabain in the root medium for 20 h increased stomatal opening and transpiration rate. On the other hand, stomatal closing was not affected by the presence of ouabain. These results suggested that Na+ might play a role in stomatal movements. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

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 state of activation of ribulose-1,5-bisphosphate carboxylase in wheat leaves

In light and in darkness, exposure of leaf segments to CO₂-free atmospheres caused a marked reduction in extractable RuBP carboxylase activity. By contrast, darkness caused a relatively small decrease in carboxylase activity in extracts from leaf segments kept in air containing CO₂. Recovery of carboxylase activity in leaves during illumination in air after exposure to CO₂-free conditions paralleled recovery of capacity for photosynthesis; in darkness recovery of carboxylase activity in leaves was slower than in the light. Extracts from leaves exposed to CO₂-free conditions recovered activity when provided with CO₂ and Mg²⁺; there were clearly, however, substances in the extracts that modified the activity achieved and caused anomalous decreases and increases with time after extraction. Studies of the effect of orthophosphate on the activity of purified wheat carboxylase in vitro were consistent with the view that many of the effects observed on the activity of crude leaf extracts were due to orthophosphate content.     

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.     

Photosynthesis of Ivy Leaves (Hedera helix) after Heat Stress I. CO2-Gas Exchange and Diffusion Resistances

For an analysis of the inhibition of the photosynthetic CO₂-uptake after heat stress attached leaves of Hedera helix L. were heat-stressed for 30 min at various temperatures. Subsequently their photosynthetic CO₂-uptake, transpiration, respiration in light and darkness, and CO₂-compensation concentration were measured under optimal conditions. After heat stress the stomatal resistance increased only corresponding to the raised CO₂-concentration inside the leaves (due to the reduced CO₂-uptake). The physical resistance between the mesophyll cell walls and the chloroplasts remained unchanged after heat stress. A non-stomatal inhibition of the CO₂-uptake is indicated by a strong increase of the CO₂-compensation concentration after heat stress. This is hardly due to a stimulation of the respiration in light, as the CO₂-evolution into CO₂-free air in light was even reduced. Therefore, it must be concluded that the photosynthetic process itself is impaired after heat stress.     

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.     

Inhibition of stomatal opening in sunflower leaves by carbon monoxide,and reversal of inhibition by light

When leaves of Helianthus annuus, whose stomates had been opened in the dark in the absence of CO₂, were exposed to 25% carbon monoxide (CO), stomatal conductivity for water vapor decreased from about 0.4 to 0.2 cm·s^-1. The CO effect on stomatal aperture required a CO/O₂ ratio of about 25. As this ratio was decreased the stomata opened, indicating that inhibitio of cytochrome-c oxidase by CO is competitive in respect to O₂. Photosynthetically active red light was unable to reverse CO-induced stomatal closure even at high irradiances, when CO₂ was absent. When it was present, stomatal opening was occasionally, but not consistently observed. Carbon monoxide did not inhibit photosynthetic carbon reduction in leaves of Helianthus.In contrast to red light, very weak blue light (405 nm) increased the stomatal aperture in the presence of CO. It also increased leaf ATP/ADP ratios which had been decreased in the presence of CO. The blue-light effect was not related to photosynthesis. Neither could it be explained by photodissociation of the cytochrome a ₃-CO complex which has an absorption maximum at 430 nm. The data indicate that ATP derived from mitochondrial oxidative phosphorylation provides energy for stomatal opening in sunflower leaves in the dark as well as in the light. Indirect transfer of ATP from chloroplasts to the cytosol via the triose phosphate/phosphoglycerate exchange which is mediated by the phosphate translocator of the chloroplast envelope can support stomatal opening only if metabolite concentrations are high enough for efficient shuttle transfer of ATP. Blue light causes stomatal opening in the presence of CO by stimulating ATP synthesis.     

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.