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.     

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.     

Osmotically Induced Water Stress, Potassium Uptake, and Stomatal Aperture in Epidermal Strips of Vicia faba L.

Uptake of potassium ions, estimated microdensitometrically,and stomatal opening in isolated epidermal strips from Viciafaba L. cv. Aquadulce were both inhibited by incubation of thestrips in solutions of low solute potential. The inhibitionof potassium uptake was substantially reduced by preincubationof the strips in benzylaminopurine¹ solution. The data are discussedin terms of the induction of abscisic acid synthesis by waterstress in the epidermal strips and the antagonism to its effectsby BAP.     

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.

     

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.     

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.     

Stomatal responses to light and CO2 are dependent on KCI concentration

Abstract. The responses of stomata on detached epidermis of Commelina communis to light and CO₂ have been shown to be strongly dependent on the concentration of KCI in the incubation medium. There was a high sensitivity to the two stimuli in 50 mM KCI, but there were much reduced responses at lower and higher concentrations. It is considered that an appropriate choice of medium is essential if useful physiological studies of stomata are to be made using epidermal strips. At lower KCI concentrations, the ability of the stomata to open is thought to be limited by the availability of K⁺ ions, and at higher concentrations their ability to close may be affected because of an inhibition of the net efflux of K⁺. The production of malate was related to KCI concentration, and was largest in the medium containing zero KCI which supported poor stomatal responses to light and CO₂It is concluded that malate metabolism is unlikely to play a central part in the changes in guard cell turgor that are brought about by light and CO₂.     

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

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

Light and Stomatal Metabolism : II. Effects of Sulfite and Arsenite on Stomatal Opening and Light Modulation of Enzymes in Epidermis

The effect of sulfite and arsenite on stomatal opening and light modulation of enzymes was examined in isolated epidermal strips of Pisum sativum L. var Little Marvel leaves. Sulfite or arsenite at 10 micromolar rapidly inhibited the stomatal opening process in light. Light activation of phosphoenolpyruvate carboxylase and NADP-malate dehydrogenase was completely diminished when the epidermal strips were incubated for 2 hours in light with either sulfite or arsenite at 10 micromolar. The data obtained suggest that the inhibition of stomatal opening by sulfite or arsenite in light might result from the inhibition of light modulation of key enzymes in guard cells.     

Cell Potentials and Turgor Pressures in Epidermal Cells of Tradescantia and Commelina

Cell membrane potentials have been measured both in epidermalstrips and intact leaf sections of Tradescantia virginiana andCommelina communis, and in epidermal cells over green and overalbino mesophyll cells of T. albiflora var. albovittata. Membranepotentials (_cell) in strips were considerably lower than thosein intact sections and were insensitive to light and to theabsence or presence of calcium. Their response to external cationlevels was indifferent to ionic species. However, in intactleaf sections incubated with calcium present, membrane potentialsresponded to K⁺ levels but not to Na⁺. were more negative thancells in epidermal strips, and responded to changes in illumination. Long-term recordings of _cell and vacuolar K⁺ levels in T. virginianaduring stomatal closure suggest that the fluctuations of _cellwere unrelated to K⁺ movement (which we could not detect) andthus probably to stomatal movement as well. Turgor pressures measured in epidermal cells of intact leafsections of T. virginiana were found to be of the same magnitudeas those previously reported for epidermal strips. It is concludedthat epidermal cells maintain their solute contents during strippingwithout the involvement of an electrophysiological transportsystem. With the possible exception of lateral subsidiary cells,there was no evidence suggesting that ordinary epidermal cellsare capable of osmotic adjustment even when additional KCI wassupplied in the osmoticum. Absolute turgor levels in intactleaf sections kept at constant external KCI were unrelated tosteady state _cell.     

Light and scanning electron microscopy of SO2-induced injury to leaf surfaces of field bean (Vicia faba L.)

Abstract. Exposure to low concentration of SO₂ increases stomatal conductance in field bean by c 20%. Microscopy of epidermal strips showed that this response was associated with extensive destruction of epidermal cells adjacent to the stomata. Guard cell survival was not affected signficantly. Collapsed epidermal cells were also observed in intact leaf samples using scanning electron miscroscopy, thus confirming that the damage seen in epidermal strips was not caused by the stripping procedure. These observations are discussed in relation to the hypothesis that the increased stomatal opening induced by low SO₂ concentrations results from preferential injury to the adjacent cells.     

The effect of blue light on energy levels in epidermal strips

Red light applied together with blue enhanced stomatal opening in epidermal strips of Commelina communis L. more than red light alone. In red light, stomatal opening was enhanced by exogenously applied ATP and was inhibited by 3-(3,4-dichlorophe-nyl)-l,l-dimethylurea (DCMU), while in the presence of blue light external ATP was almost without effect, and DCMU stimulated stomatal opening. Blue light increased the ATP levels in the epidermal strips. DCMU diminished the amount of ATP in both red light and red + blue light treatments, but did not abolish the stimulatory effect of blue light. Blue light also stimulated the respiration rate of the epidermal strips. Rotenone, which inhibited stomatal opening and respiration rate, abolished the effect of blue light in both processes. These results imply that blue light increases the ATP levels by stimulation of oxidative phosphorylation.     

Evidence for the Uptake of Large Anions through Stomatal Pores

Experiments were conducted with leek (Allium porrum L.) leaves to investigate whether aqueous solutions are able to penetrate stomata. Epidermal strips were used for the determination of transport rates. Stomata were opened by fusicoccin or closed by darkness or abscisic acid. A droplet containing the anionic fluorescent dye, uranine, was placed on the physiologically outer side of the epidermis and allowed to dry. With open stomata 30 times more uranine penetrated through the epidermal strips than with closed stomata (comparison of medians). In another experiment droplets of uranine solution were placed on leaf segments and epidermal strips were removed after drying of the droplets. Penetration of uranine through stomata was detectable under the microscope both with epidermal strips from the transport experiments and with strips obtained after application on leaf segments. As maximum uptake rates occurred during the drying process, it is concluded that penetration took place via water films. These results show that the physical restrictions preventing stomatal penetration of static droplets are not decisive for drying droplets and that stomatal uptake of dissolved ionic substances occurs under natural conditions, i.e. without surfactants or applied pressure.     

Ion-Stimulated Stomatal Opening Induced by Preillumination in Epidermal Strips of Commelina communis

The effects of preillumination were investigated on ion-stimulated stomatal opening of epidermal strips isolated from Commelina communis L. leaves, which are dark-starved 24 hours or more. The rate and the extent of ion-stimulated stomatal openings were increased by preexposure of epidermal strips to light. The evidences are interpreted as indicating that the energy induced by preillumination can be conserved in guard cells for considerable time periods and then used for a delayed stomatal opening in the presence of higher concentration of potassium or sodium ions. Action spectrum showed two peaks, one in blue and one in the red light region. The ratio of the blue peak to the red peak is 1.2; which is the smallest reported value in action spectra of stomatal movements. 3-(4-chlorophenyl)-1,-1-Dimethylurea suppressed the ion-stimulated stomatal opening induced by the preillumination. We conclude that the photosynthetic electron transport system, containing photosystem II, in guard cell chloroplasts is a basic system of energy acquirement for stomatal opening.     

Circadian Rhythm of Stomatal Movements in Epidermal Strips

Measurements were made of changes in stomatal pore widths inepidermal strips of leaves ofVicia faba and Commelina communis.Strips were incubated in dilute KCI solutions (1 and 10 molm^–3) flowing through a perfusion chamber on the stageof a microscope and kept for 4 d in continuous light. Circadianrhythms of stomatal apertures were detected in both species.Although the amplitude was small it was statistically significant.It is concluded that at least partof the mechanism for the stomatalrhythm resides in the epidermis, probably in the guard cells. Key words: Cireadian rhythm, epidermal strips, stomata     

Tyrosine phosphorylation increases Ca2+ sensitivity of vascular smooth muscle contraction

In order to elucidate the role of tyrosine phosphorylation in vasoconstriction, we investigated the effects of inhibitors of tyrosine kinase (genistein, 30 microM) and phosphatase (sodium o-vanadate, 5 microM) on the contraction of aorta isolated from guinea pig. Genistein significantly inhibited norepinephrine-induced contraction, but it did not affect that induced by KCI. Thus, tyrosine phosphorylation may not be involved in the contractile response to KCI alone. The aortic contraction elicited by KCl was significantly augmented by sodium o-vanadate, which increased both the maximum force and pD2 values of KCl contraction. In the presence of verapamil, KCl-induced contraction was abolished even after pretreatment with sodium o-vanadate. Sodium o-vanadate also augmented Ca2+-induced contraction in the aortic strips depolarized with KCl, increasing both its maximum force and pD2 values. Neither basal 45Ca2+ uptake nor verapamil-sensitive 45Ca2+ uptake induced by KCl were affected by pretreatment with sodium o-vanadate. These results suggest that tyrosine phosphorylation is involved in the contraction of guinea-pig aorta not through transplasmalemmal Ca2+ entry but through increased Ca2+ sensitivity of the intracellular contractile pathway.     

Proton-stimulated opening of stomata in relation to chloride uptake by guard cells

The concentration of potassium chloride required in the incubation medium to open stomata in isolated epidermal tissues of Commelina communis L. and Vicia faba L. could be lowered from 100 mM to 10 mM if the proton concentration of the ambient solution was increased from pH 5.6 to pH 3.5. This acidification effect was formerly attributed to the destruction of epidermal and subsidiary cells resulting in a relief of back pressure upon guard cells. While guard cells remain viable at pH 3.5, as demonstrated by their susceptibility to inhibition by uptake of glucose or to uncoupling by DNP, incipient destruction of the cells surrounding them could first be observed 30 min after the onset of the incubation experiment. By this time, however, the stomata had already opened; the time course of stomatal opening at pH 3.5 did not show any lag phase corresponding to the time required for damaging epidermal cells and showed no difference to that at pH 5.6 Thus, the acid-stimulated opening of stomata appears to be a biphasic phenomenon consisting of a physiologic effect onto which the physical effect of the relief of back pressure is superimposed over longer periods of incubation. To interpret the physiologic role of an increased proton concentration in the ambient solution of isolated epidermal strips, it is suggested that guard cells take up protons and chloride ions in an electroneutral symport. While protons are extruded again to generate the negative membrane potential required for potassium influx, chloride ions are retained to maintain electroneutrality.     

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.     

A Critical Assessment of the Role of Potassium and Osmolarity in Stomatal Opening

An attempt has been made to separate the osmotic effect fromthe ionic effect of KCI in stomatal responses. For this purposeisolated illuminated epidermis from species with and withoutsubsidiary cells were treated with KCI (0-250 mOs kg^–1)and with mannitol (0-250 mOs kg^–1). Since osmolarity wasmade the basis of comparison, the effect of mannitol had tobe observed immediately, before guard cell contents could haveleached into the incubation medium. When plotting aperturesagainst osmolarity sigmoid curves were obtained with KCI, butwith mannitol straight lines resulted provided that prior tostripping and incubation leaves were briefly illuminated. Whilst in lower concentrations (60 mOs kg^–1 for Viciafaba; 90 mOs kg^–1 for Zantedeschia aethiopica; 190 mOskg^–1 for Commelina communis) pores were wider in mannitolthan in KCI, in concentrations above these values the situationwas reversed. It appeared therefore that KCI had either an inhibitoryor a promoting effect. Inhibition was most pronounced when atthe beginning of incubation stomata were closed; the inhibitoryeffect on stomata without subsidiary cells occurred at low concentrations(0-60 mOs kg^–1) whereas when subsidiary cells were presentinhibition occurred at up to 190mOs kg^–1. Other experiments started with KCI solutions of 50 mOs kg^–1for Vicia faba, 85 mOs kg^–1 for Zantedeschia aethiopicaand 115 mOs kg^–1 for Commelina communism; mannitol wasadditionally used to give the progressive increases in osmolarity.Degrees of opening were then reached which with KCI alone couldonly be attained at the very highest concentrations. Starch disappearance was followed using the periodic-acid-silvertest; by using either ⁸⁶Rb or ⁴³K it was shown that ion uptakewas restricted to guard cells alone only at osmolarities exceeding200 mOs kg^–1. On the basis of these observations it was concluded that K transportdoes not represent the major mechanism of stomatal regulation. Key words: Stomata, Potassium, Osmolarity     

Effect of Ethephon on stomatal opening in detached epidermal strips of tobacco leaves (Nicotiana tabacum L. cv. Samsun)

The effect of Ethephon preparations upon stomatal openings in detached epidermal strips of tobacco leaves floated on 0.1 M KC1 was measured microscopically after different times of exposure. Addition of 0.01 v/v % or 0.1 v/v % Camposan, Ethrel, or E-BOH resulted in the opening or closing of the stomata depending upon the time of exposure and the concentration of Ethephon. It is evident from the results obtained in these studies that an increase in stomatal opening was induced by the acidity of the preparations and the released ethylene too. Prolonged times of action resulted in decreased stomatal openings or in stomata being closed, respectively. High concentrations of Ethephon also resulted in major damage being caused to guard cells after having been permitted to act upon them for an extended period of time.