Relationship of Temperature to Stomatal Aperture and Potassium Accumulation in Guard Cells of Vicia faba

Epidermal strips of Vicia faba were floated on 10 millimolar KCl at various temperatures and for several time periods. The diameter of the stomatal aperture was determined microscopically and K⁺ content was estimated and expressed as the per cent of the guard cell stained. Stomatal opening was associated with increased K⁺ in guard cells, but the quantitative association was modified both by time and temperature. At low temperatures (0-20 C) there was a prolonged Spannungsphase while at higher temperatures (30-45 C) motorphase was exhibited. During the motorphase there was a rapid opening of the stomates which was highly correlated with K⁺ influx. At treatment periods of 360 minutes and temperatures higher than 25 C there appeared to be a maintenance phase during which K⁺ concentration of the guard cells decreased without an equivalent decrease in aperture.     

The evolution,structure and functioning of stomata

Summary

The anatomy and ultrastructure of guard cells from a range of species varying from the primitive types, such as mosses, to the advanced grasses and orchids are described. An attempt is made to trace the lines along which stomata developed and to define what might be considered advanced stomata. Additionally, the differentiation of guard cells from guard mother cells is discussed. Of particular note is the preprophase band of microtubules which marks the zone where the future cell wall will form and the movement of the spindle and developing cell plate through 45 degrees. The structure and function of guard cells are intimately linked. Stomata are turgor regulated valves; the osmotica for absorbing water during opening are K⁺, Cl^- and malate anions which accumulate in the guard cell vacuoles. Upon stomatal closure, K⁺ and Cl^- exit from the guard cells while at least some of the carbon from malate is channelled into starch and there is a resultant loss of guard cell turgor. The Calvin cycle may be absent or of low activity in guard cell chloroplasts and under those circumstances a source of carbon and energy to sustain the guard cells is needed. Hence it is believed that sucrose is transported into the guard cells from mesophyll cells. A brief consideration of the mechanism by which the ions are transported across the plasma membrane and tonoplast is made: the driving force for the K⁺, Cl^- and malate movement across the membranes is the proton motive force set up by proton-pumping ATPases.     

Two Transduction Pathways Mediate Rapid Effects of Abscisic Acid in Commelina Guard Cells

Commelina guard cells can be rapidly closed by abscisic acid (ABA), and it is thought that this signal is always transduced through increases in cytosolic calcium. However, when Commelina plants were grown at 10 to 17[deg]C, most guard cells failed to exhibit any ABA-induced increase in cytosolic calcium even though all of these cells closed. At growth temperatures of 25[deg]C or above, ABA-induced closure was always associated with an increase in cytosolic calcium. This suggests that there may be two transduction routes for ABA in guard cells; only one involves increases in cytosolic calcium. Activation of either pathway on its own appears to be sufficient to cause closure. Because the rates of ABA accumulation and transport in plants grown at different temperatures are likely to be different, we synthesized and microinjected caged ABA directly into guard cells. ABA was released internally by UV photolysis and subsequently caused stomatal closure. This result suggests a possible intracellular locale for the hypothesized ABA receptor.     

Cellular origin and development of secondary somatic embryos in Oncidium leaf cultures

We report here for the first time that fully differentiated stomatal guard cells have the ability to form directly secondary somatic embryos. Histological and scanning electron microscopy studies reveal that the stomata are formed on primary embryos of both Oncidium cultivars Gower Ramsey and Sweet Sugar. Secondary embryogenesis from these guard cells could be induced by several plant growth regulators (PGRs), including N⁶-benzyladenine, kinetin, thidiazuron, 1-aminocyclopropane-1carboxylic acid (ACC), ancymidol and 2,3,5-triiodobenzoic acid. When compared with other PGRs, ACC at concentration 1 mg dm⁻³ resulted in highest number of secondary embryos that were derived from guard cells. The present communication provides a model system for studying factors and mechanism affecting totipotency or embryogenetic capacity of guard cells.     

Cellular osmotic phenomena during stomatal movements ofCommelina communis

Summary The accuracy of most of the published values for guard cell osmotic pressures is disputed and it is considered that many values are grossly in error. Since most of the values were obtained from incipient plasmolysis experiments limitations of the technique were investigated. It was concluded that it is not possible to use the incipient plasmolysis method for accurately determining guard cell osmotic pressures since all concentrations of plasmolytica (concentrations down to 0.1 M sucrose or calcium nitrate were used) bring about incipient plasmolysis depending on the period of time the tissue is immersed in the plasmolytica. In other words, the concentration of a plasmolyticum at which incipient plasmolysis occurs continues to decrease as the plasmolysing time increases. Furthermore, the time taken for incipient plasmolysis to occur varies according to the solutes in the plasmolyticum and the extent of stomatal aperture.A reason for the changing values of guard cell osmotic pressures was the loss of K⁺, and to a lesser extent, Cl^–, Ca²⁺ and Na⁺, and sugars and organic acids from the tissue during exposure to graded concentrations of plasmolytica (sucrose and calcium nitrate). A good correlation between loss of solutes from the epidermal tissue and decrease in guard cell osmotic pressure was not observed, however.Histochemical tests for K⁺ support the view that leakage of K⁺ from the guard cells occurs while the tissue is immersed in the plasmolytica except when high concentrations of sucrose (2.0 M) and calcium nitrate (greater than 1.0 M) were used and then leakage was minimal. However, these high concentrations of plasmolytica caused cell damage.The osmotic relationships of the various cell types within the epidermis ofCommelina communis were investigated during stomatal movements. Although absolute values for the osmotic pressures of the various cell types could not be evaluated it was apparent from the rates of changes of the osmotic pressures that when stomata closed guard cell osmotic pressures decreased while epidermal and subsidiary cell osmotic pressures increased to almost the same values as the guard cells.     

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.     

Guard cell zeaxanthin tracks photosynthetically active radiation and stomatal apertures in Vicia faba leaves*

Zeaxanthin, antheraxanthin and violaxanthin concentrations in guard cells from sonicated abaxial epidermal peels of Vicia faba were measured from dawn to dusk, and compared with concentrations in mesophyll tissue of the same leaves. Measured changes in guard cell zeaxanthin and violaxanthin concentrations indicate that guard cells operate the xanthophyll cycle throughout the day. Mesophyll tissue had no detectable zeaxanthin at dawn, whereas guard cells had 30–50 mmol mol^?1 chlorophyll a+b. On a chlorophyll basis, maximal zeaxanthin levels were 3–4 fold higher in guard cells than in mesophyll cells. Zeaxanthin concentrations tracked levels of photosynthetically active radiation (PAR) in both mesophyll and guard cells. In the mesophyll, most of the zeaxanthin changes occurred in mid-morning and mid-afternoon. In guard cells, zeaxanthin concentrations changed nearly linearly with PAR in the early morning and late afternoon, and closely tracked PAR levels throughout the day. Guard cell zeaxanthin concentrations were also closely correlated with stomatal apertures. The close relationship between zeaxanthin concentrations and PAR levels in guard cells indicates that zeaxanthin is well suited to function as a molecular photosensor in stomatal movements.     

Stomatal movement in Zea mays: Shuttle of potassium and chloride between guard cells and subsidiary cells

Summary When stomates of Zea mays open K and Cl migrate from the subsidiary cells into the guard cells; when the stomates close both elements return to the subsidiary cells. Subsidiary cells function as reservoirs for K and Cl. Import of K and Cl into the guard cells and loss of both elements from the guard cells become observable 1 or 2 min after light is turned on or off, both when histochemical methods and the electron-probe microanalyzer are used for detection. Each stomatal complex of maize contains on the average 10±3×10^-13 gram equivalents (eq) of K and 4±1×10^-13 eq of Cl. Guard cells accumulate K in the light and CO₂-free air at an average rate of 10×10^-15 eq K per minute, and Cl at approximately half that rate.     

No uptake of anions required by opening stomata of Vicia faba: Guard cells release hydrogen ions

Summary Epidermal strips from leaves of Vicia faba L. with ruptured epidermal cells and intact guard cells were exposed to solutions of K⁺ in association with non-absorbable anions. KCl served as control. Stomata exposed to a range of concentrations of K iminodiacetate, K 4,4-dimethyl-4,7-diazadecane-1,10-disulfonate and K benzene sulfonate opened as widely as on KCl, indicating that K⁺ can be taken up by guard cells without the necessity of an anion traveling along. Electroneutrality was maintained by an exchange of K⁺ for H⁺. Release of H⁺ from guard cells was recorded as a drop in the pH of the solution on which the epidermal samples floated. Formation of acid equivalents by the guard cells was also recorded by automatic titration of the bathing solution at constant pH while CO₂ was continuously being removed. A considerable amount of H⁺ was released from the epidermis by ion exchange (about 8x10^-10 eq/mm²). Subtracting this quantity from the total amount of H⁺ titrated resulted in an estimate of acid production during stomatal opening of 1.2 to 7x10^-10 eq/mm² or 1.5 to 8.5x10^-12 eq/stoma. These amounts are equivalent to the known capacity of the guard cells of Vicia faba to absorb K⁺.     

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.     

Histochemical Approach to Properties of Vicia faba Guard Cell Phosphoenolpyruvate Carboxylase

Properties of phosphoenolpyruvate carboxylase in guard cells dissected from frozen-dried Vicia faba L. leaflets were studied using quantitative histochemical techniques. Control experiments with palisade cells and whole leaflet extract proved that the single cell approach was valid. Most characteristics of enzyme activity in guard cells were identical to those in the leaflet extract. The activities were highly dependent on temperature, with maximum activity at 25 to 35 C. Half-maximum activity (with 1 millimolar phosphoenolpyruvate [PEP]) was observed at 0.1 millimolar Mg²⁺. Two-hundred millimolar NaCl inhibited the reaction by 50%. With frozen-dried leaflet extract, the apparent K_m(PEP) was 0.15 millimolar at pH 7.7; with guard cells, the values were 1.49, 0.5 to 0.8, and 0.24 millimolar in three successive experiments. Additional experiments showed that apparent K_m(PEP) of guard cell activity from plants within a single growth lot was reproducible and did not change during stomatal opening. Mixed extract experiments proved that soluble compounds were not responsible for the difference observed between leaflet and guard cell activities. The differences in apparent K_m(PEP) of guard cell activity could not be unambiguously interpreted. The physiological implications of the properties of this enzyme in guard cells are discussed.     

The chemiosmotic model of stomatal opening revisited

Stomata are light‐activated biological valves in the otherwise gas‐impermeable epidermis of aerial organs of higher plants. Stomata often regulate rates of photosynthesis and transpiration in ways that optimize whole‐plant carbon gain against water loss. Each stoma is flanked by a pair of opposing guard cells. Stomatal opening occurs by light‐activated increases in the turgor pressure of guard cells, which causes them to change shape so that the stomatal pore between them widens. These increases in turgor pressure oppose increases in cellular osmotic pressure that result from uptake of K⁺. K⁺ uptake occurs by a chemiosmotic mechanism in response to light‐activated extrusion of H⁺ outward across the plasma membrane of the guard cell. The initial changes in cellular membrane potential lead to the opening of inward‐rectifying K⁺ channels, after which K⁺ is taken up along its electrochemical gradient. Changes in membrane potential resulting from K⁺ uptake may be balanced by accumulation of Cl^?ions by guard cells and/or by synthesis of malic acid within each cell. Malic acid also acts to buffer increases in cytosolic pH caused by H⁺ extrusion. This review describes how the application of patch‐clamp technology to guard cell protoplasts has enabled investigators to elucidate the mechanisms by which H⁺ is extruded from guard cells, the types of ion channels present in the guard cell plasma membrane, how those ion channels are regulated, and the signal transduction processes that trigger stomatal opening and closing.     

Organic Acid Changes in the Epidermis of Vicia faba and Their Implication in Stomatal Movement

Considerable evidence indicates that the increase in guard cell turgor resulting in stomatal opening is brought about by active K⁺ uptake into guard cells. Only a small increase in inorganic anions appears to accompany the increase in K⁺. A plausible explanation is that organic acids are produced within guard cells and act as counterions, whereas the H⁺ produced are exchanged for K⁺.     

Involvement of chloroplasts in the programmed death of plant cells

The effect of cyanide, an apoptosis inducer, on pea leaf epidermal peels was investigated. Illumination stimulated the CN^–-induced destruction of guard cells (containing chloroplasts and mitochondria) but not of epidermal cells (containing mitochondria only). The process was prevented by antioxidants (-tocopherol, 2,5-di-tret-butyl-4-hydroxytoluene, and mannitol), by anaerobiosis, by the protein kinase C inhibitor staurosporine, and by cysteine and serine protease inhibitors. Electron acceptors (menadione, p-benzoquinone, diaminodurene, TMPD, DCPIP, and methyl viologen) suppressed CN^–-induced apoptosis of guard cells, but not epidermal cells. Methyl viologen had no influence on the removal of CN^–-induced nucleus destruction in guard cells under anaerobic conditions. The light activation of CN^–-induced apoptosis of guard cells was suppressed by DCMU (an inhibitor of the electron transfer in Photosystem II) and by DNP-INT (an antagonist of plastoquinol at the Q_o site of the chloroplast cytochrome b ₆ f complex). It is concluded that apoptosis initiation in guard cells depends on the simultaneous availability of two factors, ROS and reduced quinones of the electron transfer chain. The conditions for manifestation of programmed cell death in guard and epidermal cells of the pea leaf were significantly different.     

Identification and biochemical characterization of the plasma-membrane H+-ATPase in guard cells of Vicia faba L.

The plasma-membrane H⁺-pump in guard cells generates the driving force for the rapid ion fluxes required for stomatal opening. Since our electrophysio-logical studies revealed a two fold higher pump-current density in guard cells than in mesophyll cells of Vicia faba L. we elucidated the biochemical properties of this proton-translocating ATPase in plasma-membrane vesicles isolated from both cell types. The capability of the H⁺ —ATPase to create an H⁺ gradient is maintained in plasma-membrane vesicles derived from purified guard cells via blender maceration, high-pressure homogenization and polymer separation. The H⁺-pumping activity of these vesicles coincides with the presence of two polypeptides of approx. 100 and 92 kDa which are recognized by a monoclonal antibody raised against the plasma-membrane H⁺-ATPase from Zea mays L. coleoptiles. Comparison of H⁺-pumping activities of isolated membranes revealed an approximately two fold higher activity in guard cells than in mesophyll cells with respect to the total membrane protein content. Furthermore, we demonstrated by western blotting that the difference in pump activities resulted from a higher abundance of the electroenzyme per unit membrane protein in guard-cell plasma membranes. We suggest that the high H⁺-pump capacity is necessary to enable guard cells to respond to sudden changes in the environment by a change in stomatal aperture.     

Rubisco activity in guard cells compared with the solute requirement for stomatal opening

We investigated whether the reductive pentose phosphate path in guard cells of Pisum sativum had the capacity to contribute significantly to the production of osmotica during stomatal opening in the light. Amounts of ribulose 1,5-bisphophate carboxylase/oxygenase (Rubisco) were determined by the [¹⁴C]carboxyarabinitol bisphosphate assay. A guard cell contained about 1.2 and a mesophyll cell about 324 picograms of the enzyme; the ratio was 1:270. The specific activities of Rubisco in guard cells and in mesophyll cells were equal; there was no indication of a specific inhibitor of Rubisco in guard cells. Rubisco activity was 115 femtomol per guard-cell protoplast and hour. This value was different from zero with a probability of 0.99. After exposure of guard-cell protoplasts to ¹⁴CO₂ for 2 seconds in the light, about one-half of the radioactivity was in phosphorylated compounds and <10% in malate. Guard cells in epidermal strips produced a different labelling pattern; in the light, <10% of the label was in phosphorylated compounds and about 60% in malate. The rate of solute accumulation in intact guard cells was estimated to have been 900 femto-osmol per cell and hour. If Rubisco operated at full capacity in guard cells, and hexoses were produced as osmotica, solutes could be supplied at a rate of 19 femto-osmol per cell and hour, which would constitute 2% of the estimated requirement. The capacity of guard-cell Rubisco to meet the solute requirement for stomatal opening in leaves of Pisum sativum is insignificant.     

Participation of Chloroplasts in Plant Apoptosis

Mitochondria are known to participate in the initiation of programmed cell death (PCD) in animals and in plants. The role of chloroplasts in PCD is still unknown. We describe a new system to study PCD in plants; namely, leaf epidermal peels. The peel represents a monolayer consisting of cells of two types: phototrophic (guard cells) and chemotrophic (epidermal cells). The peels from pea (Pisum sativum L.) leaves were treated by cyanide as an inducer of PCD. We found an apoptosis-enhancing effect of illumination on chloroplast-containing guard cells, but not on chloroplastless epidermal cells. Antioxidants and anaerobiosis prevented the CN^–-induced apoptosis of cells of both types in the dark and in the light. On the other hand, methyl viologen and menadione known as ROS-generating reagents as well as the Hill reaction electron acceptors (BQ, DAD, TMPD, or DPIP) that are not oxidized spontaneously by O₂ were shown to prevent the CN^–-induced nucleus destruction in guard cells. Apoptosis of epidermal cells was potentiated by these reagents, and they had no influence on the CN^– effect. The light-dependent activation of CN^–-induced apoptosis of guard cells was suppressed by DCMU, stigmatellin or DNP-INT, by a protein kinase inhibitor staurosporine as well as by cysteine and serine protease inhibitors. The above data suggest that apoptosis of guard cells is initiated upon a combined action of two factors, i.e., ROS and reduced plastoquinone of the photosynthetic electron transfer chain. As to reduction of ubiquinone in the mitochondrial respiratory chain, it seems to be antiapoptotic for the guard cell.     

Enzymic and substrate basis for the anaplerotic step in guard cells

From the maximum rate of malate accumulation in Vicia faba L. guard cells during stomatal opening the maximum rate of organic anion synthesis is calculated to be 200 millimoles per kilogram dry weight per hour. A minimum estimate for the phosphoenolpyruvate (PEP) carboxylase-catalyzed reaction in guard cells is 650 millimoles per kilogram dry weight per hour which is significantly higher than in any other leaf tissue. The apparent K_mpep of the guard cell enzyme is 60 μm at pH 8.7, but is probably higher at lower pH. The concentration of PEP in guard cells was 270μm (=2.2 × 10⁻¹⁵ moles/guard cell pair) during anion synthesis. These results support the possibility that the carboxylation of PEP is the anaplerotic step in guard cells.     

Guard cells undergo constitutive and pressure-driven membrane turnover

Summary. During stomatal movement, guard cells undergo large and reversible changes in cell volume and consequently surface area. These alterations in surface area require addition and removal of plasma membrane material. How this is achieved is largely unknown. Here we summarize recent studies of membrane turnover in guard cells using electrophysiology and fluorescent imaging techniques. The results implicate that membrane turnover in guard cells and most likely in plant cells in general is sensitive to changes in membrane tension. We suggest that this provides a mechanism for the adaptation of surface area of guard cells to osmotically driven changes in cell volume. In addition, guard cells also exhibit constitutive membrane turnover. Constitutive and pressure-driven membrane turnover were found to be associated with addition and removal of K⁺ channels. This implies that some of the exo- and endocytic vesicles carry K⁺ channels. Together the results demonstrate that exo- and endocytosis is an essential process in guard cell functioning. Correspondence and reprints: Institute of Botany, Darmstadt University of Technology, Schnittspahnstrasse 3, 64287 Darmstadt, Federal Republic of Germany.     

Potassium Chloride as Stomatal Osmoticum in Allium cepa L., a Species Devoid of Starch in Guard Cells

K⁺ and Cl⁻ contents of guard cells and of ordinary epidermal cells were determined in epidermal samples of Allium cepa L. by electron probe microanalysis; malate contents of the same samples were determined by enzymic oxidation. KCl was, in general, the major osmoticum in guard cells, irrespective of whether stomata had opened on leaves or in epidermal strips floating on solutions. The solute requirement varied between 50 and 110 femtomoles KCl per micrometer increase in aperture per pair of guard cells. Stomata did not open on solutions of K iminodiacetate, presumably because its anion could not be taken up. Stomata opened if KCl or KBr was provided. Taken together, the results indicate that the absence of starch from guard cells deprived them of the ability to produce malate in amounts of osmotic consequence and that the presence of absorbable Cl⁻ (or Br⁻) was necessary for stomatal opening.