Differences in the way potassium chloride and sucrose solutions effect osmotic potential of significance to stomata aperture modulation

Guard cell solution osmotic potential changes resulting in the opening and closing of stomata apertures follow an initial influx of potassium ions, their substitution with sucrose molecules and the subsequent reduction of the latter. To provide an insight into the osmotic mechanism of the changes, the new equation for calculating osmotic pressure, which equates the difference between the energy of pure water across a semi-permeable membrane interface with that of solution water, was used to compare the osmotic properties of KCl and sucrose. For sucrose solutions, the effect of the sucrose molecules in increasing the spacing of the solution water was mainly responsible for osmotic potential; this contrasted with K⁺ + Cl^? ions where their spacing effect was only a little higher to that of water held to those ions. At solute concentrations giving an osmotic potential level of ?3.0 MPa near that of turgid guard cells, the spacing effect on the potential of the unattached solution water molecules caused by sucrose, but in its theoretical absence, was estimated as ?2.203 MPa compared with ?1.431 MPa for KCl. In contrast, the potential attributed to water molecules firmly held to the K⁺ + Cl^? ions was ?1.212 MPa versus zero for sucrose. The potential to keep the sucrose molecules in solution was ?0.797 MPa compared with ?0.357 MPa for KCl. The findings illustrate that the way KCl effects osmotic pressure is very different to that of sucrose. It is concluded that stomata aperture modulation is closely linked to the osmotic properties of its guard cell solution solutes.     

The Effect of Epidermal Cell Water Potential on Stomatal Response to Illumination of Leaf Discs of Vicia faba

Illuminated leaf discs of Vicia faba were brought into equilibrium with a series of mannitol solutions. The width of stomatal aperture and the osmotic potential of guard cells and epidermal cells were determined. It was found that the maximal aperture was obtained when epidermal cells were at about incipient plasmolysis and that any increase in their turgor pressure brought about a decrease in stomatal aperture. These findings emphasize the importance of epidermal cells in determining the width of the stomatal pore.     

The Effect of Osmotic Stress on the Solutes in Guard Cells of Vicia faba L.

We investigated the changes in the levels of solutes in guardcells under osmotic stress. Epidermal strips peeled from Viciafaba L. leaflets were sonicated and incubated in 0.4 M mannitolsolution (osmotic stress) in either light or dark. Stomata wereclosed by osmotic stress. Under osmotic stress, malate accumulatedlight-dependently and sucrose accumulated light-independentlyin the guard cells. The level of K⁺ in guard cells increasedslightly under osmotic stress in the light, although withoutstatistical significance. The levels of all these solutes werereduced by 10 µM ABA treatment. These results suggestthat osmotic stress affects carbon metabolism in guard cells;this metabolic change is different from that caused by ABA alone.Respiratory activity of guard cells decreased under osmoticstress. Therefore, the accumulation of malate and sucrose maybe caused by reduced respiration under osmotic stress. Accumulationof solutes in guard cells by osmotic stress may result in increasedosmotic pressure of guard cells and may play a role in protectionof guard cells from osmotic stress. (Received December 17, 1998; Accepted May 28, 1999)     

The Role of the Epidermal Cells in the Stomatal Movements

The water deficit of the leaves, the osmotic values of the stomatal cells and epidermal cells at incipiment plasmolysis, as well as the width of the stomatal apparatus and pore opening, were measured every hour from 6-17 o'clock under natural environmental conditions. During the noon hours, the intensity of light in clear weather ranged from 40,000-55,000 lux in the open position, and from 15,000-20,000 lux in the shade. The temperature was usually 15–20°C. The experimental object was Vicia Faba growing in a field, both plants freely rooted and plants in pots buried in the soil. The experiments resulted in the following observations and conclusions: 1. When leaves are exposed to strong light, the osmotic value at incipient plasmolysis changes not only in the guard cells, but also in the epidermal cells. If the epidermal cells' osmotic value rises, water is sucked from the guard cells and their uptake of water by suction is decreased, which promotes closure and counteracts opening, respectively. If the value falls, the effect is the reverse. The guard cells react passively to these epidermal changes. The passive stomatal movement eliciteed in this way has therefore been denoted as “osmopassive”, in contrast to the long known passive movement caused by a change in turgor of the epidermal cells, and which has therefore been denoted as “turgorpasslve”. The osmopassive component of stomatal closure has an earlier and more rapid onset than the hydroactive closing reaction, which consists of a decrease in the guard cells' osmotic value. Stomatat closure often starts with the osmopassive rapid process, and is completed and stabilized by the hydroactive process. It has not been possible to determine whether the osmopassive closing reaction is identical with the rapid reaction previously described, and interpreted as of adenoid nature, and tlius belonging to the active group. 2. The osmotic potential of the guard cells - i.e., the difference between the osmotic value of guard cells and epidermal cells at incipient plasmolysis - is, therefore, formed not only by a cbange in the osmotic value of the former cells, but also by a cbange in that of the latter. 3. Although the pore width runs largely parallel to the osmotic value of the guard cells, there is greater agreement between pore width and osmotic potential. When the water deficit of the leaf exceeds a certain threshold value, potential and stomatal width start to decrease. Closure is completed when the fall in potential approaches the zero value. If the water deficit subsequently continues to increase, the potential becomes negative and the stomata remain closed. 4. The stomatal movements are regulated by physiological processes which form two kinds of equilibrium between increase and decrease of the osmotic potential of the guard cells, i.e. the osmopassive increase - osmopassive decrease and the photoactive increase - hydroactive decrease. These equilibria complement each other in rate and stability. The osmopassive processes start rapidly and as soon as the deficit cbanges; hydroactive closure and sometimes also photoactive opening, are, on the contrary, time-consuming. When the water deficit is suboptimal, turgorpassive opening and closing are superadded, but only in those cases in which the osmotic potential of the guard cetls is positive.     

Accumulation of an apoplastic solute in the guard-cell wall is sufficient to exert a significant effect on transpiration in Vicia faba leaflets

Accumulation of recently photosynthesized sucrose in the guard‐cell wall is the empirical foundation for a hypothesis that links the rates of photosynthesis, translocation, and transpiration (Plant Physiology 114, 109–118). Critical assumptions of this hypothesis were tested by use of Vicia faba, an apoplastic phloem loader. Following measurements of the leaflet‐apoplastic‐water volume (by P–V isotherm analysis) and the guard‐cell wall volume (by 3‐D analysis), intact leaflets were fed dilute solutions of mannitol, an impermeant non‐toxic osmolyte. Even at bulk‐leaflet mannitol concentrations that would have only a negligible osmotic effect on stomata, transpiration at constant temperature, water‐vapour pressure, air movement and irradiance was diminished up to 25%, compared with controls. This effect on transpiration, a manifestation of smaller stomatal aperture size, was explained by accumulation of mannitol, up to 350 mol m ^{? 3}, in the estimated aqueous volume of the guard‐cell wall. The conclusion is that mannitol, a xenobiotic with structural similarity to sucrose, can move throughout the apoplast of a transpiring leaflet and accumulate in an osmotically significant concentration in the guard‐cell wall. These data therefore provide support for a new role for sucrose as a signal metabolite that integrates essential functions of the whole leaf. In addition, the results raise questions about the physiological or experimental accumulation of other guard‐cell‐targeted apoplastic solutes such as plant growth regulators, particularly abscisic acid, and ions.     

Effect of different sugars on stomatal behaviour inMerremia aegyptia (L.) Urban andM. dissecta Hallier F.

The effect of mannitol, glucose and sucrose on the stomatal behaviour of two desert species,Merremia aegyptia andM. dissecta has been studied. Stomatal opening did not uniformly depend on the decrease in turgor of the epidermal and subsidiary cells caused by the different osmotic potential of the sugars. Sucrose caused plasmolysis of the subsidiary cells only but this was not accompanied by the opening of the stomatal pore. InM. aegyptia, no plasmolysis was seen either in epidermal or subsidiary cells, even the stomata opened; inM. dissecta, on the other hand, plasmolysis occurred in these cells without any stomatal opening, after incubation in glucose or mannitol. Mannitol is least absorbed, glucose slightly more and sucrose is absorbed to a very large extent in the guard cells when the materials were inoubated in the respective sugar solutions. However, the absorption of these three sugars was almost always larger in isolated epidermal strips than in discs; in detached intact leaves it was still more reduced.     

Die Abhängigkeit des osmotischen Potentials der Stomatazellen vom Wasserzustand der Pflanze

Summary The osmotic value (incipient plasmolysis) of the epidermal cells ofVicia Faba rises with a water deficit, if it is of several days' duration, and sometimes leads to transient wilting. The stomatal cells are an exception, because their osmotic value undergoes little change. Consequently, the osmotic potential of the stomatal cells is strongly negative in relation to that of the epidermal cells. This potential decreases and finally disappears after the plant has been watered, since the osmotic value of the epidermal cells falls; it reaches that of the guard cells after 12–14 hours.Owing to the negative osmotic potential of the guard cells, stomatal opening is prevented as long as the deficit lasts, as well as during the time required for restoring the deficit. Even if it has been restored, the impediment to opening persists for a certain time, because of the after-effect exerted by the water deficit on hydroactive closure.The expenses of the investigation were defrayed by a grant from the Science Research Council of Sweden.Valuable help in carrying out the investigation has been given by Fil. kand. Gösta Stenbeck.     

Endogenous Diurnal Rhythm in the Osmotic Surplus of the Guard Cells

The osmotic surplus of the guard cells —i.e., the difference between the guard cells’and epidermal cells’omotic value at incipient plasmolysis (Vicia Faba) —exhibits an endogenous diurnal-periodic variation that is synchronous with the endogenous diurnal periodicity in the movements of the guard cells. The osmotic surplus of the guard cells is found to arise and drop by changes not only in these cells, but also in the epidermal cells.     

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

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

Storage of Osmotically Active Compounds in the Taproot of Daucus carota L.

The osmotic potential of cell sap from the storage root, lateralroots and shoots of carrot (Daucus carota L., cv. AmsterdamseBak) was calculated from the concentration of osmotically activecompounds in these tissues. The osmotic potential of the taprootdid not change with age prior to and during the storage of osmoticallyactive sugars, as sucrose and reducing sugars. The increased contribution of soluble sugars in the osmoticpotential was accompanied by a proportionally decreased contributionof potassium and organic acids. Before storage of soluble sugarsin the taproot occurred, potassium and organic acids contributed80% to the total osmotic value, in contrast with lateral roottissue, where potassium and nitrate were the main osmotic solutes.The concentration of osmotically active solutes was lower inlateral root tissue than in storage root tissue. Shoot tissueresembled taproot tissue before storage, in having potassiumand organic acids as the main osmotic solutes. The concentrationof osmotically active solutes was highest in shoot tissue andit increased towards the end of the experimental period. The calculated osmotic potentials were in good agreement withthe experimental values, as determined from the molecular depressionof the freezing point of cell sap. Storage of reducing sugarsand sucrose is discussed in relation to acid and neutral invertaseactivities. Key words: Daucus carota, Osmotic solutes, Sugar storage, Invertase activity     

Solute distribution in sugar beet leaves in relation to Phloem loading and translocation

The distribution of solutes in the various cells of sugar beet (Beta vulgaris L.) source leaves, petioles, and sink leaves was studied in tissue prepared by freeze-substitution. The differences in degree of cryoprotection indicated that sieve elements and companion cells of the source leaf, petiole, and sink leaf contain a high concentration of solute. The osmotic pressure of various types of cells was measured by observing incipient plasmolysis in freeze-substituted tissues equilibrated with a series of mannitol solutions prior to rapid freezing. Analysis of source leaf tissue revealed osmotic pressure values of 13 bars for the mesophyll and 30 bars for the sieve elements and companion cells. The osmotic pressure of the mesophyll of sink leaves was somewhat higher.     

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

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

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.     

Abscisic Acid-Induced Stomatal Closure in Vicia faba Epidermal Strips. Excretion of Solutes from Guard Cells and Increase in Elastic Modulus of Guard Cell Wall

The effects of abscisic acid (ABA) on the size of the apertureof stomata on epidermal strips of Vicia faba were studied inincubation media with different pH values. The osmotic potentialof guard cells, as determined by the limiting plasmolysis method,was higher at pH 4.0 than at pH 6.0, although the size of thestomatal apertures was almost identical at both pH values. AtpH 4.0, ABA effectively caused stomatal closure but had onlya small effect on the osmotic potential, whereas, at pH 6.0,ABA significantly increased the osmotic potential. ABA promotedthe efflux of Cl^– and malate from epidermal strips intothe incubation medium, an effect which was more marked at pH6.0, with a concomitant efflux of K⁺ to balance the charge onthe exported anions. From these results, it is suggested thatABA may cause an increase in the elastic modulus of the cellwalls of guard cells. ³ Present address: Nagano Prefectural Vegetable and OrnamentalCrops Experimental Station, 2206 Oomuro, Matsusiro-machi, Nagano381-12, Japan (Received September 30, 1986; Accepted January 9, 1987)     

Regulation of H Excretion : EFFECTS OF OSMOTIC SHOCK

Osmotic shock, a 15-minute plasmolysis followed by a 15-minute rehydration in the cold, is a nondestructive technique which inhibits fusicoccin-stimulated H⁺ excretion from oat mesophyll cells (Avena sativa L.). Osmotic shock also causes a loss of intracellular solutes and stimulates H⁺ uptake, but osmoregulation can still occur, and enhanced H⁺ uptake is observed only at low external pH. It is concluded that osmotic shock interferes directly with the excretion of H⁺ rather than affecting only H⁺ or counter ion uptake.     

The mechanism of stomatal movement in Allium cepa L.

In the guard cells of Allium cepa leaves, no starch was found either when the stomata were open or closed. The lack of other soluble polysaccharides that could be hydrolyzed during the opening reaction of the stomata (Schnabl, Planta 1977, in press) leads to the question, how is the osmotic effect, which is the basis of the stomatal movement, achieved in Allium? It is shown in this paper, by histochemical and microprobe analyses, that in Allium — as in other plant species—the K⁺ concentration of the guard cells increases during stomatal opening. The charges of the K⁺ ions in the guard cells seem to be fully compensated by imported Cl^- ions. This could mean that if starch is present in the guard cells, as in the majority of plant species, its major role in the mechanism of stomatal movement is to deliver the cuunteranions for the imported K⁺ ions.     

The role of organic and inorganic solutes in the osmotic adjustment of drought-stressed Jatropha curcas plants

This study aimed to assess the accumulation of organic and inorganic solutes and their relative contribution to osmotic adjustment in roots and leaves of Jatropha curcas subjected to different water deficit intensity. Plants were grown in vermiculite 50% (control), 40%, 30%, 20% and 10% expressed in gravimetric water content. The water potential, osmotic potential and turgor potential of leaves decreased progressively in parallel to CO₂ photosynthetic assimilation, transpiration and stomatal conductance, as the water deficit increased. However, the relative water content, succulence and water content in the leaves did not show differences between the control and stressed plants, indicating osmotic adjustment associated with an efficient mechanisms to prevent water loss by transpiration through stomatal closure. The K⁺ ions had greater quantitative participation in the osmotic adjustment in both leaves and roots followed by Na⁺ and Cl⁻, while the NO₃⁻ ion only showed minor involvement. Of the organic solutes studied, the total soluble sugars showed the highest relative contribution to the osmotic adjustment in both organs and its concentration positively increased with more severe water deficit. The free amino acids and glycinebetaine also effectively contributed to the osmotic potential reduction of both the root and leaves. The role of proline was quantitatively insignificant in terms of osmotic adjustment, in both the control and stressed roots and leaves. Our data reveal that roots and leaves of J. curcas young plants display osmotic adjustment in response to drought stress linked with mechanisms to prevent water loss by transpiration by means of the participation of inorganic and organic solutes and stomatal closure. Of all the solutes studied, soluble sugars uniquely display a prominent drought-induced synthesis and/or accumulation in both roots and leaves.     

Solute Accumulation in Tobacco Cells Adapted to NaCl

Cells of Nicotiana tabacum L. var Wisconsin 38 adapted to NaCl (up to 428 millimolar) which have undergone extensive osmotic adjustment accumulated Na⁺ and Cl⁻ as principal solutes for this adjustment. Although the intracellular concentrations of Na⁺ and Cl⁻ correlated well with the level of adaptation, these ions apparently did not contribute to the osmotic adjustment which occurred during a culture growth cycle, because the concentrations of Na⁺ and Cl⁻ did not increase during the period of most active osmotic adjustment. The average intracellular concentrations of soluble sugars and total free amino acids increased as a function of the level of adaptation; however, the levels of these solutes did not approach those observed for Na⁺ and Cl⁻. The concentration of proline was positively correlated with cell osmotic potential, accumulating to an average concentration of 129 millimolar in cells adapted to 428 millimolar NaCl and representing about 80% of the total free amino acid pool as compared to an average of 0.29 millimolar and about 4% of the pool in unadapted cells. These results indicate that although Na⁺ and Cl⁻ are principal components of osmotic adjustment, organic solutes also may make significant contributions.