The Components of the CO2-Indluced Stomatal Movements

Leaf discs of Vicia Faba were allowed to float on water in glass dishes placed in vessels containing KOH. The vessels were kept in darkness at constant temperature. The stomatal width and osmotic values of the guard cells and epidermal cells were measured, generally at one-hour intervals. When the CO₂ content of the air surrounding the leaf specimen falls, it causes a disturbance in the osmotic equilibrium between guard cells and epidermal cells. Sometimes the changes start in the form of falling osmotic values in both kinds of cell. In other cases the values rise, and in still others the changes may be confined chiefly to one of these kinds of cell. Since the changes are not the same in guard cells and epidermal cells, the osmotic difference between them rises or falls. The difference rises during the time immediately after removal of CO₂ from the surrounding air. This causes an osmotic surplus to arise or increase in the guard cells. Later, this change may take place in the opposite direction. The stomatal movements occurring simultaneously follow, on broad lines, the osmotic surplus of the guard cells. Consequently, the CO₂-induced stomatal movement is the result of an interaction between an active component—i.e., the intrinsic osmotic changes in the guard cells—and an osmopassive component, by which is meant here the osmotic changes in the epidermal cells.     

PHYSIOLOGICAL IMPLICATIONS OF VICIA FABA AND NICOTIANA TABACUM GUARD-CELL ULTRASTRUCTURE

The guard cells of Vicia faba and Nicotiana tabacum contain numerous mitochondria, elements of endoplasmic reticulum, spherosomes, and peroxisome-like microbodies. A full ribosomal complement appears in young but not in fully mature guard cells. Numerous small lipid droplets external to the plasmalemma were noted in mature Vicia guard cells. Chloroplasts were found in both epidermal and guard cells of both species. Full photosynthetic capacity was indicated by the grana fretwork of guard-cell chloroplasts. A specialized peripheral reticulum was observed in the guard-cell chloroplasts of Vicia. Plasmodesmata were observed in both walls between sister guard cells and between guard and epidermal cells. In the latter case plasmodesmata were found primarily in pit fields of transverse walls. It is postulated that the small volume of guard cells allows them an osmotic advantage over larger neighboring cells in generating turgor.     

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.     

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.     

Dynamics of stomatal water relations during the humidity response: implications of two hypothetical mechanisms

The feasibility of two hypothetical mechanisms for the stomatal response to humidity was evaluated by identifying theoretical constraints on these mechanisms and by analysing timecourses of stomatal aperture following a step change in humidity. The two hypothetical mechanisms, which allow guard cell turgor pressure to overcome the epidermal mechanical advantage, are: (1) active regulation of guard cell osmotic pressure, requiring no hydraulic disequilibrium between guard and epidermal cells, and (2) a substantial hydraulic resistance between guard and epidermal cells, resulting in hydraulic disequilibrium between them. Numerical simulations of the system are made possible by recently published empirical relationships between guard cell pressure and volume and between stomatal aperture, guard cell turgor pressure, and epidermal cell turgor pressure; these data allow the hypothetical control variables to be inferred from stomatal aperture and evaporative demand, given physical assumptions that characterize either hypothesis. We show that hypothesis (1) predicts that steady‐state π_g is monotonically related to transpiration rate, whereas hypothesis (2) suggests that the relationship between transpiration rate and the steady‐state guard to epidermal cell hydraulic resistance may be either positive or negative, and that this resistance must change substantially during the transient phase of the stomatal response to humidity.     

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.     

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.     

A light and electron microscopy study of the epidermis of Paphiopedilum spp. with emphasis on stomatal ultrastructure

Abstract Light and fluorescence microscopy studies indicated that chlorophyll was absent from the guard cells of the lady slipper orchids, Paphiopedilum insigne (Wall.) Pfitz, P. insigne (hybrid), P. venustum (Wall.) Pfitz and P. harrisseanum Hort. In the guard cells of P. aureum hyeanum Hort., however, very slight red fluorescence suggested that chlorophyll and hence chloroplasts were present. Ultrastructural studies of the lower epidermis of P. insigne (hybrid) confirmed the absence of chloroplasts in guard and epidermal cells although plastids of an unusual structure were found in these cells. In fully developed epidermal cells the plastids contained large amounts of a fibrous, possibly proteinaceous substance, spherical, lightly staining vesicles and an electron-dense material located in reticulate and non-reticulate regions. Additionally, latticed crystalline inclusions and plasto-globuli were occasionally observed in the epidermal cell plastids. In plastids of fully developed guard cells the fibrous material, starch and plastoglobuli were present. From the earliest stages of development of the epidermal tissue starch was present in both epidermal cell and guard cell plastids. At maturity, however, starch had accumulated to greater levels in the guard cell plastids and had entirely disappeared in the epidermal cell plastids. In differentiating epidermal tissue, plasmodesmata were found between neighbouring epidermal cells and between guard and epidermal cells. At maturity, plasmodesmata between guard and epidermal cells were not observed. Mitochondria were particularly abundant in guard cells. Large oil drops developed in guard and epidermal cells, being especially abundant in the former at maturity. Our results confirm the observations of Nelson & Mayo (1975) that certain lady slipper orchids possess functional stomata the guard cells of which do not contain chloroplasts.     

A hydromechanical and biochemical model of stomatal conductance

A mathematical model of stomatal conductance is presented. It is based on whole‐plant and epidermal hydromechanics, and on two hypotheses: (1) the osmotic gradient across guard cell membranes is proportional to the concentration of ATP in the guard cells; and (2) the osmotic gradient that can be sustained per unit of ATP is proportional to the turgor pressure of adjacent epidermal cells. In the present study, guard cell [ATP] is calculated using a previously published model that is based on a widely used biochemical model of C₃ mesophyll photosynthesis. The conductance model for Vicia faba L. is parameterized and tested As with most other stomatal models, the present model correctly predicts the stomatal responses to variations in transpiration rate, irradiance and intercellular CO₂. Unlike most other models, however, this model can predict the transient stomatal opening often observed before conductance declines in response to decreases in humidity, soil water potential, or xylem conductance. The model also explicitly accommodates the mechanical advantage of the epidermis and correctly predicts that stomata are relatively insensitive to the ambient partial pressure of oxygen, as a result of the assumed dependence on ATP concentration.     

Ion content and aperture in “isolated” guard cells ofCommelina communis L.

Summary Isolated guard cells ofCommelina communis L., in epidermal strips in which all cells other than guard cells have been killed by treatment at low pH, will open to a degree dependent on the K (Rb)/Cl(Br) concentration in the bathing medium. Estimates of the changes with aperture of the ion concentrations in the guard cells were made by measurement of⁸⁶Rb uptake from RbCl, of⁸²Br uptake from K⁸²Br, and of potassium activity with a potassium-sensitive microelectrode. The osmotic effects of such changes were compared with the previous estimates of the osmotic changes required to change the aperture. The results suggest that a substantial fraction of the osmotic pressure of isolated guard cells is contributed by solutes other than KCl (or other potassium salts), and that, even in stomata opened by incubation on KCl solutions, a substantial fraction of the increase in osmotic pressure associated with opening is contributed by solutes other than KCl.     

Interpretation of an empirical model for stomatal conductance in terms of guard cell function

An empirical model for stomatal conductance (g), proposed by Leuning (1995, this issue) as a modification of Ball, Woodrow & Berry's (1987) model, is interpreted in terms of a simple, steady-state model of guard cell function. In this model, stomatal aperture is a function of the relative turgor between guard cells and epidermal cells. The correlation between g and leaf surface vapour pressure deficit in Leuning's model is interpreted in terms of stomatal sensing of the transpiration rate, via changes in the gradient of total water potential between guard cells and epidermal cells. The correlation between g, CO₂ assimilation rate and leaf surface CO₂ concentration in Leuning's model is interpreted as a relationship between the corresponding osmotic gradient, irradiance, temperature, intercellular CO₂ concentration and stomatal aperture itself. The explicit relationship between osmotic gradient and stomatal aperture (possibly describing the effect of changes in guard cell volume on the membrane permeability for ion transport) results in a decrease in the transpiration rate in sufficiently dry air. Possible extension of the guard cell model to include stomatal responses to soil water status is discussed.     

Potassium content and aperture in “intact” stomatal and epidermal cells ofCommelina communis L

Summary Measurements of potassium activity with a potassium-sensitive microelectrode have been made in the cells of the stomatal complex, and in epidermal cells, ofCommelina communis L., as a function of stomatal aperture. The estimated osmotic effects of the changing accumulation of potassium salts in the guard cell have been compared with the previous estimates of the osmotic changes required to open/close the pore. The results suggest that a significant fraction of the osmotic pressure of the guard cells, particularly when closed, is contributed by solutes other than potassium salts. The degree of potassium accumulation may determine the aperture of wide-open stomata, but the potassium changes in the early stages of opening are much too small to account for the osmotic changes required. The difference in potassium contents of intact and isolated guard cells is close to that required to overcome the previously estimated effect of subsidiary cell turgor on the water relations of the guard cell. In some tissue (but not in all) much more K is lost from epidermal cells than appears in other cells of the complex as the stomata open, and extracellular storage would be required.     

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.     

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)     

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.     

A comparative study of the effect of morphactin and Niagara on the leaf epidermis

Treatment of two-week-oldBrassica campestris andTrigonella foenum-graecum plants with morphactin andVicia faba, Antirrhinum orontium, andPapaver somniferum with Niagara, induced marked variations in the orientation and ontogeny of stomata and the epiderma cells. Morphactin—chlorflurenol at 12.5, 62.5, 125, 250, and 500 ppm, caused marked damage of the shoot apices and changes in the epidermal tissue, such as divisions of the guard cells, reduction in the size of the stomata, and epidermal cells. Niagara—ethyl-hydrogen-1-propylphosphonate at 100, 500, 1 000 5 000, and 10 000 ppm caused thickenings of the epidermal cell walls and differentiation of new meristemoids from the epidermal cells, contiguous stomata, and incomplete development of the guard cells.     

Leaf surface development and the plant fossil record: stomatal patterning in Bennettitales

Stomata play a critical ecological role as an interface between the plant and its environment. Although the guard‐cell pair is highly conserved in land plants, the development and patterning of surrounding epidermal cells follow predictable pathways in different taxa that are increasingly well understood following recent advances in the developmental genetics of the plant epidermis in model taxa. Similarly, other aspects of leaf development and evolution are benefiting from a molecular–genetic approach. Applying this understanding to extinct taxa known only from fossils requires use of extensive comparative morphological data to infer ‘fossil fingerprints’ of developmental evolution (a ‘palaeo‐evo‐devo’ perspective). The seed‐plant order Bennettitales, which flourished through the Mesozoic but became extinct in the Late Cretaceous, displayed a consistent and highly unusual combination of epidermal traits, despite their diverse leaf morphology. Based on morphological evidence (including possession of flower‐like structures), bennettites are widely inferred to be closely related to angiosperms and hence inform our understanding of early angiosperm evolution. Fossil bennettites – even purely vegetative material – can be readily identified by a combination of epidermal features, including distinctive cuticular guard‐cell thickenings, lobed abaxial epidermal cells (‘puzzle cells’), transverse orientation of stomata perpendicular to the leaf axis, and a pair of lateral subsidiary cells adjacent to each guard‐cell pair (termed paracytic stomata). Here, we review these traits and compare them with analogous features in living taxa, aiming to identify homologous – and hence phylogenetically informative – character states and to increase understanding of developmental mechanisms in land plants. We propose a range of models addressing different aspects of the bennettite epidermis. The lobed abaxial epidermal cells indicate adaxial–abaxial leaf polarity and associated differentiated mesophyll that could have optimised photosynthesis. The typical transverse orientation of the stomata probably resulted from leaf expansion similar to that of a broad‐leaved monocot such as Lapageria, but radically different from that of broad‐leafed eudicots such as Arabidopsis. Finally, the developmental origin of the paired lateral subsidiary cells – whether they are mesogene cells derived from the same cell lineage as the guard‐mother cell, as in some eudicots, or perigene cells derived from an adjacent cell lineage, as in grasses – represents an unusually lineage‐specific and well‐characterised developmental trait. We identify a close similarity between the paracytic stomata of Bennettitales and the ‘living fossil’ Gnetum, strongly indicating that (as in Gnetum) the pair of lateral subsidiary cells of bennettites are both mesogene cells. Together, these features allow us to infer development in this diverse and relatively derived lineage that co‐existed with the earliest recognisable angiosperms, and suggest that the use of these characters in phylogeny reconstruction requires revision.     

Measuring Osmotic Pressure of Sap within Live Cells by Means of a Visual Melting Point Apparatus

A freezing slide apparatus is described for visual observation of freezing water and melting ice within plant cells. The slide consists of an ordinary microscope slide glued into a Plexiglass jacket, through which cold 90% ethyl alcohol is pumped at varying rates for temperature control. Temperature is recorded by means of an iron-constantan thermocouple wire (25-micron diameter) connected to a recording potentiometer. Tissue strips were quick frozen (at a cooling rate of 33 C per ½ minute) and then warmed very slowly (at a rate of 2 C per minute) for observation of melting points. This apparatus has been used to determine osmotic pressures of cell sap of guard and adjoining epidermal cells of Chrysanthemum morifolium and Pelargonium hortorum. An accuracy of ± 1.2 atmospheres is possible. Wide variations among osmotic pressures of both guard and epidermal cells were found at any one stomatal aperture in both species.     

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.     

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)