Direct Measurements of Turgor Pressure Potentials of Guard Cells: II. THE MECHANICAL ADVANTAGE OF SUBSIDIARY CELLS, THE SPANNUNQSPHASE, AND THE OPTIMUM LEAF WATER DEFICIT

Evidence of the mechanical advantage of subsidiary cells wasobtained by simultaneous measurements of turgor pressure potentialsin adjacent subsidiary and guard cells using injection circuitswith two separate needles. In Tradescantia virginiana the mechanicaladvantage approaches two. Using the same technique evidencewas obtained that the Spannungsphase is, in the first place,a turgor relations phenomenon due to the mechanical advantageof epidermal or subsidiary cells. In addition, the evidenceindicated that the elastic properties of guard cell walls mayundergo changes during the Spannungsphase when potassium iontransport commences. During these measurements it was confirmedthat the optimum leaf water deficit for maximum stomatal openingoccurs when the epidermal turgor is near zero. Under these conditionsthe width of the stomatal pore is a function of the turgor pressureof the guard cells, since at zero turgor of the subsidiary cellstheir mechanical advantage has disappeared.     

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.     

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 mechanical diversity of stomata and its significance in gas-exchange control

Given that stomatal movement is ultimately a mechanical process and that stomata are morphologically and mechanically diverse, we explored the influence of stomatal mechanical diversity on leaf gas exchange and considered some of the constraints. Mechanical measurements were conducted on the guard cells of four different species exhibiting different stomatal morphologies, including three variants on the classical "kidney" form and one "dumb-bell" type; this information, together with gas-exchange measurements, was used to model and compare their respective operational characteristics. Based on evidence from scanning electron microscope images of cryo-sectioned leaves that were sampled under full sun and high humidity and from pressure probe measurements of the stomatal aperture versus guard cell turgor relationship at maximum and zero epidermal turgor, it was concluded that maximum stomatal apertures (and maximum leaf diffusive conductance) could not be obtained in at least one of the species (the grass Triticum aestivum) without a substantial reduction in subsidiary cell osmotic (and hence turgor) pressure during stomatal opening to overcome the large mechanical advantage of subsidiary cells. A mechanism for this is proposed, with a corollary being greatly accelerated stomatal opening and closure. Gas-exchange measurements on T. aestivum revealed the capability of very rapid stomatal movements, which may be explained by the unique morphology and mechanics of its dumb-bell-shaped stomata coupled with "see-sawing" of osmotic and turgor pressure between guard and subsidiary cells during stomatal opening or closure. Such properties might underlie the success of grasses.     

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.     

Stomatal mechanics. III. Geometric interpretation of the mechanical advantage*

Abstract. Previous mathematical analyses of stomatal mechanics have demonstrated, and experimental measurements have confirmed, that the turgor-generated force of the epidermal cells dominates that of the guard cells in determining aperture. DcMichele & Sharpe (1973) termed the phenomenon the mechanical advantage of the epidermal cells, while Cooke et al. (1976) expressed it as an antagonism ratio. Both of these formulations, however, have theoretical or practical limitations. This study presents a biophysical analysis demonstrating that the effective forces in the stomatal system may be studied in terms of simple stomatal geometry. From this analysis, the mechanical advantage can be redefined and interpreted based upon simple geometric relationships calculated from measurable anatomical dimensions.     

Cell water potential,osmotic potential,and turgor in the epidermis and mesophyll of transpiring leaves

Water potential, osmotic potential and turgor measurements obtained by using a cell pressure probe together with a nanoliter osmometer were compared with measurements obtained with an isopiestic psychrometer. Both types of measurements were conducted in the mature region of Tradescantia virginiana L. leaves under non-transpiring conditions in the dark, and gave similar values of all potentials. This finding indicates that the pressure probe and the osmometer provide accurate measurements of turgor, osmotic potentials and water potentials. Because the pressure probe does not require long equilibration times and can measure turgor of single cells in intact plants, the pressure probe together with the osmometer was used to determine in-situ cell water potentials, osmotic potentials and turgor of epidermal and mesophyll cells of transpiring leaves as functions of stomatal aperture and xylem water potential. When the xylem water potential was-0.1 MPa, the stomatal aperture was at its maximum, but turgor of both epidermal and mesophyll cells was relatively low. As the xylem water potential decreased, the stomatal aperture became gradually smaller, whereas turgor of both epidermal and mesophyll cells first increased and afterward decreased. Water potentials of the mesophyll cells were always lower than those of the epidermal cells. These findings indicate that evaporation of water is mainly occurring from mesophyll cells and that peristomatal transpiration could be less important than it has been proposed previously, although peristomatal transpiration may be directly related to regulation of turgor in the guard cells.     

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.     

A conserved functional role of pectic polymers in stomatal guard cells from a range of plant species

Guard cell walls combine exceptional strength and flexibility in order to accommodate the turgor pressure-driven changes in size and shape that underlie the opening and closing of stomatal pores. To investigate the molecular basis of these exceptional qualities, we have used a combination of compositional and functional analyses in three different plant species. We show that comparisons of FTIR spectra from stomatal guard cells and those of other epidermal cells indicate a number of clear differences in cell-wall composition. The most obvious characteristics are that stomatal guard cells are enriched in phenolic esters of pectins. This enrichment is apparent in guard cells from Vicia faba (possessing a type I cell wall) and Commelina communis and Zea mays (having a type II wall). We further show that these common defining elements of guard cell walls have conserved functional roles. As previously reported in C. communis, we show that enzymatic modification of the pectin network in guard cell walls in both V. faba and Z. mays has profound effects on stomatal function. In all three species, incubation of epidermal strips with a combination of pectin methyl esterase and endopolygalacturonase (EPG) caused an increase in stomatal aperture on opening. This effect was not seen when strips were incubated with EPG alone indicating that the methyl-esterified fraction of homogalacturonan is key to this effect. In contrast, arabinanase treatment, and incubation with feruloyl esterase both impeded stomatal opening. It therefore appears that pectins and phenolic esters have a conserved functional role in guard cell walls even in grass species with type II walls, which characteristically are composed of low levels of pectins.     

Guard cell volume and pressure measured concurrently by confocal microscopy and the cell pressure probe

Guard cell turgor pressures in epidermal peels of broad bean (Vicia faba) were measured and controlled with a pressure probe. At the same time, images of the guard cell were acquired using confocal microscopy. To obtain a clear image of guard cell volume, a fluorescent dye that labels the plasma membrane was added to the solution bathing the epidermal peel. At each pressure, 17 to 20 optical sections (each 2 microm thick) were acquired. Out-of-focus light in these images was removed using blind deconvolution, and volume was estimated using direct linear integration. As pressure was increased from as low as 0.3 MPa to as high as 5.0 MPa, guard cell volume increased in a saturating fashion. The elastic modulus was calculated from these data and was found to range from approximately 2 to 40 MPa. The data allow inference of guard cell osmotic content from stomatal aperture and facilitate accurate mechanistic modeling of epidermal water relations and stomatal functioning.     

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.     

A study of stomatal mechanics using the cell pressure probe

The relationship between stomatal aperture ( a ) and guard cell pressure ( P _g) was measured directly in four different species ( Vicia faba, Tradescantia virginiana, Ginkgo biloba and Nephrolepis exaltata ) using a special cell pressure probe technique. The effect of epidermal turgor ( P _ep) on this relationship was also measured in T. virginiana . The relationship was sigmoidal for V. faba and T. virginiana , but entirely convex for G. biloba and N. exaltata. Epidermal turgor was found to have a pronounced closing effect on stomata of T. virginiana . Maximum aperture with full epidermal turgor (0·92 MPa) was about half that with zero epidermal turgor. Also, with full epidermal turgor stomata of T. virginiana did not begin to open until P _g was more than 1·25 MPa. These characteristics were used to develop an expression for a as a function of P _g and P _ep. Results for the different species are compared and discussed in terms of possible advantages and limitations of water economy.     

Epidermal and Guard Cell Interactions on Stomatal Aperture in Epidermal Strips and Intact Leaves

Despite the observation first made by von Mohl in 1856, thatepidermal cells greatly influence stomatal aperture, subsequentstudies have failed to pay adequate attention to epidermal cellviability or to quantify the degree of its influence on aperturein epidermal strips and leaf sections. Using Vicia faba stripsand leaf sections we found the following: (i) a non-linear relationshipbetween aperture and guard cell contact with live epidermalcells; (ii) epidermal cell viability on isolated strips hada threshold at about 25 °C; (iii) epidermal strips withdead epidermal cells had wider apertures and lower variabilitythan strips with live cells or intact leaf sections; (iv) afterepidermal cell viability was accounted for, stomatal aperturesshowed no significant differences between isolated strips orstrips removed from leaf sections treated in the same manner;(v) highly variable apertures appeared to be the normal conditionof the intact leaf. Caution should therefore be used in interpretingstomatal behaviour from epidermal strips without first takinginto account mechanical interactions between the guard and surroundingepidermal cells. Vicia faba L, broad bean, epidermal strips, leaf impressions, stomata, guard cells, temperature effects     

A constraint–relaxation–recovery mechanism for stomatal dynamics

Models of guard cell dynamics, built on the OnGuard platform, have provided quantitative insights into stomatal function, demonstrating substantial predictive power. However, the kinetics of stomatal opening predicted by OnGuard models were threefold to fivefold slower than observed in vivo. No manipulations of parameters within physiological ranges yielded model kinetics substantially closer to these data, thus highlighting a missing component in model construction. One well‐documented process influencing stomata is the constraining effect of the surrounding epidermal cells on guard cell volume and stomatal aperture. Here, we introduce a mechanism to describe this effect in OnGuard2 constructed around solute release and a decline in turgor of the surrounding cells and its subsequent recovery during stomatal opening. The results show that this constraint–relaxation–recovery mechanism in OnGuard2 yields dynamics that are consistent with experimental observations in wild‐type Arabidopsis, and it predicts the altered opening kinetics of ost2 H⁺‐ATPase and slac1 Cl^? channel mutants. Thus, incorporating solute flux of the surrounding cells implicitly through their constraint on guard cell expansion provides a satisfactory representation of stomatal kinetics, and it predicts a substantial and dynamic role for solute flux across the apoplastic space between the guard cells and surrounding cells in accelerating stomatal kinetics.     

Water stress effects on guard cell anatomy and the mechanical advantage of the epidermal cells

Abstract Vicia faba plants grown under water deficit were found to have guard cells considerably smaller than those of plants grown under well-watered conditions. Stomala of plants adapted to drought conditions have been observed in past studies to maintain opening at plant water potentials lower than those of plants not so adapted. By employing the geometric interpretation of the mechanical advantage (Wu, Sharpe & Spence, 1985), an anatomical/mechanical basis was found that helps explain how such opening in drought conditions can occur. The geometry and resulting mechanical properties of small stomata, in contrast to larger stomata, give them the capability of opening or maintaining open pores with lower guard cell turgor pressures, relative to the turgor of the surrounding epidermal cells.     

Stomatal responses to humidity in isolated epidermes

The ability of guard cells to hydrate and dehydrate from the surrounding air was investigated using isolated epidermes of Tradescantia pallida and Vicia faba . Stomata were found to respond to the water vapour pressure on the outside and inside of the epidermis, but the response was more sensitive to the inside vapour pressure, and occurred in the presence or absence of living, turgid epidermal cells. Experiments using helium–oxygen air showed that guard cells hydrated and dehydrated entirely from water vapour, suggesting that there was no significant transfer of water from the epidermal tissue to the guard cells. The stomatal aperture achieved at any given vapour pressure was shown to be consistent with water potential equilibrium between the guard cells and the air near the bottom of the stomatal pore, and water vapour exchange through the external cuticle appeared to be unimportant for the responses. Although stomatal responses to humidity in isolated epidermes are the result of water potential equilibrium between the guard cells and the air near the bottom of the stomatal pore, stomatal responses to humidity in leaves are unlikely to be the result of a similar equilibrium.     

Structure,Ontogeny and Taxonomic Significance of Stomata in Some Cucurbitaceae

Stomatal structure, ontogeny in vegetative and floral organs of 9 genera and 12 species of Cucurbitaceae are described. The stomatal types conform to aperigenous, monoperigenous, diperigenous, hemipara-mesoperigenous and para-mesoperigenous types of Fryns-Claessens & Van Cotthem (1973). Stomatal abnormalities such as contiguous stomata, single guard cells with or without pore, one and a half stomata, degeneration of one or both the guard cells, cytoplasmic connections between guard cells of neighbouring stomata and a guard cell of a stoma and an adjacent epidermal cell, and division of guard cells are described. Stomata index, frequency of stomata, epidermal cells, size of guard and epidermal cells and organwise distribution of stomata are given. Stomatal studies does not support the view that the Cucurbitaceae are related to the Passifloraceae. The inclusion of 9 genera and 12 species studied in the tribe Cucumerineae is justified.     

Stomatal action directly feeds back on leaf turgor: new insights into the regulation of the plant water status from non‐invasive pressure probe measurements

Uptake of CO₂ by the leaf is associated with loss of water. Control of stomatal aperture by volume changes of guard cell pairs optimizes the efficiency of water use. Under water stress, the protein kinase OPEN STOMATA 1 (OST1) activates the guard‐cell anion release channel SLOW ANION CHANNEL‐ASSOCIATED 1 (SLAC1), and thereby triggers stomatal closure. Plants with mutated OST1 and SLAC1 are defective in guard‐cell turgor regulation. To study the effect of stomatal movement on leaf turgor using intact leaves of Arabidopsis, we used a new pressure probe to monitor transpiration and turgor pressure simultaneously and non‐invasively. This probe permits routine easy access to parameters related to water status and stomatal conductance under physiological conditions using the model plant Arabidopsis thaliana. Long‐term leaf turgor pressure recordings over several weeks showed a drop in turgor during the day and recovery at night. Thus pressure changes directly correlated with the degree of plant transpiration. Leaf turgor of wild‐type plants responded to CO₂, light, humidity, ozone and abscisic acid (ABA) in a guard cell‐specific manner. Pressure probe measurements of mutants lacking OST1 and SLAC1 function indicated impairment in stomatal responses to light and humidity. In contrast to wild‐type plants, leaves from well‐watered ost1 plants exposed to a dry atmosphere wilted after light‐induced stomatal opening. Experiments with open stomata mutants indicated that the hydraulic conductance of leaf stomata is higher than that of the root–shoot continuum. Thus leaf turgor appears to rely to a large extent on the anion channel activity of autonomously regulated stomatal guard cells.     

The function of guard cells does not require an intact array of cortical microtubules

The development of stomatal guard cells is known to require cortical microtubules; however, it is not known if microtubules are also required by mature guard cells for stomatal function. To study the role of microtubules in guard cell function, epidermal peels of Vicia faba were subjected to conditions known to open or close stomata in the presence or absence of microtubule inhibitors. To verify the action of the inhibitors, microtubules in appropriately treated epidermal peels were localized by cryofixation followed by freeze substitution and embedding in butyl-methyl methacrylate. Mature guard cells had a radial array of microtubules, focused toward the thick cell wall of the pore, and the appearance of this array was the same for stomata remaining closed in darkness or induced to open by light. Treatment of epidermal peels with 1 mM colchicine for 1 h depolymerized nearly all cortical microtubules. Measurements of stomatal aperture showed that neither 1 mM colchicine nor 20 M taxol affected any of the responses tested: remaining closed in the dark, opening in response to light or fusicoccin, and closing in response to calcium and darkness. We conclude that intact microtubule arrays are not invariably required for guard cell function.