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.     

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.     

An analysis of the mechanics of guard cell motion

This paper presents a mechanical analysis of the cellular deformations which occur during the opening and closing of stomata. The aperture of the stomatal pore is shown to be a result of opposing pressures of the guard and adjacent epidermal cells. The analysis indicates that the epidermal cells have a mechanical advantage over the guard cells. With no mechanical advantage, an equal reduction in the turgor pressure of both guard and epidermal cells would have a neglible effect upon stomatal aperture. However, due to the mechanical advantage of the surrounding cells, the stomatal aperture increases with equal reductions in turgor, until the adjacent epidermal cells become flaccid. The minimum diffusion resistance of the pore occurs at this point. Further reductions in guard cell turgor lead to closure of the pore. The analysis further demonstrates how the shape, size, wall thickness and material properties of the guard cell walls influence their behavior.     

Effects of humidity on light-induced stomatal opening: evidence for hydraulic coupling among stomata

A mechanism for co-ordinating behaviour of stomata within an areole during patchy stomatal conductance has recently been proposed. This mechanism depends on hydraulic interactions among stomata that are mediated by transpiration-induced changes in epidermal turgor. One testable prediction that arises from this proposed mechanism is that the strength of hydraulic coupling among stomata should be proportional to evaporative demand and, therefore, inversely proportional to humidity. When a leaf is illuminated following a period of darkness, there is typically a period of time, termed the Spannungsphase, during which guard cell osmotic and turgor pressure are increasing, but the pore remains closed. If hydraulic coupling is proportional to evaporative demand, then variation among stomata in the duration of the Spannungsphase should be lower for leaves at low humidity than for leaves at high humidity. A similar prediction emerged from a computer model based on the proposed hydraulic mechanisms. These predictions were tested by measuring individual stomatal apertures on intact transpiring leaves at low and high humidity and on vacuum-infiltrated leaf pieces (to eliminate transpiration) as PFD was increased to high values from either darkness or a low value. Results showed that the range of Spannungsphasenamong stomata was reduced at low humidity compared to high humidities. Experiments that began at low PFD, rather than at darkness, showed no delay in stomatal opening. These results are discussed in the context of the proposed hydraulic coupling mechanisms.     

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.     

Leaf hydraulic conductivity and stomatal responses to humidity in amphistomatous leaves

The response of stomata to changes in humidity for a single surface of an amphistomatous leaf was investigated in Xanthium strumarium and Vicia faba using gas exchange and direct observation of stomatal apertures. The stomatal response to humidity for a given surface was found to be the same whether or not the humidity for the opposite surface was changed concurrently. Stomata on the surface for which humidity was constant showed no response to changes in humidity for the opposite surface. Despite large changes in epidermal turgor on the surface for which humidity was changed, there was no change in epidermal turgor for the surface with constant humidity. Measurements of transpiration and epidermal turgor as functions of the mole fraction gradient of water between leaf and air were used to calculate a value for leaf hydraulic resistance. The results suggest that in these species, the mechanism for the stomatal response to humidity resides in the epidermis or the mesophyll very close to the epidermis, and that most of the hydraulic resistance of the leaf occurs between the xylem and the evaporating sites.     

In Situ Measurement of Epidermal Cell Turgor, Leaf Water Potential, and Gas Exchange in Tradescantia virginiana L

A combined system has been developed in which epidermal cell turgor, leaf water potential, and gas exchange were determined for transpiring leaves of Tradescantia virginiana L. Uniform and stable values of turgor were observed in epidermal cells (stomatal complex cells were not studied) under stable environmental conditions for both upper and lower epidermises. The changes in epidermal cell turgor that were associated with changes in leaf transpiration were larger than the changes in leaf water potential, indicating the presence of transpirationally induced within-leaf water potential gradients. Estimates of 3 to 5 millimoles per square meter per second per megapascal were obtained for the value of within-leaf hydraulic conductivity. Step changes in atmospheric humidity caused rapid changes in epidermal cell turgor with little or no initial change in stomatal conductance, indicating little direct relation between stomatal humidity response and epidermal water status. The significance of within-leaf water potential gradients to measurements of plant water potential and to current hypotheses regarding stomatal response to humidity is discussed.     

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.     

Plant response to atmospheric humidity

Abstract. Plants growing in environments differing in prevailing humidity exhibit variations in traits associated with regulation of water loss, particularly cuticular and stomatal properties. Expansive growth is also typically reduced by low humidity. Nevertheless, there is little evidence in plants for a specific sensor for humidity, analogous to the blue light or phytochrome photoreceptors. The detailed mechanism of the stomatal response to humidity remains unknown. Available data suggest mediation by fluxes of water vapour, with evaporation rate assuming the role of sensor. This implies that stomata respond to the driving force for diffusional water loss, leaf-air vapour pressure difference. Induction of metabolic stomatal response to humidity may involve signal metabolites, such as abscisic acid, that are present in the transpiration stream. These materials may accumulate in the vicinity of guard cells according to the magnitude and location of cuticular transpiration, both of which could change with humidity. Such a mechanism remains hypothetical, but is suggested to account for feedforward responses in which transpiration decreases with increasing evaporative demand, and for the apparent insensitivity of stomatal aperture in isolated epidermis to epidermal water status. Other responses of plants to humidity may involve similar indirect response mechanisms, in the absence of specific humidity sensors.     

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.     

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 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.     

The kinetics of stomatal responses to VPD in Vicia faba: electrophysiological and water relations models

Abstract. An Ohm's law analogy is frequently employed to calculate parameters of leaf gas exchange. For example, resistance to water vapour loss is calculated as the quotient of vapour pressure difference (VPD) and vapour loss by transpiration. In the present research, this electrical analogy was extended. Steady-state transpiration as a function of VPD, assayed in leaflets of Vicia faba using gas exchange techniques, was compared with steady-state K⁺ current magnitude as a function of voltage in isolated guard cell protoplasts of Vicia faba, assayed using the patch clamping technique in the whole cell configuration. An electrophysiological model originally developed to explain the kinetics of current changes following step changes in voltage across a cell membrane was used to fit the kinetics of transpiration changes following step changes in VPD applied to leaflets of Vicia faba. Following step increases in VPD, transpiration exhibited an initial increase, reflecting the increased driving force for water loss and, for large step increases in VPD, a transient decrease in stomatal resistance. Transpiration subsequently declined, reflecting stomatal closure. By analogy to electrophysiological responses, it is hypothesized that the humidity parameter that is sensed by guard cells is VPD. Two models based on epidermal water relations were also applied to transpiration kinetics. In the first model, the transient increase in transpiration following a step increase in VPD was attributed partially to an increase in the Physical driving force (VPD) and partially to a transient decrease in stomatal resistance resulting from reduced epidermal backpressure. In the second model, the transient decrease in stomatal resistance was attributed to a direct response of the guard cells to VPD. Both models based on water relations gave good fits of the data, emphasizing the need for further study regarding the metabolic nature of the guard cell response to humidity.     

Stomatal responses to light and leaf-air water vapor pressure difference show similar kinetics in sugarcane and soybean

Stomatal responses to light and humidity (vapor pressure difference, VPD) are important determinants of stomatal conductance. Stomatal movements induced by light are the result of a transduction of the light stimulus into modulated ion fluxes in guard cells and concomitant osmotic adjustments and turgor changes. It is generally assumed that this transduction process is a general stomatal property, with different environmental stimuli integrated into guard cell metabolism through their modulation of ion fluxes. In contrast with this notion, the VPD response, which is unique because both its triggering signal and the turgor changes required for aperture modulations involve water molecules, has been considered to be hydropassive and thus independent of guard cell metabolism. We used a kinetic approach to compare the light and VPD responses in order to test the hypothesis that hydropassive changes in guard cell turgor could be faster than the metabolism-dependent light responses. Changes in stomatal conductance in intact leaves of sugarcane and soybean were measured after application of step changes in VPD and in light. In spite of a 5-fold difference in overall rates between the two species, the response rates following light or VPD steps were similar. Although a coincidental kinetic similarity between two mechanistically different responses cannot be ruled out, the data suggest a common mechanism controlling stomatal movements, with the VPD stimulus inducing metabolic modulations of ion fluxes analogous to other stomatal responses.     

Effects of manipulation of water and nitrogen regime on the water relations of the desert shrub Larrea tridentata

Summary Water and nitrogen regimes of Larrea tridentata shrubs growing in the field were manipulated during an annual cycle. Patterns of leaf water status, leaf water relations characteristics, and stomatal behavior were followed concurrently. Large variations in leaf water status in both irrigated and nonirrigated individuals were observed. Predawn and midday leaf water potentials of nonirrigated shrubs were lowest except when measurements had been preceded by significant rainfall. Despite the large seasonal variation in leaf water status, reasonably constant, high levels of turgor were maintained. Pressure-volume curve analysis suggested that changes in the bulk leaf osmotic potential at full turgor were small and that nearly all of the turgor adjustment was due to tissue elastic adjustment. The increase in tissue elasticity with increasing water deficit manifested itself as a decrease in the relative water content at zero turgor and as a decrease in the tissue bulk elastic modulus. Because of large hydration-induced displacement in the osmotic potential and relative water content at zero turgor, it was necessary to use shoots in their natural state of hydration for pressure-volume curve determinations. Large diurnal and seasonal differences in maximum stomatal conductance were observed, but could not easily be attributed to variations in leaf water potential or leaf water relations characteristics such as the turgor loss point. The single factor which seemed to account for most of the diurnal and seasonal differences in maximum stomatal conductance between individual shrubs was an index of soil/root/ shoot hydraulic resistance. Daily maximum stomatal conductance was found to decrease with increasing soil/root/ shoot hydraulic resistance. This pattern was most consistent if the hydraulic resistance calculation was based on an estimate of total canopy transpiration rather than the more commonly used transpiration per unit leaf area. The reasons for this are discussed. It is suggested that while stomatal aperture necessarily represents a major physical resistance controlling transpiration, plant hydraulic resistance may represent the functional resistance through its effects on stomatal aperture.     

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.     

Stomatal response to humidity in sugarcane and soybean: effect of vapour pressure difference on the kinetics of the blue light response

Abstract. The effect of atmospheric humidity on the kinetics of stomatal responses was quantified in gas exchange experiments using sugarcane ( Saccharum spp. hybrid) and soybean ( Glycine max ). Pulses of blue light were used to elicit pulses of stomatal conductance that were mediated by the specific blue light response of guard cells. Kinetic parameters of the conductance response were more closely related to leaf-air vapour pressure difference (VPD) than to relative humidity or transpiration. Increasing VPD significantly accelerated stomatal opening in both sugarcane and soybean, despite an approximately five-fold faster response in sugarcane. In contrast, the kinetics of stomatal recovery (closure) following the pulse were similar in the two species. Acceleration of opening by high VPD was observed even under conditions where soybean exhibited a feedforward response of decreasing transpiration (E) with increasing evaporative demand (VPD). This result suggests that epidermal, rather than bulk leaf, water status mediates the VPD effect on stomatal kinetics. The data are consistent with the hypothesis that increased cpidermal water loss at high VPD decreases the backpressure exerted by neighbouring cells on guard cells. allowing more rapid stomatal opening per unit of guard cell metabolic response to blue light.     

ABA-deficient (aba1) and ABA-insensitive (abi1-1, abi2-1) mutants of Arabidopsis have a wild-type stomatal response to humidity

In most plant species, a decrease in atmospheric humidity at the leaf surface triggers a decrease in stomatal conductance. While guard cells appear to respond to humidity‐induced changes in transpiration rate, as opposed to relative humidity or vapour pressure difference, the underlying cellular mechanisms for this response remain unknown. In the present set of experiments, abscisic acid (ABA)‐deficient (aba1) and ABA‐insensitive (abi1‐1 and abi2‐1) mutants of Arabidopsis thaliana were used to test the hypothesis that the humidity signal is transduced by changes in the flux or concentration of ABA delivered to the stomatal complex in the transpiration stream. In gas exchange experiments, stomatal conductance was as sensitive to changes in vapour pressure difference in aba1, abi1‐1 and abi2‐1 mutant plants as in wild‐type plants. These experiments appear to rule out an obligate role for either the concentration or flux of ABA or ABA conjugates as mediators of the guard cell response to atmospheric water potential. The results stand in contrast to the well‐established role of ABA in mediating guard cell responses to decreases in soil water potential.     

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.     

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.