Stomatal patchiness and cellular computing.

Control of carbon dioxide and water vapor exchange between a leaf's interior and the surrounding air is accomplished by variations in the turgor pressures in the small epidermal and guard cells that cover the leaf's surface. These pressures respond to changes in light intensity and wavelength, temperature, CO2 concentration, and air humidity. The dynamical equations that describe such processes are formally identical to those that define computation in a two-layer, adaptive, cellular nonlinear network. This exact identification suggests that leaf gas-exchange processes can be understood as analog computation and that exploiting the output of two-layer, adaptive, cellular nonlinear networks might provide new tools in applied plant research.

Effects of the mesophyll on stomatal responses in amphistomatous leaves.

The role of the mesophyll in stomatal functioning in thin amphistomatous leaves was investigated by altering gas exchange for one surface and observing the effects on stomatal conductance for the other surface. Three methods of perturbing gas exchange on the adaxial surface were used. First, gas exchange for the adaxial surface was completely blocked with plastic wrap or vacuum grease. Second, leaves were inverted to induce closure of the adaxial stomata. And third, ambient humidity for the adaxial surface was reduced to induce stomatal closure on that surface. Experiments were performed at low light intensity and three different CO2 concentrations to test the idea that stomatal responses in thin amphistomatous leaves are partially controlled by a signal from the mesophyll that varies with light and CO2 . In general, stomata on the abaxial surface opened when gas exchange on the adaxial surface was reduced, with the largest increases in conductance occurring at high CO2 concentration. The data are discussed with respect to role of a purported signal from the mesophyll and the partitioning of that signal between the two surfaces of the leaf.

Stomatal heterogeneity in responses to humidity and temperature: Testing a mechanistic model.

The role of stomatal heterogeneity in the response of stomatal conductance (gs ) to the mole fraction difference in water vapour between the inside of the leaf and the ambient air (Δw) was determined using thermography and gas exchange for 3 species. The value of Δw for the leaf was varied in 2 different ways: first by varying air humidity while holding leaf temperature constant and second by varying leaf temperature while holding air humidity constant. Stomatal heterogeneity was explored by examining the response of gs in small areas of the leaf (as determined by thermography) and comparing them to each other and to the average value of gs (as determined by gas exchange). These analyses show that despite substantial heterogeneity in gs values, the response of gs to Δw was qualitatively similar in all areas of the leaf, and all responses of gs to Δw were well predicted by a recently proposed, vapour-phase mechanism for stomatal responses to temperature and humidity. Remarkably, the 2 model parameters, Θ and Z, that depend on leaf anatomy were constant for a given species, and only the maximum conductance varied in different regions of the leaf.

Is the signal from the mesophyll to the guard cells a vapour-phase ion?

Previous studies have suggested that the red light and CO2 responses of stomata are caused by a signal from the mesophyll to the guard cells. Experiments were conducted to test the idea that this signal is a vapour-phase ion. Stomata in isolated epidermes of Tradescantia pallida were found to respond to air ions created by an electrode that was positioned under the epidermes. Anthocyanins in the epidermes of this species were observed to change colour in response to these air ions, and this change in colour was attributed to changes in pH. A similar change in lower epidermal colour was observed in intact leaves upon illumination and with changes in CO2 concentration. Based on the change in epidermal colour, the pH of the epidermis was estimated to be approximately 7.0 in darkness and 6.5 in the light. Stomata in isolated epidermes responded to pH when suspended over (but not in contact with) solutions of different pH. We speculate that stomatal responses to CO2 and light are caused by vapour-phase ions, possibly hydronium ions that change the pH of the epidermis.

Modelling stomatal conductance in response to environmental factors.

Stomata are an attractive system for modellers for many reasons, and the literature contains a large number of papers describing models that predict stomatal conductance as a function of environmental factors. The approaches and goals of these models vary considerably. This review summarizes these different approaches and discusses their strengths and weaknesses with a focus on mechanistically based models. The critical unresolved questions are highlighted and placed in the context of current research on stomatal physiology. Finally, directions for future research are considered.

Testing a vapour-phase model of stomatal responses to humidity.

This study tests two predictions from a recently proposed model for stomatal responses to humidity and temperature. The model is based on water potential equilibrium between the guard cells and the air at the bottom of the stomatal pore and contains three independent variables: gs(0), Z and Θ. gs(0) is the value of stomatal conductance that would occur at saturating humidity and will vary among leaves and with CO2 and light. The value of Z is determined primarily by the resistance to heat transfer from the epidermis to the evaporating site and the value of Θ is determined primarily by the resistance to water vapour diffusion from the evaporating site to the guard cells. This leads to the two predictions that were tested. Firstly, the values of Z and Θ should be constant for leaves of a given species grown under given conditions, although gs(0) should vary among leaves and with light and CO2. And secondly, the ratio of Z to Θ should be higher in leaves having their stomata in crypts because the distance for heat transfer is greater than that for water vapour diffusion. Data from three species, Nerium oleander, Pastinaca sativum and Xanthium strumarium support these two predictions.

Alternative perspective on the control of transpiration by radiation.

Stomatal responses to light are important determinants for plant water use efficiency and for general circulation models, but a mechanistic understanding of these responses remains elusive. A recent study [Pieruschka R, Huber G, Berry JA (2010) Proc Natl Acad Sci USA 107:13372-13377] concluded that stomata respond to total absorbed radiation rather than red and blue light as previously thought. We tested this idea by reexamining stomatal responses to red and blue light and to IR radiation. We show that responses to red and blue light are not consistent with a response to total absorbed radiation and that apparent stomatal responses to IR radiation are explainable as experimental artifacts. In addition, our data and analysis provide a method for accurately determining the internal temperature of a leaf.

A new, vapour-phase mechanism for stomatal responses to humidity and temperature.

A new mechanism for stomatal responses to humidity and temperature is proposed. Unlike previously-proposed mechanisms, which rely on liquid water transport to create water potential gradients within the leaf, the new mechanism assumes that water transport to the guard cells is primarily through the vapour phase. Under steady-state conditions, guard cells are assumed to be in near-equilibrium with the water vapour in the air near the bottom of the stomatal pore. As the water potential of this air varies with changing air humidity and leaf temperature, the resultant changes in guard cell water potential produce stomatal movements. A simple, closed-form, mathematical model based on this idea is derived. The new model is parameterized for a previously published set of data and is shown to fit the data as well as or better than existing models. The model contains mathematical elements that are consistent with previously-proposed mechanistic models based on liquid flow as well as empirical models based on relative humidity. As such, it provides a mechanistic explanation for the realm of validity for each of these approaches.

Stomatal responses to flooding of the intercellular air spaces suggest a vapor-phase signal between the mesophyll and the guard cells.

Flooding the intercellular air spaces of leaves with water was shown to cause rapid closure of stomata in Tradescantia pallida, Lactuca serriola, Helianthus annuus, and Oenothera caespitosa. The response occurred when water was injected into the intercellular spaces, vacuum infiltrated into the intercellular spaces, or forced into the intercellular spaces by pressurizing the xylem. Injecting 50 mm KCl or silicone oil into the intercellular spaces also caused stomata to close, but the response was slower than with distilled water. Epidermis-mesophyll grafts for T. pallida were created by placing the epidermis of one leaf onto the exposed mesophyll of another leaf. Stomata in these grafts opened under light but closed rapidly when water was allowed to wick between epidermis and the mesophyll. When epidermis-mesophyll grafts were constructed with a thin hydrophobic filter between the mesophyll and epidermis stomata responded normally to light and CO(2). These data, when taken together, suggest that the effect of water on stomata is caused partly by dilution of K(+) in the guard cell and partly by the existence of a vapor-phase signal that originates in the mesophyll and causes stomata to open in the light.

Stomatal responses to humidity and temperature in darkness.

Stomatal responses to leaf temperature (T(l)) and to the mole fractions of water vapour in the ambient air (w(a)) and the leaf intercellular air spaces (w(i)) were determined in darkness to remove the potential effects of changes in photosynthesis and intercellular CO(2) concentration. Both the steady-state and kinetic responses of stomatal conductance (g(s)) to w(a) in darkness were found to be indistinguishable from those in the light. g(s) showed a steep response to the difference (Deltaw) between w(a) and w(i) when w(a) was varied. The response was much less steep when w(i) was varied. Although stomatal apertures responded steeply to T(l) when Deltaw was held constant at 17 mmol mol(-1), the response was much less steep when Deltaw was held constant at about zero. Similar results were obtained in the light for Deltaw = 15 mmol mol(-1) and Deltaw approximately 0 mmol mol(-1). These results are discussed in the context of mechanisms for the stomatal response to humidity.

Opinion: stomatal responses to light and CO(2) depend on the mesophyll.

The mechanisms by which stomata respond to red light and CO(2) are unknown, but much of the current literature assumes that these mechanisms reside wholly within the guard cells. However, responses of guard cells in isolated epidermes are typically much smaller than those in leaves, and there are several lines of evidence in the literature suggesting that the mesophyll is necessary for these responses in leaves. This paper advances the opinion that although guard cells may have small direct responses to red light and CO(2), most of the stomatal response to these factors in leaves is caused by an unknown signal that originates in the mesophyll.

The role of the mesophyll in stomatal responses to light and CO2.

Stomatal responses to light and CO(2) were investigated using isolated epidermes of Tradescantia pallida, Vicia faba and Pisum sativum. Stomata in leaves of T. pallida and P. sativum responded to light and CO(2), but those from V. faba did not. Stomata in isolated epidermes of all three species could be opened on KCl solutions, but they showed no response to light or CO(2). However, when isolated epidermes of T. pallida and P. sativum were placed on an exposed mesophyll from a leaf of the same species or a different species, they regained responsiveness to light and CO(2). Stomatal responses in these epidermes were similar to those in leaves in that they responded rapidly and reversibly to changes in light and CO(2). Epidermes from V. faba did not respond to light or CO(2) when placed on mesophyll from any of the three species. Experiments with single optic fibres suggest that stomata were being regulated via signals from the mesophyll produced in response to light and CO(2) rather than being sensitized to light and CO(2) by the mesophyll. The data suggest that most of the stomatal response to CO(2) and light occurs in response to a signal generated by the mesophyll.

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.

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.

Stomatal patchiness and task-performing networks.

BACKGROUND: Patchy stomatal conductance is a poorly understood and little-studied phenomenon. It is relatively common, yet it appears to be detrimental to water-use efficiency under some conditions and has no immediately obvious physiological function of any kind. Much of the difficulty in studying patchy stomatal conductance is tied to its unpredictability, both in occurrence and in characteristics. SCOPE AND CONCLUSIONS: Statistical analyses of the variability of stomatal patchiness reveal remarkable similarities to structures and behaviours found in locally connected networks of dynamic units that perform tasks. Such systems solve problems that reside at the level of the entire network despite the absence of a central processor or a mechanism for directly sharing information over the entire system. Frequently, task performance is emergent, in the sense that no unit independently performs the task. Because each unit in the network can communicate with only its immediate neighbours, problem solving is accomplished by the states of the individual units self-organizing into synchronized, collective patterns. In some cases, patches of states form and move coherently over the network, thus providing a means for distantly separated parts of the network to communicate. Often, exactly what form these patches take and how they move as the units synchronize is highly unpredictable. In analogy with such networks, it is suggested that stomatal patchiness may be a signature that plants optimize gas exchange in a more sophisticated and adaptive manner than if performed by their individual stomata independently.

Membrane trafficking and osmotically induced volume changes in guard cells.

Guard cells rapidly adjust their plasma membrane surface area while responding to osmotically induced volume changes. Previous studies have shown that this process is associated with membrane internalization and remobilization. To investigate how guard cells maintain membrane integrity during rapid volume changes, the effects of two membrane trafficking inhibitors on the response of intact guard cells of Vicia faba to osmotic treatments were studied. Using confocal microscopy and epidermal peels, the relationship between the area of a medial paradermal guard-cell section and guard-cell volume was determined. This allowed estimates of guard-cell volume to be made from single paradermal confocal images, and therefore allowed rapid determination of volume as cells responded to osmotic treatments. Volume changes in control cells showed exponential kinetics, and it was possible to calculate an apparent value for guard-cell hydraulic conductivity from these kinetics. Wortmannin and cytochalasin D inhibited the rate of volume loss following a 0-1.5 MPa osmotic treatment. Cytochalasin D also inhibited volume increases following a change from 1.5 MPa to 0 MPa, but wortmannin had no effect. Previous studies showing that treatment with arabinanase inhibits changes in guard-cell volume in response to osmotic treatments were confirmed. However, pressure volume curves show that the effects of arabinanase and the cytochalasin D were not due to changes in cell wall elasticity. It is suggested that arabinanase, cytochalasin D, and wortmannin cause reductions in the hydraulic conductivity of the plasma membrane, possibly via gating of aquaporins. A possible role for aquaporins in co-ordinating volume changes with membrane trafficking is discussed.

Evidence for involvement of photosynthetic processes in the stomatal response to CO2.

Stomatal conductance (gs) typically declines in response to increasing intercellular CO2 concentration (ci). However, the mechanisms underlying this response are not fully understood. Recent work suggests that stomatal responses to ci and red light (RL) are linked to photosynthetic electron transport. We investigated the role of photosynthetic electron transport in the stomatal response to ci in intact leaves of cocklebur (Xanthium strumarium) plants by examining the responses of gs and net CO2 assimilation rate to ci in light and darkness, in the presence and absence of the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and at 2% and 21% ambient oxygen. Our results indicate that (1) gs and assimilation rate decline concurrently and with similar spatial patterns in response to DCMU; (2) the response of gs to ci changes slope in concert with the transition from Rubisco- to electron transport-limited photosynthesis at various irradiances and oxygen concentrations; (3) the response of gs to ci is similar in darkness and in DCMU-treated leaves, whereas the response in light in non-DCMU-treated leaves is much larger and has a different shape; (4) the response of gs to ci is insensitive to oxygen in DCMU-treated leaves or in darkness; and (5) stomata respond normally to RL when ci is held constant, indicating the RL response does not require a reduction in ci by mesophyll photosynthesis. Together, these results suggest that part of the stomatal response to ci involves the balance between photosynthetic electron transport and carbon reduction either in the mesophyll or in guard cell chloroplasts.

Evidence for complex, collective dynamics and emergent, distributed computation in plants.

It has been suggested that some biological processes are equivalent to computation, but quantitative evidence for that view is weak. Plants must solve the problem of adjusting stomatal apertures to allow sufficient CO(2) uptake for photosynthesis while preventing excessive water loss. Under some conditions, stomatal apertures become synchronized into patches that exhibit richly complicated dynamics, similar to behaviors found in cellular automata that perform computational tasks. Using sequences of chlorophyll fluorescence images from leaves of Xanthium strumarium L. (cocklebur), we quantified spatial and temporal correlations in stomatal dynamics. Our values are statistically indistinguishable from those of the same correlations found in the dynamics of automata that compute. These results are consistent with the proposition that a plant solves its optimal gas exchange problem through an emergent, distributed computation performed by its leaves.

Changes in surface area of intact guard cells are correlated with membrane internalization.

Guard cells must maintain the integrity of the plasma membrane as they undergo large, rapid changes in volume. It has been assumed that changes in volume are accompanied by changes in surface area, but mechanisms for regulating plasma membrane surface area have not been identified in intact guard cells, and the extent to which surface area of the guard cells changes with volume has never been determined. The alternative hypothesis-that surface area remains approximately constant because of changes in shape-has not been investigated. To address these questions, we determined surface area for intact guard cells of Vicia faba as they underwent changes in volume in response to changes in the external osmotic potential. We also estimated membrane internalization for these cells. Epidermal peels were subjected to external solutions of varying osmotic potential to shrink and swell the guard cells. A membrane-specific fluorescent dye was used to identify the plasma membrane, and confocal microscopy was used to acquire a series of optical paradermal sections of the guard cell pair at each osmotic potential. Solid digital objects representing the guard cells were created from the membrane outlines identified in these paradermal sections, and surface area, volume, and various linear dimensions were determined for these solid objects. Surface area decreased by as much as 40% when external osmotic potential was increased from 0 to 1.5 MPa, and surface area varied linearly with volume. Membrane internalization was approximated by determining the amount of the fluorescence in the cell's interior. This value was shown to increase approximately linearly with decreases in the cell's surface area. The changes in surface area, volume, and membrane internalization were reversible when the guard cells were returned to a buffer solution with an osmotic potential of approximately zero. The data show that intact guard cells undergo changes in surface area that are too large to be accommodated by plasma membrane stretching and shrinkage and suggest that membrane is reversibly internalized to maintain cell integrity.