The Transmembrane Region of Guard Cell SLAC1 Channels Perceives CO2 Signals via an ABA-Independent Pathway in Arabidopsis

The guard cell S-type anion channel, SLOW ANION CHANNEL1 (SLAC1), a key component in the control of stomatal movements, is activated in response to CO₂ and abscisic acid (ABA). Several amino acids existing in the N-terminal region of SLAC1 are involved in regulating its activity via phosphorylation in the ABA response. However, little is known about sites involved in CO₂ signal perception. To dissect sites that are necessary for the stomatal CO₂ response, we performed slac1 complementation experiments using transgenic plants expressing truncated SLAC1 proteins. Measurements of gas exchange and stomatal apertures in the truncated transgenic lines in response to CO₂ and ABA revealed that sites involved in the stomatal CO₂ response exist in the transmembrane region and do not require the SLAC1 N and C termini. CO₂ and ABA regulation of S-type anion channel activity in guard cells of the transgenic lines confirmed these results. In vivo site-directed mutagenesis experiments targeted to amino acids within the transmembrane region of SLAC1 raise the possibility that two tyrosine residues exposed on the membrane are involved in the stomatal CO₂ response.     

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 CO₂. 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 the environment have been studied in leaves for well over 100 years. More recently, the mechanisms for these responses have been investigated using isolated epidermes or isolated guard cell protoplasts. Despite the combination of these two approaches, the mechanisms by which stomata respond to environmental signals are not well understood. Since stomata control CO₂ uptake and water loss from leaves, the responses of stomata to environmental factors are important determinants of terrestrial productivity and water use. It is therefore critical that we understand the mechanisms by which stomata respond to the environment if we are to accurately predict the effects of future climates on productivity and water cycles (Randall et al., 1996).There are two assumptions about stomata that are implicit in much of the recent literature: (1) that stomatal responses result from sensory mechanisms that reside within the guard cells, and (2) that stomata in isolated epidermes respond similarly to those in a leaf. The exception to this generalization is the stomatal response to humidity, which has been suggested to be the result of changes in guard cell water potential (Dewar, 1995, 2002) or of signaling from other cells in the leaf to the guard cells (Buckley et al., 2003). The assumption that guard cells directly sense CO₂ and light is largely based on data from isolated epidermes that show effects of light and CO₂ on stomatal apertures. As pointed out by Mott (2009), however, stomatal responses to light and CO₂ in isolated epidermes are generally much different from those observed in leaves; e.g. responses in isolated epidermes are generally smaller than those in leaves, opening in response to light is slower, and closing in darkness is rarely observed. These observations were used to suggest that the mesophyll is somehow involved in stomatal responses to red light and CO₂. This idea is supported by several recent studies that suggest that guard cells do not respond directly to red light. In the first of these studies it was shown that guard cells in an intact leaf do not show hyperpolarization of the plasma membrane in response to red light if the red light is applied to only the guard cell (Roelfsema et al., 2002). In contrast, blue light applied only to the guard cell does cause hyperpolarization, and red light does cause hyperpolarization if applied to the guard cell and the underlying mesophyll. The second study showed that stomata in albino areas of a leaf do not respond to red light, although they contain chloroplasts and do respond to blue light (Roelfsema et al., 2006). Finally, a third study has shown that isolated epidermes are much more sensitive to light and CO₂ when placed in close contact with an exposed mesophyll from a leaf from the same or a different species (Mott et al., 2008). These epidermis-mesophyll grafts showed stomatal responses to light and CO₂ that were indistinguishable from those in an intact leaf—a sharp contrast to the behavior of stomata in isolated epidermes that are floating on buffer solutions. In that study, illumination of a single stoma in a leaf using a small-diameter fiber optic did not produce stomatal opening, but opening did occur if several stomata and the underlying mesophyll were illuminated. Furthermore, this treatment actually caused opening of adjacent, but unilluminated, stomata (Mott et al., 2008).In constructing the epidermis-mesophyll grafts in the study described above (Mott et al., 2008), it was noticed that functional grafts could be produced only if both the mesophyll and the epidermis were blotted completely dry of any free water before placing them together. Although the tissues were apparently still fully hydrated, there was very little free water present (i.e. water not contained within the walls of the leaf cells), and both the mesophyll and epidermis felt and looked dry prior to assembly. In addition, even when free water was blotted away initially, stomata did not open in grafts that ended up with visible water on the epidermis or mesophyll that was caused by condensation during the experiment. These observations suggest that the presence of free water somehow prevented the stomata in the grafts from opening. Assuming that the mechanisms operating in the grafts were similar to those in an intact leaf, this result also suggests that free water may have an effect on stomata in leaves as well. In addition, it seems possible that the effect of free water on stomata could be related to the disruption of the signal from the mesophyll that was proposed in an earlier study (Mott et al., 2008). We hypothesize that disruption of this signal could be caused by (1) dilution of some solute that is necessary for opening (such as K⁺) in the guard cell walls, (2) dilution of an apoplastic, liquid-phase opening signal from the mesophyll to the guard cells, and (3) blockage of a vapor-phase opening signal from the mesophyll to the guard cells. This study was initiated to test these three hypotheses by examining the effect of free water and other liquids on stomatal functioning.     

Stomatal movement and potassium transport in epidermal strips of Zea mays: The effect of CO2

Summary The correlation between stomatal action and potassium movement in the epidermis of Zea mays was examined in isolated epidermal strips floated on distilled water. Stomatal opening in the isolated epidermis is reversible in response to alternate periods of light or darkness, and is always correlated with a shift in the potassium content of the guard cells. K accumulates in guard cells during stomatal opening, and moves from the guard cells into the subsidiary cells during rapid stomatal closure. When epidermal strips are illuminated in normal air, as against CO₂-free air, the stomata do not open and there is a virtually complete depletion of K from the stomatal apparatus. In darkness CO₂-containing air inhibits stomatal opening and K accumulation in guard cells, but does not lead to a depletion of K from the stomata as observed in the light.     

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 responses to changes in atmospheric humidity and water supply: Experiments with leaf sections of Zea mays in CO2-free air

Summary Leaf sections were exposed to CO₂-free air, thus excluding interference by the CO₂-sensitive system in the guard cells. Stomates did not close in response to change from moist to dry air, whether it passed over the leaf or was forced through the intercelluar spaces. In contrast, the stomatal apertures became narrower if the water potential in the liquid supplying the leaf was lowered. Of solutions with the same osmolality, those with the higher viscosity produced the larger responses.Transient stomatal movements in the opposite direction to the final response were observed upon any sudden change in the water status of the leaf sections, whether caused by varying the moisture content of the air passing around or through the leaf sections, or by varying the water supply. Increased load on the water supply caused temporary opening movements, while improvements in water supply led to closing movements of varying duration. When dry air was forced through the leaf sections, non-sinusoidal oscillations with large amplitudes were sometimes observed.It is concluded that the guard cells are tightly coupled to the water-supply system of the leaf and only indirectly to the conditions in the atmosphere by a negative feedback of transpiration on the water potential in the water-conducting system.     

Stomatal Opening in Isolated Epidermal Strips of Vicia faba. I. Response to Light and to CO(2)-free Air

This paper reports a consistent and large opening response to light + CO₂-free air in living stomata of isolated epidermal strips of Vicia faba. The response was compared to that of non-isolated stomata in leaf discs floating on water; stomatal apertures, guard cell solute potentials and starch contents were similar in the 2 situations. To obtain such stomatal behavior, it was necessary to float epidermal strips on dilute KCl solutions. This suggests that solute uptake is necessary for stomatal opening.

The demonstration of normal stomatal behavior in isolated epidermal strips provides a very useful system in which to investigate the mechanism of stomatal opening. It was possible to show independent responses in stomatal aperture to light and to CO₂-free air.

     

Jasmonate‐mediated stomatal closure under elevated CO2 revealed by time‐resolved metabolomics

Foliar stomatal movements are critical for regulating plant water loss and gas exchange. Elevated carbon dioxide (CO₂) levels are known to induce stomatal closure. However, the current knowledge on CO₂ signal transduction in stomatal guard cells is limited. Here we report metabolomic responses of Brassica napus guard cells to elevated CO₂ using three hyphenated metabolomics platforms: gas chromatography‐mass spectrometry (MS); liquid chromatography (LC)‐multiple reaction monitoring‐MS; and ultra‐high‐performance LC‐quadrupole time‐of‐flight‐MS. A total of 358 metabolites from guard cells were quantified in a time‐course response to elevated CO₂ level. Most metabolites increased under elevated CO₂, showing the most significant differences at 10 min. In addition, reactive oxygen species production increased and stomatal aperture decreased with time. Major alterations in flavonoid, organic acid, sugar, fatty acid, phenylpropanoid and amino acid metabolic pathways indicated changes in both primary and specialized metabolic pathways in guard cells. Most interestingly, the jasmonic acid (JA) biosynthesis pathway was significantly altered in the course of elevated CO₂ treatment. Together with results obtained from JA biosynthesis and signaling mutants as well as CO₂ signaling mutants, we discovered that CO₂‐induced stomatal closure is mediated by JA signaling.     

Guard Cell Chloroplasts Are Essential for Blue Light-Dependent Stomatal Opening in Arabidopsis

Blue light (BL) induces stomatal opening through the activation of H⁺-ATPases with subsequent ion accumulation in guard cells. In most plant species, red light (RL) enhances BL-dependent stomatal opening. This RL effect is attributable to the chloroplasts of guard cell, the only cells in the epidermis possessing this organelle. To clarify the role of chloroplasts in stomatal regulation, we investigated the effects of RL on BL-dependent stomatal opening in isolated epidermis, guard cell protoplasts, and intact leaves of Arabidopsis thaliana. In isolated epidermal tissues and intact leaves, weak BL superimposed on RL enhanced stomatal opening while BL alone was less effective. In guard cell protoplasts, RL enhanced BL-dependent H⁺-pumping and DCMU, a photosynthetic electron transport inhibitor, eliminated this effect. RL enhanced phosphorylation levels of the H⁺-ATPase in response to BL, but this RL effect was not suppressed by DCMU. Furthermore, DCMU inhibited both RL-induced and BL-dependent stomatal opening in intact leaves. The photosynthetic rate in leaves correlated positively with BL-dependent stomatal opening in the presence of DCMU. We conclude that guard cell chloroplasts provide ATP and/or reducing equivalents that fuel BL-dependent stomatal opening, and that they indirectly monitor photosynthetic CO₂ fixation in mesophyll chloroplasts by absorbing PAR in the epidermis.     

Responses of Stomata in Epidermal Strips of Vicia fabato Carbon Dioxide and Growth Hormones when Incubated on Potassium Chloride and Potassium Iminodiacetate

The effect of various K⁺ levels in combination with Cl^− or iminodiacetate (IDA^{& minus;}) on stomatal responsesin isolated epidermal strips of Vicia faba L. were examinedin order to determine the role of malate during guard cell movements.Responses of guard cells to ABA, kinetin, and varying CO₂ levelswere similar when epidermal strips were floated on KCL or KIDAat 10 mM; such responses were typical in that ABA caused closure,kinetin stimulated opening in ambient air, and apertures weregreater in CO₂-free than ambient air. Maximal stomatal openingwas observed in both ambient and CO₂-free air with KCL at 100mM. The transfer of epidermal strips from 100 mM KCL to solutionsof 100 mM KCL supplemented with ABA or kinetin did not bringabout changes in stomatal aperture. KCL at 100 mM supporteda greater degree of stomatal opening than did 100 mM KIDA irrespectiveof the CO₂ content of the air. In CO₂-free air transfer of epidermalstrips from 100 mM KIDA to solutions containing 100 mM KIDAsupplemented with ABA or kinetin caused little change in stomatalaperture, whereas, in ambient air, the same treatments resultedin stomatal opening. The results are discussed in relation tothe role of malate during guard cell movements.     

Effect of Potassium Levels on the Stomatal Behavior of the Hemi-Parasite Striga hermonthica

The hemi-parasite Striga hermonthica, exhibits an anomalous pattern of stomatal response, stomata remaining open in darkness and when subjected to water stress. This suggests irregularity in stomatal response due to malfunction of the stomatal mechanism. To test this suggestion guard cells were isolated from the effects of surrounding cells, by incubating epidermal strips at low pH. These stomata responded rapidly to low CO₂ concentrations, darkness, and ABA. Thus, a paradox exists between stomatal behavior observed in whole leaves and that in isolated guard cells. However, when incubated in the presence of high potassium concentrations (>200 millimolar KCl) stomatal responses in epidermal strips resembled those found in whole leaves, with enhanced opening and reduced closing responses. It is suggested that the anomalous behavior of stomata in Striga and other leafy hemiparasites can be explained by the modulatory effects of high potassium concentrations which accumulate in the leaves as a consequence of high transpiration rates and the lack of a retranslocation system.     

Insights into the Molecular Mechanisms of CO2-Mediated Regulation of Stomatal Movements

Plants must continually balance the influx of CO₂ for photosynthesis against the loss of water vapor through stomatal pores in their leaves. This balance can be achieved by controlling the aperture of the stomatal pores in response to several environmental stimuli. Elevation in atmospheric [CO₂] induces stomatal closure and further impacts leaf temperatures, plant growth and water-use efficiency, and global crop productivity. Here, we review recent advances in understanding CO₂-perception mechanisms and CO₂-mediated signal transduction in the regulation of stomatal movements, and we explore how these mechanisms are integrated with other signaling pathways in guard cells.     

Multi-level Modeling of Light-Induced Stomatal Opening Offers New Insights into Its Regulation by Drought

Plant guard cells gate CO₂ uptake and transpirational water loss through stomatal pores. As a result of decades of experimental investigation, there is an abundance of information on the involvement of specific proteins and secondary messengers in the regulation of stomatal movements and on the pairwise relationships between guard cell components. We constructed a multi-level dynamic model of guard cell signal transduction during light-induced stomatal opening and of the effect of the plant hormone abscisic acid (ABA) on this process. The model integrates into a coherent network the direct and indirect biological evidence regarding the regulation of seventy components implicated in stomatal opening. Analysis of this signal transduction network identified robust cross-talk between blue light and ABA, in which [Ca²⁺]_c plays a key role, and indicated an absence of cross-talk between red light and ABA. The dynamic model captured more than 10³¹ distinct states for the system and yielded outcomes that were in qualitative agreement with a wide variety of previous experimental results. We obtained novel model predictions by simulating single component knockout phenotypes. We found that under white light or blue light, over 60%, and under red light, over 90% of all simulated knockouts had similar opening responses as wild type, showing that the system is robust against single node loss. The model revealed an open question concerning the effect of ABA on red light-induced stomatal opening. We experimentally showed that ABA is able to inhibit red light-induced stomatal opening, and our model offers possible hypotheses for the underlying mechanism, which point to potential future experiments. Our modelling methodology combines simplicity and flexibility with dynamic richness, making it well suited for a wide class of biological regulatory systems.     

Temperature dependencies and apparent activation energies of stomatal opening and closing

Summary Stomatal opening movements in response to illumination, and stomatal closure following darkening were studied in leaf sections of Zea mays, using air-flow porometers. Stomatal opening is characterized by a phase of linear increase of air flow through the leaf (slope = opening velocity); stomatal closure follows a relaxation curve from which a time constant (closing coefficient) can be derived.Apparent energies of activation, , were computed for the opening velocity and for the closing coefficient from stomatal movements recorded at tissue temperatures between 5° and 50°. It was assumed that the closing coefficient can be used as a measure of the closing force, and that the opening force has to exceed the closing force in order to bring about stomatal opening. is about 7 kcal mole^-1 for the closing coefficient and between 12 and 18 kcal mole^-1 for the opening force. Thus, during stomatal opening, metabolism must provide energy to build up a pressure difference between guard cells and the surrounding tissue.The process controlling the velocity of closure is essentially a passive loss of water (and solutes?) from the guard cells. The of 7 kcal mole^-1 found for the closing coefficient is, however, higher than that for the viscosity of water or for the coefficient of self diffusion of water. It is, therefore, concluded either that water interacts with the cell structures which it has to permeate during stomatal closure, or that the rate of salt loss from guard cells controls the velocity of stomatal closure.The closing force decreases when leaf temperature rises above 35° or falls below 15°. Therefore, stomata of maize open relatively faster and wider above 35° and below 15°, and the 's of the opening velocity appear to be very large above 35° (up to 50 kcal mole^-1) while they have a negative sign below 15°.Research supported by the U.SS. Atomic Energy Commission under contract AT (11-1) 1338. I thank Miss Freia Schulz-Baldes for interested technical assistance.     

Guard cell photosynthesis is critical for stomatal turgor production,yet does not directly mediate CO2‐ and ABA‐induced stomatal closing

Stomata mediate gas exchange between the inter‐cellular spaces of leaves and the atmosphere. CO₂ levels in leaves (Ci) are determined by respiration, photosynthesis, stomatal conductance and atmospheric [CO₂]. [CO₂] in leaves mediates stomatal movements. The role of guard cell photosynthesis in stomatal conductance responses is a matter of debate, and genetic approaches are needed. We have generated transgenic Arabidopsis plants that are chlorophyll‐deficient in guard cells only, expressing a constitutively active chlorophyllase in a guard cell specific enhancer trap line. Our data show that more than 90% of guard cells were chlorophyll‐deficient. Interestingly, approximately 45% of stomata had an unusual, previously not‐described, morphology of thin‐shaped chlorophyll‐less stomata. Nevertheless, stomatal size, stomatal index, plant morphology, and whole‐leaf photosynthetic parameters (PSII, qP, qN, F_V′/F_M′) were comparable with wild‐type plants. Time‐resolved intact leaf gas‐exchange analyses showed a reduction in stomatal conductance and CO₂‐assimilation rates of the transgenic plants. Normalization of CO₂ responses showed that stomata of transgenic plants respond to [CO₂] shifts. Detailed stomatal aperture measurements of normal kidney‐shaped stomata, which lack chlorophyll, showed stomatal closing responses to [CO₂] elevation and abscisic acid (ABA), while thin‐shaped stomata were continuously closed. Our present findings show that stomatal movement responses to [CO₂] and ABA are functional in guard cells that lack chlorophyll. These data suggest that guard cell CO₂ and ABA signal transduction are not directly modulated by guard cell photosynthesis/electron transport. Moreover, the finding that chlorophyll‐less stomata cause a ‘deflated’ thin‐shaped phenotype, suggests that photosynthesis in guard cells is critical for energization and guard cell turgor production.     

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.     

Enhancement of the Stomatal Response to Blue Light by Red Light, Reduced Intercellular Concentrations of CO(2), and Low Vapor Pressure Differences

The effects of environmental parameters on the blue light response of stomata were studied by quantifying transient increases in stomatal conductance in Commelina communis following 15 seconds by 0.100 millimole per square meter per second pulses of blue light. Because conductance increases were not observed following red light pulses of the same or greater (30 seconds by 0.200 millimole per square meter per second) fluences, the responses observed could be reliably attributed to the specific blue light response of the guard cells, rather than to guard cell chlorophyll. In both Paphiopedilum harrisianum, which lacks guard cell chloroplasts, and Commelina, the blue light response was enhanced by 0.263 millimole per square meter per second continuous background red light. Thus, the blue light response and its enhancement do not require energy derived from red-light-driven photophosphorylation by the guard cell chloroplasts. In Commelina, reduction of the intercellular concentration of CO₂ by manipulation of ambient CO₂ concentrations resulted in an enhanced blue light response. In both Commelina and Paphiopedilum, the blue light response was decreased by an increased vapor pressure difference. The magnitude of blue-light-specific stomatal opening thus appears to be sensitive to environmental conditions that affect the carbon and water status of the plant.     

Systems dynamic modeling of the stomatal guard cell predicts emergent behaviors in transport, signaling, and volume control

The dynamics of stomatal movements and their consequences for photosynthesis and transpirational water loss have long been incorporated into mathematical models, but none have been developed from the bottom up that are widely applicable in predicting stomatal behavior at a cellular level. We previously established a systems dynamic model incorporating explicitly the wealth of biophysical and kinetic knowledge available for guard cell transport, signaling, and homeostasis. Here we describe the behavior of the model in response to experimentally documented changes in primary pump activities and malate (Mal) synthesis imposed over a diurnal cycle. We show that the model successfully recapitulates the cyclic variations in H⁺, K⁺, Cl⁻, and Mal concentrations in the cytosol and vacuole known for guard cells. It also yields a number of unexpected and counterintuitive outputs. Among these, we report a diurnal elevation in cytosolic-free Ca²⁺ concentration and an exchange of vacuolar Cl⁻ with Mal, both of which find substantiation in the literature but had previously been suggested to require additional and complex levels of regulation. These findings highlight the true predictive power of the OnGuard model in providing a framework for systems analysis of stomatal guard cells, and they demonstrate the utility of the OnGuard software and HoTSig library in exploring fundamental problems in cellular physiology and homeostasis.The guard cells, which surround stomatal pores in the epidermis of plant leaves, regulate the pore aperture to balance the often conflicting demands for CO₂ in photosynthesis with the need to conserve water by the plant. Stomatal transpiration accounts for much of the nearly 70% of global water usage associated with agriculture and has a profound impact on the water and carbon cycles of the world (Gedney et al., 2006; UNESCO, 2009). Recent studies have associated increases in continental water runoff with the rise in available CO₂ and decreases in stomatal transpiration (Gedney et al., 2006) and have suggested that stomatal behavior skews the impact of greenhouse gasses on fresh water resources (Betts et al., 2007). The past half century has generated a vast wealth of knowledge for guard cell transport, signaling, and homeostasis, resolving the properties of all of the major transporters and many of the signaling pathways that control them (Blatt, 2000a; Schroeder et al., 2001; Blatt et al., 2007; Wang and Song, 2008; McAinsh and Pittman, 2009). Even so, resolving many aspects of stomatal dynamics remains a challenge. These studies have yet to yield any detail about how the entire network of transporters works as a unit to modulate solute flux and regulate stomatal aperture. Quantitative systems analysis offers one approach to this problem that is now much needed. Efforts to model stomatal function to date generally have been driven by a top-down approach: The mechanics of stomatal movements are subsumed within a few empirical parameters of linear hydraulic pathways and conductances (Farquhar and Wong, 1984; Ball, 1987; Williams et al., 1996; Eamus and Shanahan, 2002; West et al., 2005). These models have proven useful at the plant and community levels; but they have not incorporated the essential detail to support an understanding of the molecular and cellular mechanics that drive stomatal movements.In the previous article (Hills et al., 2012) we introduced a computational approach to developing a dynamic model of the stomatal guard cell based on the HoTSig library and OnGuard software. We resolved an OnGuard model that takes account of all of the fundamental properties for transporters at the plasma membrane and tonoplast, the salient features of osmolite metabolism, and key homeostatic and dynamic signaling characteristics that have been described in the literature. The model successfully integrated a number of the steady-state characteristics of guard cells, recapitulating the patterns in guard cell response to the extracellular variables of KCl and CaCl₂ concentrations and to extracellular pH. Here we explore the capacity of the model to reproduce diurnal oscillations in guard cell membrane transport and malate (Mal) metabolism, and its consequences for the dynamics of guard cell volume, turgor pressure, and stomatal aperture. We demonstrate the true predictive power of the OnGuard model in generating a number of unexpected and counterintuitive outputs. Among these, the model yields counterintuitive changes in cytosolic-free [Ca²⁺] ([Ca2+]_i) and a daily exchange of Cl⁻ with Mal that are well documented in the literature, but have been suggested to require additional and complex levels of regulation. These behaviors are accounted for entirely by the known kinetic features of the transporters encoded in the model. Thus, the results demonstrate the predictive power of the OnGuard model as a framework from which to test the basic tenets of the stomatal behavior and to explore the interactions of transport and metabolism in the guard cell system.