In stomatal guard cells of higher-plant leaves, abscisic acid (ABA) evokes increases in cytosolic free Ca 2؉ concentration ( (5), and nodulation (6) and are central to hormonal physiology (7-9). Changes in [Ca 2ϩ ] i influence ion channel gating (1, 9, 10), light-mediated gene expression (11), cell differentiation, elongation, and tip growth (3).In stomatal guard cells, one of the best-characterized plant cell models, increasing [Ca 2ϩ ] i is known to inactivate inwardrectifying K ϩ channels and to activate Cl Ϫ channels, biasing the plasma membrane for solute efflux, which drives stomatal closure (9). Changes in [Ca 2ϩ ] i have been associated with stimuli that lead to stomatal closure, notably abscisic acid (ABA) and CO 2 (9, 12). These changes in [Ca 2ϩ ] i depend on Ca 2ϩ release from intracellular stores (13-16) and on Ca 2ϩ entry across the plasma membrane (17, 18). Nonetheless, direct evidence for channels that could mediate Ca 2ϩ influx has been lacking. Indeed, little evidence has come forth for Ca 2ϩ channels at the plasma membrane of higher-plant cells until recently (19)(20)(21)(22)(23).One clue to a major pathway for Ca 2ϩ entry into guard cells has come from measurements of [Ca 2ϩ ] i and its elevation by ABA under voltage clamp (17). These studies indicated a voltage dependence to the [Ca 2ϩ ] i rise, suggesting that ABA stimulated a Ca 2ϩ channel, but that its activity also required membrane hyperpolarization. We have recorded single-channel currents from Vicia guard cell protoplasts under conditions that eliminate the background of current through K ϩ and Cl Ϫ channels. The results reported here demonstrate the presence of Ca 2ϩ channels at the plasma membrane that open on membrane hyperpolarization and are activated by ABA. Materials and MethodsPlant Material. Epidermal strips of Vicia faba L., cv. Bunyard Exhibition, were obtained and protoplasts were prepared as described (24,25). All operations were carried out on a Zeiss Axiovert microscope with 40ϫ LWD Nomarski DIC optics at 20-22°C. Solution was added (Ӎ20 chamber vol͞min) by gravity feed and removed by aspiration.Electrophysiology. Pipettes were pulled with a Narishige (Tokyo) PP-81 puller modified for three-stage pulls (input resistances, 30-50 M⍀) to reduce the number of channels under a patch. Pipettes were coated with Sigmacote (Sigma) to reduce capacitance. Connections to amplifier and bath were by a 0.1 M KCl͞Ag-AgCl liquid junctions, and junction potentials were taken into account (26). Single-channel currents were recorded with an Axopatch 200B patch amplifier (Axon Instruments, Foster City, CA) after filtering at 5 kHz and sampled at 44 kHz for analysis. Data were filtered at 1 kHz (Kemo, Beckenham, U.K.) offline and analyzed with N-PRO (Wye Science, Wye, Kent, U.K.), P/V CLAMP V. 6 (CED, Cambridge, U.K.) software. Channel amplitudes were calculated from point-amplitude histograms estimated from open events Ն5-ms duration (Fig. 1) beyond closed levels determined from periods of no channel activity (27). Channel numbers w...

Abscisic acid (ABA) triggers a complex sequence of signaling events that lead to concerted modulation of ion channels at the plasma membrane of guard cells and solute efflux to drive stomatal closure in plant leaves. Recent work has indicated that nitric oxide (NO) and its synthesis are a prerequisite for ABA signal transduction in Arabidopsis and Vicia guard cells. Its mechanism(s) of action is not well defined in guard cells and, generally, in higher plants. Here we show directly that NO selectively regulates Ca 2؉ -sensitive ion channels of Vicia guard cells by promoting Ca 2؉ release from intracellular stores to raise cytosolic-free [Ca 2؉ ]. NO-sensitive Ca 2؉ release was blocked by antagonists of guanylate cyclase and cyclic ADP ribose-dependent endomembrane Ca 2؉ channels, implying an action mediated via a cGMP-dependent cascade. NO did not recapitulate ABA-evoked control of plasma membrane Ca 2؉ channels and Ca 2؉ -insensitive K ؉ channels, and NO scavengers failed to block the activation of these K ؉ channels evoked by ABA. These results place NO action firmly within one branch of the Ca 2؉ -signaling pathways engaged by ABA and define the boundaries of parallel signaling events in the control of guard cell movements.cGMP-mediated signaling ͉ stress physiology ͉ cyclic ADP ribose ͉ cytosolic-free [Ca 2ϩ ] elevation ͉ Vicia

Stomatal guard cells play a key role in gas exchange for photosynthesis while minimizing transpirational water loss from plants by opening and closing the stomatal pore. Foliar gas exchange has long been incorporated into mathematical models, several of which are robust enough to recapitulate transpirational characteristics at the whole-plant and community levels. Few models of stomata have been developed from the bottom up, however, and none are sufficiently generalized to be widely applicable in predicting stomatal behavior at a cellular level. We describe here the construction of computational models for the guard cell, building on the wealth of biophysical and kinetic knowledge available for guard cell transport, signaling, and homeostasis. The OnGuard software was constructed with the HoTSig library to incorporate explicitly all of the fundamental properties for transporters at the plasma membrane and tonoplast, the salient features of osmolite metabolism, and the major controls of cytosolic-free Ca 2+ concentration and pH. The library engenders a structured approach to tier and interrelate computational elements, and the OnGuard software allows ready access to parameters and equations 'on the fly' while enabling the network of components within each model to interact computationally. We show that an OnGuard model readily achieves stability in a set of physiologically sensible baseline or Reference States; we also show the robustness of these Reference States in adjusting to changes in environmental parameters and the activities of major groups of transporters both at the tonoplast and plasma membrane. The following article addresses the predictive power of the OnGuard model to generate unexpected and counterintuitive outputs.Stomatal guard cells surround pores in the epidermis of plant leaves and regulate the pore aperture. They open the pore in response to low CO 2 and light to facilitate CO 2 access for photosynthesis, and they close the pore in the dark, under drought stress, and in the presence of the water-stress hormone abscisic acid to minimize water loss through transpiration. Stomata have a profound impact on the water and carbon cycles of the world (Gedney et al., 2006;Betts et al., 2007). Their dynamics have been incorporated into models for transpiration and water use efficiency (Farquhar and Wong, 1984;Ball, 1987;Williams et al., 1996;Eamus and Shanahan, 2002;West et al., 2005), successfully reproducing the gas exchange, CO 2 , and transpirational characteristics of experiments at the plant and community levels. To date, these models have taken a top-down approach. They subsume stomatal movements within a few empirical parameters of linear hydraulic pathways and conductances without reference to the molecular mechanics of the guard cell. No generalized guard cell model has yet to be developed from the bottom up, drawing on the wealth of knowledge available for guard cell transport, signaling, and homeostasis. It is clear that such a model is now needed. The depth and breadth of information ava...

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 2, 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 Ca 2+ concentration and an exchange of vacuolar Cl 2 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.