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Stomata can be regarded as hydraulically driven valves in the leaf surface, which open to allow CO2 uptake and close to prevent excessive loss of water. Movement of these 'Watergates' is regulated by environmental conditions, such as light, CO2 and humidity. Guard cells can sense environmental conditions and function as motor cells within the stomatal complex. Stomatal movement results from the transport of K+ salts across the guard cell membranes. In this review, we discuss the biophysical principles and mechanisms of stomatal movement and relate these to ion transport at the plasma membrane and vacuolar membrane. Studies with isolated guard cells, combined with recordings on single guard cells in intact plants, revealed that light stimulates stomatal opening via blue light-specific and photosynthetic-active radiation-dependent pathways. In addition, guard cells sense changes in air humidity and the water status of distant tissues via the stress hormone abscisic acid (ABA). Guard cells thus provide an excellent system to study cross-talk, as multiple signaling pathways induce both short- and long-term responses in these sensory cells.

The Arabidopsis genome encodes for 20 members of putative ligand-gated channels, termed glutamate receptors (GLR). Despite the fact that initial studies suggested a role for GLRs in various aspects of photomorphogenesis, calcium homeostasis or aluminium toxicity, their functional properties and physiological role in plants remain elusive. Here, we have focussed on AtGLR3.4, which is ubiquitously expressed in Arabidopsis including roots, vascular bundles, mesophyll cells and guard cells. AtGLR3.4 encodes a glutamate-, touch-, and cold-sensitive member of this gene family. Abiotic stress stimuli such as touch, osmotic stress or cold stimulated AtGLR3.4 expression in an abscisic acid-independent, but calcium-dependent manner. In plants expressing the Ca(2+) -reporter apoaequorin, glutamate as well as cold elicited cytosolic calcium elevations. Upon glutamate treatment of mesophyll cells, the plasma membrane depolarised by about 120 mV. Both glutamate responses were transient in nature, sensitive to glutamate receptor antagonists, and were subject to desensitisation. One hour after eliciting the first calcium signal, a 50% recovery from desensitisation was observed, reflecting the stimulus-induced fast activation of AtGLR3.4 transcription. We thus conclude that AtGLR3.4 in particular and GLRs in general could play an important role in the Ca(2+) -based, fast transmission of environmental stress.

The phytohormone abscisic acid (ABA) reports on the water status of the plant and induces stomatal closure. Guard cell anion channels play a central role in this response, because they mediate anion efflux, and in turn, cause a depolarization-induced K+ release. We recorded early steps in ABA signaling, introducing multibarreled microelectrodes in guard cells of intact plants. Upon external ABA treatment, anion channels transiently activated after a lag phase of 2 min. As expected for a cytosolic ABA receptor, iontophoretic ABA loading into the cytoplasm initiated a rise in anion current without delay. These ABA responses could be elicited repetitively at resting and at largely depolarized potentials (e.g., 0 mV), ruling out signal transduction by means of hyperpolarization-activated calcium channels. Likewise, ABA stimulation did not induce a rise in the cytosolic free-calcium concentration. However, the presence of 100 nM background Ca2+ was required for anion channel function, because the action of ABA on anion channels was repressed after loading of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate. The chain of events appears very direct, because none of the tested putative ABA-signaling intermediates (inositol 1,4,5 trisphosphate, inositol hexakisphosphate, nicotinic acid adenine dinucleotide phosphate, and cyclic ADP-ribose), could mimic ABA as anion channel activator. In patch-clamp experiments, cytosolic ABA also evoked anion current transients carried by R- and S-type anion channels. The response was dose-dependent with half-maximum activation at 2.6 micromolar ABA. Our studies point to an ABA pathway initiated by ABA binding to a cytosolic receptor that within seconds activates anion channels, and in turn, leads to depolarization of the plasma membrane.

Despite the availability of many mutants for signal transduction, Arabidopsis thaliana guard cells have so far not been used in electrophysiological research. Problems with the isolation of epidermal strips and the small size of A. thaliana guard cells were often prohibiting. In the present study these difficulties were overcome and guard cells were impaled with double-barreled microelectrodes. Membrane-potential recordings were often stable for over half an hour and voltage-clamp measurements could be conducted. The guard cells were found to exhibit two states. The majority of the guard cells had depolarized membrane potentials, which were largely dependent on external K+ concentrations. Other cells displayed spontaneous transitions to a more hyperpolarized state, at which the free-running membrane potential (Em) was not sensitive to the external K concentration. Two outward-rectifying conductances were identified in cells in the depolarized state. A slow outward-rectifying channel (s-ORC) had properties resembling the K+-selective ORC of Vicia faba guard cells (Blatt, 1988, J Membr Biol 102: 235-246). The activation and inactivation times and the activation potential, all depended on the reversal potential (Erev) of the s-ORC conductance. The s-ORC was blocked by Ba2+ (K 1/2 = 0.3 - 1.3 mM) and verapamil (K 1/2 = 15-20 micromolar). A second rapid outward-rectifying conductance (r-ORC) activated instantaneously upon stepping the voltage to positive values and was stimulated by Ba2+. Inward-rectifying channels (IRC) were only observed in cells in the hyperpolarized state. The activation time and activation potential of this channel were not sensitive to the external K+ concentration. The slow activation of the IRC (t 1/2 approximately 0.5 s) and its negative activation potential (V threshold = - 155 mV) resemble the values found for the KAT1 channel expressed in Saccharomyces cerevisiae (Bertl et al., 1995, Proc Natl Acad Sci USA 92: 2701-2705). The results indicate that A. thaliana guard cells provide an excellent system for the study of signal transduction processes.

The apoplastic pH of guard cells probably acidifies in response to light, since light induces proton extrusion by both guard cells and epidermal leaf cells. From the data presented here, it is concluded that these apoplastic pH changes will affect K+ fluxes in guard cells of Arabidopsis thaliana (L.) Heynh. Guard cells of this species were impaled with double-barrelled microelectrodes, to measure the membrane potential (Em) and the plasma-membrane conductance. Guard cells were found to exhibit two states with respect to their Em, a depolarized and a hyperpolarized state. Apoplastic acidification depolarized Em in both states, though the origin of the depolarization differed for each state. In the depolarized state, the change in Em was the result of a combined pH effect on instantaneously activating conductances and on the slow outward rectifying K + channel (s-ORC). At a more acidic apoplastic pH, the current through instantaneously activated conductances became more inwardly directed, while the maximum conductance of s-ORC decreased. The effect on s-ORC was accompanied by an acceleration of activation and deactivation of the channel. Experiments with acid loading of guard cells indicated that the effect on sORC was due to a lowered intracellular pH, caused by apoplastic acidification. In the hyperpolarized state, the pH-induced depolarization was due to a direct effect of the apoplastic pH on the inward rectifying K+ channel. Acidification shifted the threshold potential of the channel to more positive values. This effect was accompanied by a decrease in activation times and an increase of deactivation times, of the channel. From the changes in Em and membrane conductance, the expected effect of acidification on K + fluxes was calculated. It was concluded that apoplastic acidification will increase the K+-efflux in the depolarized state and reduce the K+-influx in the hyperpolarized state.

The Arabidopsis tandem-pore K+ (TPK) channels displaying four transmembrane domains and two pore regions share structural homologies with their animal counterparts of the KCNK family. In contrast to the Shaker-like Arabidopsis channels (six transmembrane domains/one pore region), the functional properties and the biological role of plant TPK channels have not been elucidated yet. Here, we show that AtTPK4 (KCO4) localizes to the plasma membrane and is predominantly expressed in pollen. AtTPK4 (KCO4) resembles the electrical properties of a voltage-independent K+ channel after expression in Xenopus oocytes and yeast. Hyperpolarizing as well as depolarizing membrane voltages elicited instantaneous K+ currents, which were blocked by extracellular calcium and cytoplasmic protons. Functional complementation assays using a K+ transport-deficient yeast confirmed the biophysical and pharmacological properties of the AtTPK4 channel. The features of AtTPK4 point toward a role in potassium homeostasis and membrane voltage control of the growing pollen tube. Thus, AtTPK4 represents a member of plant tandem-pore- K+ channels, resembling the characteristics of its animal counterparts as well as plant-specific features with respect to modulation of channel activity by acidosis and calcium.

It is generally accepted that K+ uptake into guard cells via inward-rectifying K+ channels is required for stomatal opening. To test whether the guard cell K+ channel KAT1 is essential for stomatal opening, a knockout mutant, KAT1::En-1, was isolated from an En-1 mutagenized Arabidopsis thaliana population. Stomatal action and K+ uptake, however, were not impaired in KAT1-deficient plants. Reverse transcription-PCR experiments with isolated guard cell protoplasts showed that in addition to KAT1, the K+ channels AKT1, AKT2/3, AtKC1, and KAT2 were expressed in this cell type. In impalement measurements, intact guard cells exhibited inward-rectifying K+ currents across the plasma membrane of both wild-type and KAT1::En-1 plants. This study demonstrates that multiple K+ channel transcripts exist in guard cells and that KAT1 is not essential for stomatal action.

The use of a discontinuous single electrode voltage-clamp (dSEVC) offers an attractive alternative to the patch-clamp technique, since whole-cell measurements can be performed with a single sharp electrode. Comparison of current-voltage relations, however, revealed a weaker voltage dependence of channels measured with the dSEVC compared to patch clamp. The accuracy of the dSEVC was tested on Vicia faba guard cells impaled with double-barrelled electrodes. The actual clamp potential was measured independently of the dSEVC, at the second barrel. The weaker voltage dependence of ion channels appeared to be due to an overestimation of the clamp potential by the dSEVC. The deviation between the intended and actual clamp potential showed a linear relationship with the injected current; on average a 126 mV deviation was found for a clamp current of 1 nA. The deviation was probably caused by a slow settling capacity at the electrode, not compensated by the dSEVC amplifier. It is concluded that the dSEVC method in its current state is only suited for the study of small ion conductances in plant cells.

The use of a discontinuous single electrode voltage‐clamp (dSEVC) offers an attractive alternative to the patch‐clamp technique, since whole‐cell measurements can be performed with a single sharp electrode. Comparison of current–voltage relations, however, revealed a weaker voltage dependence of channels measured with the dSEVC compared to patch clamp. The accuracy of the dSEVC was tested on Vicia faba guard cells impaled with double‐barrelled electrodes. The actual clamp potential was measured independently of the dSEVC, at the second barrel. The weaker voltage dependence of ion channels appeared to be due to an overestimation of the clamp potential by the dSEVC. The deviation between the intended and actual clamp potential showed a linear relationship with the injected current; on average a 126 mV deviation was found for a clamp current of 1 nA. The deviation was probably caused by a slow settling capacity at the electrode, not compensated by the dSEVC amplifier. It is concluded that the dSEVC method in its current state is only suited for the study of small ion conductances in plant cells.