Here we report on the molecular identification, guard cell expression and functional characterization of AtGORK, an Arabidopsis thaliana guard cell outward rectifying K + channel. GORK represents a new member of the plant Shaker K + channel superfamily. When heterologously expressed in Xenopus oocytes the gene product of GORK mediated depolarization-activated K + currents. In agreement with the delayed outward rectifier in intact guard cells and protoplasts thereof, GORK is activated in a voltage-and potassium-dependent manner. Furthermore, the single channel conductance and regulation of GORK in response to pH changes resembles the biophysical properties of the guard cell delayed outward rectifier. Thus GORK very likely represents the molecular entity for depolarization-induced potassium release from guard cells. ß 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

It is generally accepted that K ؉ uptake into guard cells via inwardrectifying 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, AKT2͞3, AtKC1, and KAT2 were expressed in this cell type. In impalement measurements, intact guard cells exhibited inwardrectifying K ؉ currents across the plasma membrane of both wildtype 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.

Auxin is a key regulator of plant growth and development, but the causal relationship between hormone transport and root responses remains unresolved. Here we describe auxin uptake, together with early steps in signaling, in Arabidopsis root hairs. Using intracellular microelectrodes we show membrane depolarization, in response to IAA in a concentration- and pH-dependent manner. This depolarization is strongly impaired in aux1 mutants, indicating that AUX1 is the major transporter for auxin uptake in root hairs. Local intracellular auxin application triggers Ca2+ signals that propagate as long-distance waves between root cells and modulate their auxin responses. AUX1-mediated IAA transport, as well as IAA- triggered calcium signals, are blocked by treatment with the SCFTIR1/AFB - inhibitor auxinole. Further, they are strongly reduced in the tir1afb2afb3 and the cngc14 mutant. Our study reveals that the AUX1 transporter, the SCFTIR1/AFB receptor and the CNGC14 Ca2+ channel, mediate fast auxin signaling in roots.

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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 Ϸ2 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 hyperpolarizationactivated calcium channels. Likewise, ABA stimulation did not induce a rise in the cytosolic free-calcium concentration. However, the presence of Ϸ100 nM background Ca 2؉ was required for anion channel function, because the action of ABA on anion channels was repressed after loading of the Ca 2؉ 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 patchclamp 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 M 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. stomatal closure T he plant hormone abscisic acid (ABA) provides a developmental signal serving as a chemical switch between, for instance, dormancy and growth of seeds and buds (1). Furthermore, this sesquiterpene is involved in the transmission of environmental changes like drought, saline, and cold periods into stress-adaptation processes (2). Based on the time scale of the individual ABA responses, they can be subdivided into fast (seconds up to minutes) and slow (hours up to days, or even months) signaling processes. Stomatal closure represents the fastest ABA response known so far, characterized by a half-time of Ϸ5 min, and presumably does not involve gene activation. Fast stomatal closure is accomplished by the release of potassium and chloride via voltage-dependent ion channels and by metabolic degradation of the major organic anion malate (3, 4).In search for ABA signaling intermediates, guard cells have been challenged with well characterized modulators operating in signal transduction pathways of animal cells (5-8). To a large extent, the respective substances were injected into guard cells of excised epidermal peels, and the corresponding responses were monitored (9-12). After microinjection, photolysis of caged inositol 1,4,...

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 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 domains͞one 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.

SummaryThe putative two-pore Ca 2+ channel TPC1 has been suggested to be involved in responses to abiotic and biotic stresses. We show that AtTPC1 co-localizes with the K + -selective channel AtTPK1 in the vacuolar membrane. Loss of AtTPC1 abolished Ca 2+-activated slow vacuolar (SV) currents, which were increased in AtTPC1-overexpressing Arabidopsis compared to the wild-type. A Ca 2+ -insensitive vacuolar cation channel, as yet uncharacterized, could be resolved in tpc1-2 knockout plants. The kinetics of ABA-and CO 2 -induced stomatal closure were similar in wild-type and tpc1-2 knockout plants, excluding a role of SV channels in guard-cell signalling in response to these physiological stimuli. ABA-, K + -, and Ca 2+-dependent root growth phenotypes were not changed in tpc1-2 compared to wild-type plants. Given the permeability of SV channels to mono-and divalent cations, the question arises as to whether TPC1 in vivo represents a pathway for Ca 2+ entry into the cytosol. Ca 2+ responses as measured in aequorin-expressing wild-type, tpc1-2 knockout and TPC1-overexpressing plants disprove a contribution of TPC1 to any of the stimulus-induced Ca 2+ signals tested, including abiotic stresses (cold, hyperosmotic, salt and oxidative), elevation in extracellular Ca 2+ concentration and biotic factors (elf18, flg22). In good agreement, stimulus-and Ca 2+ -dependent gene activation was not affected by alterations in TPC1 expression. Together with our finding that the loss of TPC1 did not change the activity of hyperpolarization-activated Ca 2+-permeable channels in the plasma membrane, we conclude that TPC1, under physiological conditions, functions as a vacuolar cation channel without a major impact on cytosolic Ca 2+ homeostasis.