In response to drought stress the phytohormone ABA (abscisic acid) induces stomatal closure and, therein, activates guard cell anion channels in a calcium-dependent as well as-independent manner. Two key components of the ABA signaling pathway are the protein kinase OST1 (open stomata 1) and the protein phosphatase ABI1 (ABA insensitive 1). The recently identified guard cell anion channel SLAC1 appeared to be the key ion channel in this signaling pathway but remained electrically silent when expressed heterologously. Using split YFP assays, we identified OST1 as an interaction partner of SLAC1 and ABI1. Upon coexpression of SLAC1 with OST1 in Xenopus oocytes, SLAC1-related anion currents appeared similar to those observed in guard cells. Integration of ABI1 into the SLAC1/OST1 complex, however, prevented SLAC1 activation. Our studies demonstrate that SLAC1 represents the slow, deactivating, weak voltage-dependent anion channel of guard cells controlled by phosphorylation/dephosphorylation.ABA signaling ͉ S-type anion channel ͉ OST1/ABI1

In response to drought stress, the phytohormone abscisic acid (ABA) induces stomatal closure. Thereby the stress hormone activates guard cell anion channels in a calcium-dependent, as well as -independent, manner. Open stomata 1 protein kinase (OST1) and ABI1 protein phosphatase (ABA insensitive 1) represent key components of calcium-independent ABA signaling. Recently, the guard cell anion channel SLAC1 was identified. When expressed heterologously SLAC1 remained electrically silent. Upon coexpression with Ca 2+ -independent OST1, however, SLAC1 anion channels appear activated in an ABI1-dependent manner. Mutants lacking distinct calcium-dependent protein kinases (CPKs) appeared impaired in ABA stimulation of guard cell ion channels, too. To study SLAC1 activation via the calcium-dependent ABA pathway, we studied the SLAC1 response to CPKs in the Xenopus laevis oocyte system. Split YFP-based protein-protein interaction assays, using SLAC1 as the bait, identified guard cell expressed CPK21 and 23 as major interacting partners. Upon coexpression of SLAC1 with CPK21 and 23, anion currents document SLAC1 stimulation by these guard cell protein kinases. Ca 2+ -sensitive activation of SLAC1, however, could be assigned to the CPK21 pathway only because CPK23 turned out to be rather Ca 2+ -insensitive. In line with activation by OST1, CPK activation of the guard cell anion channel was suppressed by ABI1. Thus the CPK and OST1 branch of ABA signal transduction in guard cells seem to converge on the level of SLAC1 under the control of the ABI1/ABA-receptor complex.abscisic acid signaling | drought stress | guard cell | S-type anion channel T he drought hormone abscisic acid (ABA) triggers release of K + and anions from guard cells and thereby causes stomatal closure (1, 2). Recently, SLAC1, a guard cell anion channel, was identified (3-5). In guard cells of these ABA-and CO 2 /O 3 -insensitive mutant plants, anion currents appeared largely suppressed. When SLAC1 was expressed with the open stomata 1 protein kinase (OST1) in Xenopus oocytes, SLAC1-related anion currents, similar to those observed in guard cells, appeared (6). The presence of ABI1, however, prevented SLAC1 activation. This ABA pathway resembles the Ca 2+ -independent activation of SLAC-type anion currents in guard cells. ABA signal transduction, however, has been shown to activate guard cell anion channels in a calcium-independent as well as -dependent manner (7-10). This became evident in abi1-1 mutant plants, where anion channels do not respond to ABA anymore (11) but still activate with calcium (12). Furthermore the described mutant growth controlled by abscisic acid (gca2) (13-14), isolated from the Arabidopsis ecotype Landsberg erecta, was shown to be impaired in ABA-induced stomatal closure in a Ca 2+ -dependent manner. Moreover [Ca 2+ ] cyt elevation was shown to result in activation of S-type anion channels via phosphorylation (12, 15), suggesting a role of phosphorylation events in [Ca 2+ ] cyt signaling.CDPKs resemble Ca 2+ -dependent Ser/Thr pr...

S-type anion channels are direct targets of abscisic acid (ABA) signaling and contribute to chloride and nitrate release from guard cells, which in turn initiates stomatal closure. SLAC1 was the first component of the guard cell S-type anion channel identified. However, we found that guard cells of Arabidopsis SLAC1 mutants exhibited nitrate conductance. SLAH3 (SLAC1 homolog 3) was also present in guard cells, and coexpression of SLAH3 with the calcium ion (Ca2+)-dependent kinase CPK21 in Xenopus oocytes mediated nitrate-induced anion currents. Nitrate, calcium, and phosphorylation regulated SLAH3 activity. CPK21-dependent SLAH3 phosphorylation and activation were blocked by ABI1, a PP2C-type protein phosphatase that is inhibited by ABA and inhibits the ABA signaling pathway in guard cells. We reconstituted the ABA-stimulated phosphorylation of the SLAH3 amino-terminal domain by CPK21 in vitro by including the ABA receptor-phosphatase complex RCAR1-ABI1 in the reactions. We propose that ABA perception by the complex consisting of ABA receptors of the RCAR/PYR/PYL family and ABI1 releases CPK21 from inhibition by ABI1, and then CPK21 is further activated by an increase in the cytosolic Ca2+ concentration, leading to its phosphorylation of SLAH3. Thus, the identification of SLAH3 as the nitrate-, calcium-, and ABA-sensitive guard cell anion channel provides insights into the relationship among stomatal response to drought, signaling by nitrate, and nitrate metabolism.

SUMMARYStomatal pores formed by a pair of guard cells in the leaf epidermis control gas exchange and transpirational water loss. Stomatal closure is mediated by the release of potassium and anions from guard cells. Anion efflux from guard cells involves slow (S-type) and rapid (R-type) anion channels. Recently the SLAC1 gene has been shown to encode the slow, voltage-independent anion channel component in guard cells. In contrast, the R-type channel still awaits identification. Here, we show that AtALMT12, a member of the aluminum activated malate transporter family in Arabidopsis, represents a guard cell R-type anion channel. AtALMT12 is highly expressed in guard cells and is targeted to the plasma membrane. Plants lacking AtALMT12 are impaired in dark-and CO 2 -induced stomatal closure, as well as in response to the drought-stress hormone abscisic acid. Patch-clamp studies on guard cell protoplasts isolated from atalmt12 mutants revealed reduced R-type currents compared with wild-type plants when malate is present in the bath media. Following expression of AtALMT12 in Xenopus oocytes, voltage-dependent anion currents reminiscent to R-type channels could be activated. In line with the features of the R-type channel, the activity of heterologously expressed AtALMT12 depends on extracellular malate. Thereby this key metabolite and osmolite of guard cells shifts the threshold for voltage activation of AtALMT12 towards more hyperpolarized potentials. R-Type channels, like voltagedependent cation channels in nerve cells, are capable of transiently depolarizing guard cells, and thus could trigger membrane potential oscillations, action potentials and initiate long-term anion and K + efflux via SLAC1and GORK, respectively.

The productivity of higher plants as a major source of food and energy is linked to their ability to buffer changes in the concentrations of essential and toxic ions. Transport across the tonoplast is energized by two proton pumps, the vacuolar H + -ATPase (VATPase) and the vacuolar H + -pyrophosphatase (V-PPase); however, their functional relation and relative contributions to ion storage and detoxification are unclear. We have identified an Arabidopsis mutant in which energization of vacuolar transport solely relies on the activity of the V-PPase. The vha-a2 vha-a3 double mutant, which lacks the two tonoplast-localized isoforms of the membrane-integral V-ATPase subunit VHA-a, is viable but shows day-length-dependent growth retardation. Nitrate content is reduced whereas nitrate assimilation is increased in the vha-a2 vha-a3 mutant, indicating that vacuolar nitrate storage represents a major growth-limiting factor. Zinc is an essential micronutrient that is toxic at excess concentrations and is detoxified via a vacuolar Zn 2+ /H + -antiport system. Accordingly, the double mutant shows reduced zinc tolerance. In the same way the vacuolar Na + /H + -antiport system is assumed to be an important component of the system that removes sodium from the cytosol. Unexpectedly, salt tolerance and accumulation are not affected in the vhaa2 vha-a3 double mutant. In contrast, reduction of V-ATPase activity in the trans-Golgi network/early endosome (TGN/EE) leads to increased salt sensitivity. Taken together, our results show that during gametophyte and embryo development V-PPase activity at the tonoplast is sufficient whereas tonoplast V-ATPase activity is limiting for nutrient storage but not for sodium tolerance during vegetative and reproductive growth.proton-pump | pH-homeostasis | vacuole | nitrate | salt tolerance T he productivity of higher plants as a major source of food and renewable energy is linked to their ability to cope with fluctuations in essential as well as toxic ion and metabolite concentrations. In plants the large central vacuoles function as reservoirs for ions and metabolites that allow buffering of changes in nutrients as well as challenges by toxic components that plants, as sessile, photoautotrophic organisms, frequently encounter. Furthermore, vacuoles are essential for plant growth and development. Expansion of plant cells is achieved by osmotically driven water influx into the vacuole that, in combination with the cell wall, generates turgor, the driving force for hydraulic stiffness and plant growth.All vacuolar functions require massive fluxes of ions and metabolites that are channeled by a battery of vacuolar transport proteins, many of which have been well characterized physiologically, but the nature of several transporters remains to be identified (1). Among those characterized on the molecular level are, e.g., those transporting NO 3 − , Na + , and protons. Nitrate, a major plant nutrient, is accumulated and stored in the vacuole from where it can be retrieved according to metabolic deman...