Uptake, allocation and signaling of nitrate

Plants need to acquire nitrogen (N) efficiently from the soil for growth. Nitrate is one of the major N sources for higher plants. Therefore, nitrate uptake and allocation are key factors in efficient N utilization. Membrane-bound transporters are required for nitrate uptake from the soil and for the inter- and intracellular movement of nitrate inside the plants. Four gene families, nitrate transporter 1/peptide transporter (NRT1/PTR), NRT2, chloride channel (CLC), and slow anion channel-associated 1 homolog 3 (SLAC1/SLAH), are involved in nitrate uptake, allocation, and storage in higher plants. Recent studies of these transporters or channels have provided new insights into the molecular mechanisms of nitrate uptake and allocation. Interestingly, several of these transporters also play versatile roles in nitrate sensing, plant development, pathogen defense, and/or stress response.     

Anion channel SLAH3 functions in nitrate‐dependent alleviation of ammonium toxicity in Arabidopsis

Slow anion channels (SLAC/SLAH) are efflux channels previously shown to be critical for stomatal regulation. However, detailed analysis using the β‐glucuronidase reporter gene showed that members of the SLAC/SLAH gene family are predominantly expressed in roots, in addition to stomatal guard cells, implicating distinct function(s) of SLAC/SLAH in the roots. Comprehensive mutant analyses of all slac/slah mutants indicated that slah3 plants showed a greater growth defect than wild‐type plants when ammonium was supplied as the sole nitrogen source. Ammonium toxicity was mimicked by acidic pH in nitrogen‐free external medium, suggesting that medium acidification by ammonium‐fed plants may underlie ammonium toxicity. Interestingly, such toxicity was more severe in slah3 mutants and, particularly in wild‐type plants, was alleviated by supplementing the media with micromolar levels of nitrate. These data thus provide evidence that SLAH3, a nitrate efflux channel, plays a role in nitrate‐dependent alleviation of ammonium toxicity in plants.     

An S-Type Anion Channel SLAC1 Is Involved in Cryptogein-Induced Ion Fluxes and Modulates Hypersensitive Responses in Tobacco BY-2 Cells

Pharmacological evidence suggests that anion channel-mediated plasma membrane anion effluxes are crucial in early defense signaling to induce immune responses and hypersensitive cell death in plants. However, their molecular bases and regulation remain largely unknown. We overexpressed Arabidopsis SLAC1, an S-type anion channel involved in stomatal closure, in cultured tobacco BY-2 cells and analyzed the effect on cryptogein-induced defense responses including fluxes of Cl⁻ and other ions, production of reactive oxygen species (ROS), gene expression and hypersensitive responses. The SLAC1-GFP fusion protein was localized at the plasma membrane in BY-2 cells. Overexpression of SLAC1 enhanced cryptogein-induced Cl⁻ efflux and extracellular alkalinization as well as rapid/transient and slow/prolonged phases of NADPH oxidase-mediated ROS production, which was suppressed by an anion channel inhibitor, DIDS. The overexpressor also showed enhanced sensitivity to cryptogein to induce downstream immune responses, including the induction of defense marker genes and the hypersensitive cell death. These results suggest that SLAC1 expressed in BY-2 cells mediates cryptogein-induced plasma membrane Cl⁻ efflux to positively modulate the elicitor-triggered activation of other ion fluxes, ROS as well as a wide range of defense signaling pathways. These findings shed light on the possible involvement of the SLAC/SLAH family anion channels in cryptogein signaling to trigger the plasma membrane ion channel cascade in the plant defense signal transduction network.     

Anion channels: master switches of stress responses

During stress, plant cells activate anion channels and trigger the release of anions across the plasma membrane. Recently, two new gene families have been identified that encode major groups of anion channels. The SLAC/SLAH channels are characterized by slow voltage-dependent activation (S-type), whereas ALMT genes encode rapid-activating channels (R-type). Both S- and R-type channels are stimulated in guard cells by the stress hormone ABA, which leads to stomatal closure. Besides their role in ABA-dependent stomatal movement, anion channels are also activated by biotic stress factors such as microbe-associated molecular patterns (MAMPs). Given that anion channels occur throughout the plant kingdom, they are likely to serve a general function as master switches of stress responses.     

Combined action of guard cell plasma membrane rapid- and slow-type anion channels in stomatal regulation

Initiation of stomatal closure by various stimuli requires activation of guard cell plasma membrane anion channels, which are defined as rapid (R)- and slow (S)-type. The single-gene loss-of-function mutants of these proteins are well characterized. However, the impact of suppressing both the S- and R-type channels has not been studied. Here, by generating and studying double and triple Arabidopsis thaliana mutants of SLOW ANION CHANNEL1 (SLAC1), SLAC1 HOMOLOG3 (SLAH3), and ALUMINUM-ACTIVATED MALATE TRANSPORTER 12/QUICK-ACTIVATING ANION CHANNEL 1 (QUAC1), we show that impairment of R- and S-type channels gradually increased whole-plant steady-state stomatal conductance. Ozone-induced cell death also increased gradually in higher-order mutants with the highest levels observed in the quac1 slac1 slah3 triple mutant. Strikingly, while single mutants retained stomatal responsiveness to abscisic acid, darkness, reduced air humidity, and elevated CO₂, the double mutant lacking SLAC1 and QUAC1 was nearly insensitive to these stimuli, indicating the need for coordinated activation of both R- and S-type anion channels in stomatal closure.

Combined impairment of guard cell slow and rapid anion channels results in increased stomatal conductance and complete stomatal insensitivity to abscisic acid, darkness, and elevated CO₂.     

Ion channels in plants: From bioelectricity,via signaling,to behavioral actions

In his recent opus magnum review paper published in the October issue of Physiology Reviews, Rainer Hedrich summarized the field of plant ion channels.¹ He started from the earliest electric recordings initiated by Charles Darwin of carnivorous Dionaea muscipula,^1,2 known as Venus flytrap, and covered the topic extensively up to the most recent discoveries on Shaker-type potassium channels, anion channels of SLAC/SLAH families, and ligand-activated channels of glutamate receptor-like type (GLR) and cyclic nucleotide-gated channels (CNGC).¹     

Open stomata 1 (OST1) kinase controls R–type anion channel QUAC1 in Arabidopsis guard cells

Under drought stress, the stress hormone ABA addresses the SnR kinase OST1 via its cytosolic receptor and the protein phosphatase ABI1. Upon activation, OST1 phosphorylates the guard cell S–type anion channel SLAC1. Arabidopsis ABI1 and OST1 loss‐of‐function mutants are characterized by an extreme wilting 'open stomata′ phenotype. Given the fact that guard cells express both SLAC‐ and R–/QUAC‐type anion channels, we questioned whether OST1, besides SLAC1, also controls the QUAC1 channel. In other words, are ABI1/OST1 defects preventing both of the guard cell anion channel types from operating properly in terms of stomatal closure? The activation of the R–/QUAC‐type anion channel by ABA signaling kinase OST1 and phosphatase ABI1 was analyzed in two experimental systems: Arabidopsis guard cells and the plant cell‐free background of Xenopus oocytes. Patch‐clamp studies on guard cells show that ABA activates R–/QUAC‐type currents of wild‐type plants, but to a much lesser extent in those of abi1–1 and ost1–2 mutants. In the oocyte system the co‐expression of QUAC1 and OST1 resulted in a pronounced activation of the R–type anion channel. These studies indicate that OST1 is addressing both S–/SLAC‐ and R–/QUAC‐type guard cell anion channels, and explain why the ost1–2 mutant is much more sensitive to drought than single slac1 or quac1 mutants.     

Anion Selectivity of Slow Anion Channels in the Plasma Membrane of Guard Cells (Large Nitrate Permeability)

Closing of stomatal pores in the leaf epidermis of higher plants is mediated by long-term release of potassium and the anions chloride and malate from guard cells and by parallel metabolism of malate. Previous studies have shown that slowly activating anion channels in the plasma membrane of guard cells can provide a major pathway for anion efflux while also controlling K+ efflux during stomatal closing: Anion efflux produces depolarization of the guard cell plasma membrane that drives K+ efflux required for stomatal closing. The patch-clamp technique was applied to Vicia faba guard cells to determine the permeability of physiologically significant anions and halides through slow anion channels to assess the contribution of these anion channels to anion efflux during stomatal closing. Permeability ratio measurements showed that all tested anions were permeable with the selectivity sequence relative to Cl- of NO3- > Br- > F- ~ Cl- ~ I- > malate. Large malate concentrations in the cytosol (150 mM) produced a slow down-regulation of slow anion channel currents. Single anion channel currents were recorded that correlated with whole-cell anion currents. Single slow anion channels confirmed the large permeability ratio for nitrate over chloride ions. Furthermore, single-channel studies support previous indications of multiple conductance states of slow anion channels, suggesting cooperativity among anion channels. Anion conductances showed that slow anion channels can mediate physiological rates of Cl- and initial malate efflux required for mediation of stomatal closure. The large NO3- permeability as well as the significant permeabilities of all anions tested indicates that slow anion channels do not discriminate strongly among anions. Furthermore, these data suggest that slow anion channels can provide an efficient pathway for efflux of physiologically important anions from guard cells and possibly also from other higher plant cells that express slow anion channels.     

Anion channels in higher plants: functional characterization, molecular structure and physiological role

Anion channels are well documented in various tissues, cell types and membranes of algae and higher plants, and current evidence supports their central role in cell signaling, osmoregulation, plant nutrition and metabolism. It is the aim of this review to illustrate through a few selected examples the variety of anion channels operating in plant cells and some of their regulation properties and unique physiological functions. In contrast, information on the molecular structure of plant anion channels has only recently started to emerge. Only a few genes coding for putative plant anion channels from the large chloride channel (CLC) family have been isolated, and current molecular data on these plant CLCs are presented and discussed. A major challenge remains to identify the genes encoding the various anion channels described so far in plant cells. Future prospects along this line are briefly outlined, as well as recent advances based on the use of knockout mutants in the model plant Arabidopsis thaliana to explore the physiological functions of anion channels in planta.     

Central functions of bicarbonate in S-type anion channel activation and OST1 protein kinase in CO2 signal transduction in guard cell

Plants respond to elevated CO(2) via carbonic anhydrases that mediate stomatal closing, but little is known about the early signalling mechanisms following the initial CO(2) response. It remains unclear whether CO(2), HCO(3)(-) or a combination activates downstream signalling. Here, we demonstrate that bicarbonate functions as a small-molecule activator of SLAC1 anion channels in guard cells. Elevated intracellular [HCO(3)(-)](i) with low [CO(2)] and [H(+)] activated S-type anion currents, whereas low [HCO(3)(-)](i) at high [CO(2)] and [H(+)] did not. Bicarbonate enhanced the intracellular Ca(2+) sensitivity of S-type anion channel activation in wild-type and ht1-2 kinase mutant guard cells. ht1-2 mutant guard cells exhibited enhanced bicarbonate sensitivity of S-type anion channel activation. The OST1 protein kinase has been reported not to affect CO(2) signalling. Unexpectedly, OST1 loss-of-function alleles showed strongly impaired CO(2)-induced stomatal closing and HCO(3)(-) activation of anion channels. Moreover, PYR/RCAR abscisic acid (ABA) receptor mutants slowed but did not abolish CO(2)/HCO(3)(-) signalling, redefining the convergence point of CO(2) and ABA signalling. A new working model of the sequence of CO(2) signalling events in gas exchange regulation is presented.     

Reconstitution of CO2 Regulation of SLAC1 Anion Channel and Function of CO2-Permeable PIP2;1 Aquaporin as CARBONIC ANHYDRASE4 Interactor

Dark respiration causes an increase in leaf CO₂ concentration (Ci), and the continuing increases in atmospheric [CO₂] further increases Ci. Elevated leaf CO₂ concentration causes stomatal pores to close. Here, we demonstrate that high intracellular CO₂/HCO₃⁻ enhances currents mediated by the Arabidopsis thaliana guard cell S-type anion channel SLAC1 upon coexpression of any one of the Arabidopsis protein kinases OST1, CPK6, or CPK23 in Xenopus laevis oocytes. Split-ubiquitin screening identified the PIP2;1 aquaporin as an interactor of the βCA4 carbonic anhydrase, which was confirmed in split luciferase, bimolecular fluorescence complementation, and coimmunoprecipitation experiments. PIP2;1 exhibited CO₂ permeability. Mutation of PIP2;1 in planta alone was insufficient to impair CO₂- and abscisic acid-induced stomatal closing, likely due to redundancy. Interestingly, coexpression of βCA4 and PIP2;1 with OST1-SLAC1 or CPK6/23-SLAC1 in oocytes enabled extracellular CO₂ enhancement of SLAC1 anion channel activity. An inactive PIP2;1 point mutation was identified that abrogated water and CO₂ permeability and extracellular CO₂ regulation of SLAC1 activity. These findings identify the CO₂-permeable PIP2;1 as key interactor of βCA4 and demonstrate functional reconstitution of extracellular CO₂ signaling to ion channel regulation upon coexpression of PIP2;1, βCA4, SLAC1, and protein kinases. These data further implicate SLAC1 as a bicarbonate-responsive protein contributing to CO₂ regulation of S-type anion channels.     

AtALMT12 represents an R‐type anion channel required for stomatal movement in Arabidopsis guard cells

Stomatal 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 CO₂‐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 voltage‐dependent 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 SLAC1 and GORK, respectively.     

R type anion channel: A multifunctional channel seeking its molecular identity

Plant genomes code for channels involved in the transport of cations, anions and uncharged molecules through membranes. Although the molecular identity of channels for cations and uncharged molecules has progressed rapidly in the recent years, the molecular identity of anion channels has lagged behind. Electrophysiological studies have identified S-type (slow) and R-type (rapid) anion channels. In this brief review, we summarize the proposed functions of the R-type anion channels which, like the S-type, were first characterized by electrophysiology over 20 years ago, but unlike the S-type, have still yet to be cloned. We show that the R-type channel can play multiple roles.Key words: R-type anion channel, nitrate, sulphate, guard cell, action potentialAnion channels play a central role in signal transduction, nutrient transport and cell turgor regulation.¹ By far, their function was particularly well investigated in the guard cells of stomata using a combination of electrophysiological, pharmacological and genetic tools. In this system, anion channel activation was shown to be one of the limiting steps in the loss of cell turgor leading to stomatal closure.² In algal cells, anion channels were shown to contribute to membrane excitability through the generation of action potential.^1,3With the burst of molecular biology in the nineties, the genes coding for plant ion channels started to be unveiled. The first channel gene to be cloned in plant was the shaker-like potassium channel identified in a yeast functional expression screen.^4,5 More than ten years later, TaALMT1 and AtCLCa were characterized as the first members of two important anion channel families.^6,7 This growing group of newly identified channels, accounting for electrophysiological activity described long ago, includes the MSLs anion selective mechanosensitive channels.⁸ Recently, the well known S-type channel has been finally recognized to be encoded by members of the SLAC1 (and other SLAH) family (Slow Anion Channel-Associated 1).⁹ In agreement with electrophysiological data,^10–13 it requires phosphorylation by a Protein Kinase in order to be functional.^14,15 In contrast, the molecular identity of the R-type anion channel remains unknown. Therefore, this candidate, which has been functionally known since twenty years, remains the next challenge for plant channel physiologists.     

Functional reconstitution of a chloride channel protein from bovine trachea.

We characterized the electrophysiological properties of a chloride channel protein isolated from bovine trachea after incorporation into planar lipid bilayers, and studied the effects of thiol-modulating agents on channel regulation both in bilayers and vesicular iodide uptake studies. Our experiments showed that this protein formed perfectly anion-selective channels in the bilayer, with an anion permeability sequence of I- (2.1) > NO3- (1.7) > Br- (1.2) > Cl- (1.0). The conductance of this channel was 25-30 picosiemens in 150 mM Cl-, and saturated with increasing chloride concentration. This channel could be completely inhibited by 4,4'-bis(isothiocyano)-2,2'-stilbenedisulfonate. Immunoblot analysis, using polyclonal antibodies (anti-p38), revealed one major band at 140 kDa. Upon reduction with dithiothreitol, 64- and 38-kDa polypeptides were observed. Functional experiments showed that reduction was accompanied by loss of 125I- uptake and single-channel activity. In the presence of dithiothreitol, only the low molecular mass protein forms (64 and 38 kDa) were detected by anti-p38 antibodies on Western blots. Cross-linking of S-S bonds with Cu(2+)-o-phenanthroline led to activation of chloride channels in vesicles and bilayers. Over-aggregation of chloride channels by this S-S cross-linking reagent caused inhibition of 125I- uptake by 80-100% and the abolishment of single-channel activity. We propose that the native chloride channel from bovine trachea can exist in vivo in different structural and functional forms depending upon its thiol-disulfide oxidation reduction status. The oxidized form has a molecular mass of 140 kDa and represents a fully active chloride channel. Inactivation of this channel might occur by over-aggregation of protein subunits, or by dissociation of the 140-kDa subunit by disulfide bond reduction.     

Nitrate transporter NRT1.1 and anion channel SLAH3 form a functional unit to regulate nitrate-dependent alleviation of ammonium toxicity

Ammonium (NH₄⁺) and nitrate (NO₃⁻) are major inorganic nitrogen (N) sources for plants. When serving as the sole or dominant N supply, NH₄⁺ often causes root inhibition and shoot chlorosis in plants, known as ammonium toxicity. NO₃⁻ usually causes no toxicity and can mitigate ammonium toxicity even at low concentrations, referred to as nitrate-dependent alleviation of ammonium toxicity. Our previous studies indicated a NO₃⁻ efflux channel SLAH3 is involved in this process. However, whether additional components contribute to NO₃⁻-mediated NH₄⁺ detoxification is unknown. Previously, mutations in NO₃⁻ transporter NRT1.1 were shown to cause enhanced resistance to high concentrations of NH₄⁺. Whereas, in this study, we found when the high-NH₄⁺ medium was supplemented with low concentrations of NO₃⁻, nrt1.1 mutant plants showed hyper-sensitive phenotype instead. Furthermore, mutation in NRT1.1 caused enhanced medium acidification under high-NH₄⁺/low-NO₃⁻ condition, suggesting NRT1.1 regulates ammonium toxicity by facilitating H⁺ uptake. Moreover, NRT1.1 was shown to interact with SLAH3 to form a transporter-channel complex. Interestingly, SLAH3 appeared to affect NO₃⁻ influx while NRT1.1 influenced NO₃⁻ efflux, suggesting NRT1.1 and SLAH3 regulate each other at protein and/or gene expression levels. Our study thus revealed NRT1.1 and SLAH3 form a functional unit to regulate nitrate-dependent alleviation of ammonium toxicity through regulating NO₃⁻ transport and balancing rhizosphere acidification.     

Ozone‐triggered rapid stomatal response involves the production of reactive oxygen species,and is controlled by SLAC1 and OST1

The air pollutant ozone can be used as a tool to unravel in planta processes induced by reactive oxygen species (ROS). Here, we have utilized ozone to study ROS‐dependent stomatal signaling. We show that the ozone‐triggered rapid transient decrease (RTD) in stomatal conductance coincided with a burst of ROS in guard cells. RTD was present in 11 different Arabidopsis ecotypes, suggesting that it is a genetically robust response. To study which signaling components or ion channels were involved in RTD, we tested 44 mutants deficient in various aspects of stomatal function. This revealed that the SLAC1 protein, essential for guard cell plasma membrane S‐type anion channel function, and the protein kinase OST1 were required for the ROS‐induced fast stomatal closure. We showed a physical interaction between OST1 and SLAC1, and provide evidence that SLAC1 is phosphorylated by OST1. Phosphoproteomic experiments indicated that OST1 phosphorylated multiple amino acids in the N terminus of SLAC1. Using TILLING we identified three new slac1 alleles where predicted phosphosites were mutated. The lack of RTD in two of them, slac1‐7 (S120F) and slac1‐8 (S146F), suggested that these serine residues were important for the activation of SLAC1. Mass‐spectrometry analysis combined with site‐directed mutagenesis and phosphorylation assays, however, showed that only S120 was a specific phosphorylation site for OST1. The absence of the RTD in the dominant‐negative mutants abi1‐1 and abi2‐1 also suggested a regulatory role for the protein phosphatases ABI1 and ABI2 in the ROS‐induced activation of the S‐type anion channel.     

Identification and modulation of a voltage-dependent anion channel in the plasma membrane of guard cells by high-affinity ligands.

Guard cell anion channels (GCAC1) catalyze the release of anions across the plasma membrane during regulated volume decrease and also seem to be involved in the targeting of the plant growth hormones auxins. We have analyzed the modulation and inhibition of these voltage-dependent anion channels by different anion channel blockers. Ethacrynic acid, a structural correlate of an auxin, caused a shift in activation potential and simultaneously a transient increase in the peak current amplitude, whereas other blockers shifted and blocked the voltage-dependent activity of the channel. Comparison of dose-response curves for shift and block imposed by the inhibitor, indicate two different sites within the channel which interact with the ligand. The capability to inhibit GCAC1 increases in a dose-dependent manner in the sequence: probenecid less than A-9-C less than ethacrynic acid less than niflumic acid less than IAA-94 less than NPPB. All inhibitors reversibly blocked the anion channel from the extracellular side. Channel block on the level of single anion channels is characterized by a reduction of long open transitions into flickering bursts, indicating an interaction with the open mouth of the channel. IAA-23, a structural analog of IAA-94, was used to enrich ligand-binding polypeptides from the plasma membrane of guard cells by IAA-23 affinity chromatography. From this protein fraction a 60 kDa polypeptide crossreacted specifically with polyclonal antibodies raised against anion channels isolated from kidney membranes. In contrast to guard cells, mesophyll plasma membranes were deficient in voltage-dependent anion channels and lacked crossreactivity with the antibody.     

Plasma membrane anion channels in higher plants and their putative functions in roots

Recent years have seen considerable progress in identifying anion channel activities in higher plant cells. This review outlines the functional properties of plasma membrane anion channels in plant cells and discusses their likely roles in root function. Plant anion channels can be grouped according to their voltage dependence and kinetics: (1) depolarization-activated anion channels which mediate either anion efflux (R and S types) or anion influx (outwardly rectifying type); (2) hyperpolarization-activated anion channels which mediate anion efflux, and (3) anion channels activated by light or membrane stretch. These types of anion channel are apparent in root cells where they may function in anion homeostasis, membrane stabilization, osmoregulation, boron tolerance and regulation of passive salt loading into the xylem vessels. In addition, roots possess anion channels exhibiting unique properties which are consistent with them having specialized functions in root physiology. Most notable are the organic anion selective channels, which are regulated by extracellular Al3+ or the phosphate status of the plant. Finally, although the molecular identities of plant anion channels remain elusive, the diverse electrophysiological properties of plant anion channels suggest that large and diverse multigene families probably encode these channels.