TPK1, a Ca(2+)-regulated Arabidopsis vacuole two-pore K(+) channel is activated by 14-3-3 proteins

The vacuole represents a pivotal plant organelle for management of ion homeostasis, storage of proteins and solutes, as well as deposition of cytotoxic compounds. Ion channels, pumps and carriers in the vacuolar membrane under control of cytosolic factors provide for ionic and metabolic homeostasis between this storage organelle and the cytoplasm. Here we show that AtTPK1 (KCO1), a vacuolar membrane localized K(+) channel of the TPK family, interacts with 14-3-3 proteins (general regulating factors, GRFs). Following in planta expression TPK1 and GRF6 co-localize at the vacuolar membrane. Co-localization of wild-type TPK1, but not the TPK1-S42A mutant, indicates that phosphorylation of the 14-3-3 binding motif of TPK1 represents a prerequisite for interaction. Pull-down assays and surface plasmon resonance measurements revealed GRF6 high-affinity interaction with TPK1. Following expression of TPK1 in yeast and isolation of vacuoles, patch-clamp studies identified TPK1 as a voltage-independent and Ca(2+)-activated K(+) channel. Addition of 14-3-3 proteins strongly increased the TPK1 activity in a dose-dependent manner. However, an inverse effect of GRF6 on the activity of the slow-activating vacuolar (SV) channel was observed in mesophyll vacuoles from Arabidopsis thaliana. Thus, TPK1 seems to provide for a Ca(2+)- and 14-3-3-sensitive mechanism capable of controlling cytoplasmic potassium homeostasis in plants.     

The fou2 mutation in the major vacuolar cation channel TPC1 confers tolerance to inhibitory luminal calcium

The SV channel encoded by the TPC1 gene represents a Ca²⁺- and voltage-dependent vacuolar cation channel. Point mutation D454N within TPC1 , named fou2 for fatty acid oxygenation upregulated 2 , results in increased synthesis of the stress hormone jasmonate. As wounding causes Ca²⁺ signals and cytosolic Ca²⁺ is required for SV channel function, we here studied the Ca²⁺-dependent properties of this major vacuolar cation channel with Arabidopsis thaliana mesophyll vacuoles. In patch clamp measurements, wild-type and fou2 SV channels did not exhibit differences in cytosolic Ca²⁺ sensitivity and Ca²⁺ impermeability. K⁺ fluxes through wild-type TPC1 were reduced or even completely faded away when vacuolar Ca²⁺ reached the 0.1-m m level. The fou2 protein under these conditions, however, remained active. Thus, D454N seems to be part of a luminal Ca²⁺ recognition site. Thereby the SV channel mutant gains tolerance towards elevated luminal Ca²⁺. A three-fold higher vacuolar Ca/K ratio in the fou2 mutant relative to wild-type plants seems to indicate that fou2 can accumulate higher levels of vacuolar Ca²⁺ before SV channel activity vanishes and K⁺ homeostasis is impaired. In response to wounding fou2 plants might thus elicit strong vacuole-derived cytosolic Ca²⁺ signals resulting in overproduction of jasmonate.     

TPK1 is a vacuolar ion channel different from the slow-vacuolar cation channel

TPK1 (formerly KCO1) is the founding member of the family of two-pore domain K(+) channels in Arabidopsis (Arabidopsis thaliana), which originally was described following expression in Sf9 insect cells as a Ca(2+)- and voltage-dependent outwardly rectifying plasma membrane K(+) channel. In plants, this channel has been shown by green fluorescent protein fusion to localize to the vacuolar membrane, which led to speculations that the TPK1 gene product would be a component of the nonselective, Ca(2+) and voltage-dependent slow-vacuolar (SV) cation channel found in many plants species. Using yeast (Saccharomyces cerevisiae) as an expression system for TPK1, we show functional expression of the channel in the vacuolar membrane. In isolated vacuoles of yeast yvc1 disruption mutants, the TPK1 gene product shows ion channel activity with some characteristics very similar to the SV-type channel. The open channel conductance of TPK1 in symmetrically 100 mM KCl is slightly asymmetric with roughly 40 pS at positive membrane voltages and 75 pS at negative voltages. Similar to the SV-type channel, TPK1 is activated by cytosolic Ca(2+), requiring micromolar concentration for activation. However, in contrast to the SV-type channel, TPK1 exhibits strong selectivity for K(+) over Na(+), and its activity turned out to be independent of the membrane voltage over the range of +/-80 mV. Our data clearly demonstrate that TPK1 is a voltage-independent, Ca(2+)-activated, K(+)-selective ion channel in the vacuolar membrane that does not mediate SV-type ionic currents.     

Loss of the vacuolar cation channel, AtTPC1, does not impair Ca2+ signals induced by abiotic and biotic stresses.

The 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-over-expressing 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-over-expressing 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.     

Identification of a vacuolar sucrose transporter in barley and Arabidopsis mesophyll cells by a tonoplast proteomic approach

The vacuole is the main cellular storage pool, where sucrose (Suc) accumulates to high concentrations. While a limited number of vacuolar membrane proteins, such as V-type H(+)-ATPases and H(+)-pyrophosphatases, are well characterized, the majority of vacuolar transporters are still unidentified, among them the transporter(s) responsible for vacuolar Suc uptake and release. In search of novel tonoplast transporters, we used a proteomic approach, analyzing the tonoplast fraction of highly purified mesophyll vacuoles of the crop plant barley (Hordeum vulgare). We identified 101 proteins, including 88 vacuolar and putative vacuolar proteins. The Suc transporter (SUT) HvSUT2 was discovered among the 40 vacuolar proteins, which were previously not reported in Arabidopsis (Arabidopsis thaliana) vacuolar proteomic studies. To confirm the tonoplast localization of this Suc transporter, we constructed and expressed green fluorescent protein (GFP) fusion proteins with HvSUT2 and its closest Arabidopsis homolog, AtSUT4. Transient expression of HvSUT2-GFP and AtSUT4-GFP in Arabidopsis leaves and onion (Allium cepa) epidermal cells resulted in green fluorescence at the tonoplast, indicating that these Suc transporters are indeed located at the vacuolar membrane. Using a microcapillary, we selected mesophyll protoplasts from a leaf protoplast preparation and demonstrated unequivocally that, in contrast to the companion cell-specific AtSUC2, HvSUT2 and AtSUT4 are expressed in mesophyll protoplasts, suggesting that HvSUT2 and AtSUT4 are involved in transport and vacuolar storage of photosynthetically derived Suc.     

K+ currents through SV-type vacuolar channels are sensitive to elevated luminal sodium levels

Non-selective slow vacuolar (SV) channels mediate uptake of K+ and Na+ into vacuolar compartment. Under salt stress plant cells accumulate Na+ in the vacuole and release vacuolar K+ into the cytoplasm. It is, however, unclear how plants mediate transport of K+ from the vacuole without concomitant efflux of toxic Na+. Here we show by patch-clamp studies on isolated Arabidopsis thaliana cell culture vacuoles that SV channels do not mediate Na+ release from the vacuole as luminal Na+ blocks this channel. Gating of the SV channel is dependent on the K+ gradient across the vacuolar membrane. Under symmetrical K+ concentrations on both sides of the vacuolar membrane, SV channels mediate potassium uptake. When cytoplasmic K+ decreases, SV channels allow K+ release from the vacuole. In contrast to potassium, Na+ can be taken up by SV channels, but not released even in the presence of a 150-fold gradient (lumen to cytoplasm). Accumulation of Na+ in the vacuole shifts the activation potential of SV channels to more positive voltages and prevents gradient-driven efflux of K+. Similar to sodium, under physiological conditions, vacuolar Ca2+ is not released from vacuoles via SV channels. We suggest that a major Arabidopsis SV channel is equipped with a positively charged intrinsic gate located at the luminal side, which prevents release of Na+ and Ca2+, but permits efflux of K+. This property of the SV channel guarantees that K+ can shuttle across the vacuolar membrane while maintaining Na+ and Ca2+ stored in this organelle.     

Molecular identification and physiological characterization of a novel monosaccharide transporter from Arabidopsis involved in vacuolar sugar transport

The tonoplast monosaccharide transporter (TMT) family comprises three isoforms in Arabidopsis thaliana, and TMT-green fluorescent protein fusion proteins are targeted to the vacuolar membrane. TMT promoter-beta-glucuronidase plants revealed that the TONOPLAST MONOSACCHARIDE TRANSPORTER1 (TMT1) and TMT2 genes exhibit a tissue- and cell type-specific expression pattern, whereas TMT3 is only weakly expressed. TMT1 and TMT2 expression is induced by drought, salt, and cold treatments and by sugar. During cold adaptation, tmt knockout lines accumulated less glucose and fructose compared with wild-type plants, whereas no differences were observed for sucrose. Cold adaptation of wild-type plants substantially promoted glucose uptake into isolated leaf mesophyll vacuoles. Glucose uptake into isolated vacuoles was inhibited by NH(4)(+), fructose, and phlorizin, indicating that transport is energy-dependent and that both glucose and fructose were taken up by the same carrier. Glucose import into vacuoles from two cold-induced tmt1 knockout lines or from triple knockout plants was substantially lower than into corresponding wild-type vacuoles. Monosaccharide feeding into leaf discs revealed the strongest response to sugar in tmt1 knockout lines compared with wild-type plants, suggesting that TMT1 is required for cytosolic glucose homeostasis. Our results indicate that TMT1 is involved in vacuolar monosaccharide transport and plays a major role during stress responses.     

Members of the Arabidopsis AtTPK/KCO family form homomeric vacuolar channels in planta

The Arabidopsis thaliana K⁺ channel family of AtTPK/KCO proteins consists of six members including a 'single-pore' (K_ir-type) and five 'tandem-pore' channels. AtTPK4 is currently the only ion channel of this family for which a function has been demonstrated in planta . The protein is located at the plasma membrane forming a voltage-independent K⁺ channel that is blocked by extracellular calcium ions. In contrast, AtTPK1 is a tonoplast-localized protein, that establishes a K⁺-selective, voltage-independent ion channel activated by cytosolic calcium when expressed in a heterologous system, i.e. yeast. Here, we provide evidence that other AtTPK/KCO channel subunits, i.e. AtTPK2, AtTPK3, AtTPK5 and AtKCO3, are also targeted to the vacuolar membrane, opening the possibility that they interact at the target membrane to form heteromeric ion channels. However, when testing the cellular expression patterns of AtTPK / KCO genes we observed distinct expression domains that overlap in only a few tissues of the Arabidopsis plant, making it unlikely that different channel subunits interact to form heteromeric channels. This conclusion was substantiated by in planta expression of combinations of selected tonoplast AtTPK/KCO proteins. Fluorescence resonance energy transfer assays indicate that protein interaction occurs between identical channel subunits (most efficiently between AtTPK1 or AtKCO3) but not between different channel subunits. The finding could be confirmed by bimolecular fluorescence complementation assays. We conclude that tonoplast-located AtTPK/KCO subunits form homomeric ion channels in vivo .     

Vacuolar calcium channels

The central vacuole is the largest Ca2+ store in a mature plant cell. Ca2+ release from this store contributes to Ca2+-mediated intracellular signalling in a variety of physiological responses. However, the routes for vacuolar Ca2+ release are not well characterized. To date, at least two voltage-dependent and two ligand-gated Ca2+-permeable channels have been reported in plant vacuoles. However, the so-called VVCa (vacuolar voltage-gated Ca2+) channel most probably is not a separate channel but is identical to another voltage-dependent channel-the so-called SV (slow vacuolar) channel. Studies in the last few years have added a new dimension to our knowledge of SV channel-mediated ion transport and the mechanisms of its regulation by multiple natural factors. Recently, the SV channel was identified as the product of the TPC1 gene in Arabidopsis. In contrast, the TPC1 channel from other species was thought to be localized in the plasma membrane. A re-evaluation of this work under the assumption that the TPC1 channel is generally a vacuolar channel provides interesting insights into the physiological function of the TPC1/SV channel. Considerably less is known about vacuolar Ca2+ channels that are supposed to be activated by inositol 1,4,5-trisphosphate or cADP ribose. The major problems are controversial reports about functional characteristics, and a remarkable lack of homologues of animal ligand-gated Ca2+ channels in higher plants. To help understand Ca2+-mediated intracellular signalling in plant cells, a critical update of existing experimental evidence for vacuolar Ca2+ channels is presented.     

Processing of ATG8s, ubiquitin-like proteins, and their deconjugation by ATG4s are essential for plant autophagy

Autophagy is an intracellular process for vacuolar degradation of cytoplasmic components. Thus far, plant autophagy has been studied primarily using morphological analyses. A recent genome-wide search revealed significant conservation among autophagy genes (ATGs) in yeast and plants. It has not been proved, however, that Arabidopsis thaliana ATG genes are required for plant autophagy. To evaluate this requirement, we examined the ubiquitination-like Atg8 lipidation system, whose component genes are all found in the Arabidopsis genome. In Arabidopsis, all nine ATG8 genes and two ATG4 genes were expressed ubiquitously and were induced further by nitrogen starvation. To establish a system monitoring autophagy in whole plants, we generated transgenic Arabidopsis expressing each green fluorescent protein-ATG8 fusion (GFP-ATG8). In wild-type plants, GFP-ATG8s were observed as ring shapes in the cytoplasm and were delivered to vacuolar lumens under nitrogen-starved conditions. By contrast, in a T-DNA insertion double mutant of the ATG4s (atg4a4b-1), autophagosomes were not observed, and the GFP-ATG8s were not delivered to the vacuole under nitrogen-starved conditions. In addition, we detected autophagic bodies in the vacuoles of wild-type roots but not in those of atg4a4b-1 in the presence of concanamycin A, a V-ATPase inhibitor. Biochemical analyses also provided evidence that autophagy in higher plants requires ATG proteins. The phenotypic analysis of atg4a4b-1 indicated that plant autophagy contributes to the development of a root system under conditions of nutrient limitation.     

Functional and physiological characterization of Arabidopsis INOSITOL TRANSPORTER1, a novel tonoplast-localized transporter for myo-inositol

Arabidopsis thaliana INOSITOL TRANSPORTER1 (INT1) is a member of a small gene family with only three more genes (INT2 to INT4). INT2 and INT4 were shown to encode plasma membrane-localized transporters for different inositol epimers, and INT3 was characterized as a pseudogene. Here, we present the functional and physiological characterization of the INT1 protein, analyses of the tissue-specific expression of the INT1 gene, and analyses of phenotypic differences observed between wild-type plants and mutant lines carrying the int1.1 and int1.2 alleles. INT1 is a ubiquitously expressed gene, and Arabidopsis lines with T-DNA insertions in INT1 showed increased intracellular myo-inositol concentrations and reduced root growth. In Arabidopsis, tobacco (Nicotiana tabacum), and Saccharomyces cerevisiae, fusions of the green fluorescent protein to the C terminus of INT1 were targeted to the tonoplast membranes. Finally, patch-clamp analyses were performed on vacuoles from wild-type plants and from both int1 mutant lines to study the transport properties of INT1 at the tonoplast. In summary, the presented molecular, physiological, and functional studies demonstrate that INT1 is a tonoplast-localized H(+)/inositol symporter that mediates the efflux of inositol that is generated during the degradation of inositol-containing compounds in the vacuolar lumen.     

A Novel Calcium Binding Site in the Slow Vacuolar Cation Channel TPC1 Senses Luminal Calcium Levels

Cytosolic calcium homeostasis is pivotal for intracellular signaling and requires sensing of calcium concentrations in the cytosol and accessible stores. Numerous Ca²⁺ binding sites have been characterized in cytosolic proteins. However, little is known about Ca²⁺ binding inside organelles, like the vacuole. The slow vacuolar (SV) channel, encoded by Arabidopsis thaliana TPC1, is regulated by luminal Ca²⁺. However, the D454/fou2 mutation in TPC1 eliminates vacuolar calcium sensitivity and increases store calcium content. In a search for the luminal calcium binding site, structure modeling indicated a possible coordination site formed by residues Glu-450, Asp-454, Glu-456, and Glu-457 on the luminal side of TPC1. Each Glu residue was replaced by Gln, the modified genes were transiently expressed in loss-of-TPC1-function protoplasts, and SV channel responses to luminal calcium were recorded by patch clamp. SV channels lacking any of the four negatively charged residues appeared altered in calcium sensitivity of channel gating. Our results indicate that Glu-450 and Asp-454 are directly involved in Ca²⁺ binding, whereas Glu-456 and Glu-457 are probably involved in connecting the luminal Ca²⁺ binding site to the channel gate. This novel vacuolar calcium binding site represents a potential tool to address calcium storage in plants.     

Vacuolar ion channels in the liverwort Conocephalum conicum

As a liverwort Conocephalum conicum belongs to the oldest terrestrial plants¹ and is phylogenetically located between green algae and higher plants. Recent patch-clamp recordings on Conocephalum vacuoles^2,3 demonstrate ion channels very similar to higher plants and clearly different from vacuolar ion channels described in green algae. Here we summarize the features of a vacuolar cation channel and a vacuolar anion channel that both are common in terrestrial plants but are not detected in green algae, and we speculate about the molecular identity of these channels in the liverwort Conocephalum.Key words: vacuole, SV channel, anion channel, Conocephalum conicum, Embryophyta     

Degradation of oxidized proteins by autophagy during oxidative stress in Arabidopsis

Upon encountering oxidative stress, proteins are oxidized extensively by highly reactive and toxic reactive oxidative species, and these damaged, oxidized proteins need to be degraded rapidly and effectively. There are two major proteolytic systems for bulk degradation in eukaryotes, the proteasome and vacuolar autophagy. In mammalian cells, the 20S proteasome and a specific type of vacuolar autophagy, chaperone-mediated autophagy, are involved in the degradation of oxidized proteins in mild oxidative stress. However, little is known about how cells remove oxidized proteins when under severe oxidative stress. Using two macroautophagy markers, monodansylcadaverine and green fluorescent protein-AtATG8e, we here show that application of hydrogen peroxide or the reactive oxidative species inducer methyl viologen can induce macroautophagy in Arabidopsis (Arabidopsis thaliana) plants. Macroautophagy-defective RNAi-AtATG18a transgenic plants are more sensitive to methyl viologen treatment than wild-type plants and accumulate a higher level of oxidized proteins due to a lower degradation rate. In the presence of a vacuolar H(+)-ATPase inhibitor, concanamycin A, oxidized proteins were detected in the vacuole of wild-type root cells but not RNAi-AtATG18a root cells. Together, our results indicate that autophagy is involved in degrading oxidized proteins under oxidative stress conditions in Arabidopsis.     

Overexpression of the Arabidopsis thaliana MinD1 gene alters chloroplast size and number in transgenic tobacco plants

The Arabidopsis thaliana (L.) Heynh. minD gene (AtMinD1) was isolated and constitutively expressed in tobacco (Nicotiana tabacum L.) plants using the CaMV 35S promoter. Confocal and electron-microscopic analysis of the AtMinD1 transgenic tobacco lines revealed that the chloroplasts were abnormally large and fewer in number compared with wild-type tobacco plants. The abnormal chloroplasts were less prevalent in guard cells than in mesophyll cells. Chloroplast and nuclear gene expression was not significantly different in AtMinD1-overexpressing plants relative to wild-type tobacco plants. Chloroplast DNA copy number was not affected, based on the relative level of the rbcL gene in transgenic plants. Transgenic tobacco plants constitutively overexpressing AtMinD1 were completely normal phenotypically with respect to growth and development, and also displayed normal photosynthetic electron transport rates. These results show that the Arabidopsis MinD1 gene also functions in a heterologous system and confirm the role of the MinD protein in regulation of chloroplast division.     

Zinc transporter of Arabidopsis thaliana AtMTP1 is localized to vacuolar membranes and implicated in zinc homeostasis

Cation diffusion facilitator (CDF) proteins belong to a family of heavy metal efflux transporters that might play an essential role in homeostasis and tolerance to metal ions. We investigated the subcellular localization of Arabidopsis thaliana AtMTP1, a member of the CDF family, and its physiological role in the tolerance to Zn using MTP1-deficient mutant plants. AtMTP1 was immunochemically detected as a 43 kDa protein in the vacuolar membrane fractioned by sucrose density gradient centrifugation. The expression level of AtMTP1 in suspension-cultured cells was not affected by the Zn concentration in the medium. When AtMTP1 fused with green fluorescent protein was transiently expressed in protoplasts prepared from Arabidopsis suspension-cultured cells, green fluorescence was clearly observed in the vacuolar membrane. A T-DNA insertion mutant line for AtMTP1 displays enhanced sensitivity to high Zn concentrations ranging from 200 to 500 microM, but not to Zn-deficient conditions. Mesophyll cells of the mtp1-1 mutant plants grown in the presence of 500 microM Zn were degraded, suggesting that Zn at high concentrations causes serious damage to leaves and that AtMTP1 plays a crucial role in preventing this damage in plants. Thus we propose that AtMTP1 is localized in the vacuolar membrane and is involved in sequestration of excess Zn in the cytoplasm into vacuoles to maintain Zn homeostasis.     

Arabidopsis LHT1 is a high-affinity transporter for cellular amino acid uptake in both root epidermis and leaf mesophyll

Amino acid transport in plants is mediated by at least two large families of plasma membrane transporters. Arabidopsis thaliana, a nonmycorrhizal species, is able to grow on media containing amino acids as the sole nitrogen source. Arabidopsis amino acid permease (AAP) subfamily genes are preferentially expressed in the vascular tissue, suggesting roles in long-distance transport between organs. We show that the broad-specificity, high-affinity amino acid transporter LYSINE HISTIDINE TRANSPORTER1 (LHT1), an AAP homolog, is expressed in both the rhizodermis and mesophyll of Arabidopsis. Seedlings deficient in LHT1 cannot use Glu or Asp as sole nitrogen sources because of the severe inhibition of amino acid uptake from the medium, and uptake of amino acids into mesophyll protoplasts is inhibited. Interestingly, lht1 mutants, which show growth defects on fertilized soil, can be rescued when LHT1 is reexpressed in green tissue. These findings are consistent with two major LHT1 functions: uptake in roots and supply of leaf mesophyll with xylem-derived amino acids. The capacity for amino acid uptake, and thus nitrogen use efficiency under limited inorganic N supply, is increased severalfold by LHT1 overexpression. These results suggest that LHT1 overexpression may improve the N efficiency of plant growth under limiting nitrogen, and the mutant analyses may enhance our understanding of N cycling in plants.     

The Arabidopsis vacuolar malate channel is a member of the ALMT family

In plants, malate is a central metabolite and fulfills a large number of functions. Vacuolar malate may reach very high concentrations and fluctuate rapidly, whereas cytosolic malate is kept at a constant level allowing optimal metabolism. Recently, a vacuolar malate transporter (Arabidopsis thaliana tonoplast dicarboxylate transporter, AttDT) was identified that did not correspond to the well-characterized vacuolar malate channel. We therefore hypothesized that a member of the aluminum-activated malate transporter (ALMT) gene family could code for a vacuolar malate channel. Using GFP fusion constructs, we could show that AtALMT9 (A. thaliana ALMT9) is targeted to the vacuole. Promoter-GUS fusion constructs demonstrated that this gene is expressed in all organs, but is cell-type specific as GUS activity in leaves was detected nearly exclusively in mesophyll cells. Patch-clamp analysis of an Atalmt9 T-DNA insertion mutant exhibited strongly reduced vacuolar malate channel activity. In order to functionally characterize AtALMT9 as a malate channel, we heterologously expressed this gene in tobacco and in oocytes. Overexpression of AtALMT9-GFP in Nicotiana benthamiana leaves strongly enhanced the malate current densities across the mesophyll tonoplasts. Functional expression of AtALMT9 in Xenopus oocytes induced anion currents, which were clearly distinguishable from endogenous oocyte currents. Our results demonstrate that AtALMT9 is a vacuolar malate channel. Deletion mutants for AtALMT9 exhibit only slightly reduced malate content in mesophyll protoplasts and no visible phenotype, indicating that AttDT and the residual malate channel activity are sufficient to sustain the transport activity necessary to regulate the cytosolic malate homeostasis.     

Proton-driven sucrose symport and antiport are provided by the vacuolar transporters SUC4 and TMT1/2

The vacuolar membrane is involved in solute uptake into and release from the vacuole, which is the largest plant organelle. In addition to inorganic ions and metabolites, large quantities of protons and sugars are shuttled across this membrane. Current models suggest that the proton gradient across the membrane drives the accumulation and/or release of sugars. Recent studies have associated AtSUC4 with the vacuolar membrane. Some members of the SUC family are plasma membrane proton/sucrose symporters. In addition, the sugar transporters TMT1 and TMT2, which are localized to the vacuolar membrane, have been suggested to function in proton-driven glucose antiport. Here we used the patch-clamp technique to monitor carrier-mediated sucrose transport by AtSUC4 and AtTMTs in intact Arabidopsis thaliana mesophyll vacuoles. In the whole-vacuole configuration with wild-type material, cytosolic sucrose-induced proton currents were associated with a proton/sucrose antiport mechanism. To identify the related transporter on one hand, and to enable the recording of symporter-mediated currents on the other hand, we electrophysiologically characterized vacuolar proteins recognized by Arabidopsis mutants of partially impaired sugar compartmentation. To our surprise, the intrinsic sucrose/proton antiporter activity was greatly reduced when vacuoles were isolated from plants lacking the monosaccharide transporter AtTMT1/TMT2. Transient expression of AtSUC4 in this mutant background resulted in proton/sucrose symport activity. From these studies, we conclude that, in the natural environment within the Arabidopsis cell, AtSUC4 most likely catalyses proton-coupled sucrose export from the vacuole. However, TMT1/2 probably represents a proton-coupled antiporter capable of high-capacity loading of glucose and sucrose into the vacuole.     

New structure and function in plant K+ channels: KCO1, an outward rectifier with a steep Ca2+ dependency.

Potassium (K+) channels mediating important physiological functions are characterized by a common pore-forming (P) domain. We report the cloning and functional analysis of the first higher plant outward rectifying K+ channel (KCO1) from Arabidopsis thaliana. KCO1 belongs to a new class of ''two-pore'' K+ channels recently described in human and yeast. KCO1 has four putative transmembrane segments and tandem calcium-binding EF-hand motifs. Heterologous expression of KCO1 in baculovirus-infected insect (Spodoptera frugiperda) cells resulted in outwardly rectifying, K+-selective currents elicited by depolarizing voltage pulses in whole-cell measurements. Activation of KCO1 was strongly dependent on the presence of nanomolar concentrations of cytosolic free Ca2+ [Ca2+]cyt. No K+ currents were detected when [Ca2+]cyt was adjusted to <150 nM. However, KCO1 strongly activated at increasing [Ca2+]cyt, with a saturating activity observed at approximately 300 nM [Ca2+]cyt. KCO1 single channel analysis on excised membrane patches, resulting in a single channel conductance of 64 pS, confirmed outward rectification as well as Ca2+-dependent activation. These data suggest a direct link between calcium-mediated signaling processes and K+ ion transport in higher plants. The identification of KCO1 as the first plant K+ outward channel opens a new field of structure-function studies in plant ion channels.