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The group of voltage-independent K+ channels in Arabidopsis thaliana consists of six members, five tandem-pore channels (TPK1-TPK5) and a single K-ir-like channel (KCO3). All TPK/KCO channels are located at the vacuolar membrane except for TPK4, which was shown to be a plasma membrane channel in pollen. The vacuolar channels interact with 14-3-3 proteins (also called General Regulating Factors, GRFs), indicating regulation at the level of protein-protein interactions. Here we review current knowledge about these ion channels and their genes, and highlight open questions that need to be urgently addressed in future studies to fully appreciate the physiological functions of these ion channels.

The Arabidopsis thaliana K+ channel family of AtTPK/KCO proteins consists of six members including a 'single-pore' (Kir-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.

In a search for potassium channels involved in light- and clock-regulated leaf movements, we cloned four putative K channel genes from the leaf-moving organs, pulvini, of the legume Samanea saman. The S. saman SPOCK1 is homologous to KCO1, an Arabidopsis two-pore-domain K channel, the S. saman SPORK1 is similar to SKOR and GORK, Arabidopsis outward-rectifying Shaker-like K channels, and theS. saman SPICK1 and SPICK2 are homologous to AKT2, a weakly-inward-rectifying Shaker-like Arabidopsis K channel. All four S. saman sequences possess the universal K-channel-specific pore signature, TXXTXGYG, strongly suggesting a role in transmembrane K+ transport. The fourS. saman genes had different expression patterns within four leaf parts: ‘extensor’ and ‘flexor’ (the motor tissues), the leaf blades (mainly mesophyll), and the vascular bundle (‘rachis’). Based on northern blot analysis, their transcript level was correlated with the rhythmic leaf movements: (a) all four genes were regulated diurnally (Spick2, Spork1, andSpock1 in extensor and flexor, Spick1 in extensor and rachis); (b) Spork1 andSpock1 rhythms were inverted upon the inversion of the day-night cycle; and (c) in extensor and/or flexor, the expression ofSpork1, Spick1, and Spick2was also under a circadian control. These findings parallel the circadian rhythm shown to govern the resting membrane K+permeability in extensor and flexor protoplasts and the susceptibility of this permeability to light stimulation (Kim et al., 1993). Thus,Samanea pulvinar motor cells are the first described system combining light and circadian regulation of K channels at the level of transcript and membrane transport.

Parasitella parasitica is a facultative parasite of many Mucorales including Absidia glauca. The infection process includes the formation of a plasmatic continuum between host and parasite, which allows the invasion of the host by nuclei of the parasite. This process gives rise to interspecies recombinants. Auxotrophic A. glauca mutants are complemented by the transfer of genes from the parasite to the host. At the molecular level, we could show that plasmid-coded genes are also transferred. The successful formation of infection structures is mating-type dependent. Parasitella parasitica exclusively infects hosts belonging to the complementary mating type. The formation of infection structures is correlated with the ability to synthesise the sex pheromone trisporic acid, which is produced in mixed cultures by compatible combinations between host and parasite. Normally, trisporic acid is formed by a cooperative biosynthesis involving both mating types. Trisporic acid seems to be involved also in mediating the recognition between P. parasitica and A. glauca. To test the hypothesis of direct hormonal correlations between sex and parasitism, we have isolated one of the key enzymes for trisporic acid synthesis, dihydromethyltrisporic acid dehydrogenase, sequenced the N-termini of some proteolytic cleavage products, and have started to isolate the corresponding gene. Key words: mycoparasitism, zygomycetes, trisporic acid, Absidia glauca, Parasitella parasitica, Mucor.

The permeation pore of K(+) channels is formed by four copies of the pore domain. AtKCO3 is the only putative voltage-independent K(+) channel subunit of Arabidopsis thaliana with a single pore domain. KCO3-like proteins recently emerged in evolution and, to date, have been found only in the genus Arabidopsis (A. thaliana and A. lyrata). We show that the absence of KCO3 does not cause marked changes in growth under various conditions. Only under osmotic stress we observed reduced root growth of the kco3-1 null-allele line. This phenotype was complemented by expressing a KCO3 mutant with an inactive pore, indicating that the function of KCO3 under osmotic stress does not depend on its direct ability to transport ions. Constitutively overexpressed AtKCO3 or AtKCO3::GFP are efficiently sorted to the tonoplast indicating that the protein is approved by the endoplasmic reticulum quality control. However, vacuoles isolated from transgenic plants do not have significant alterations in current density. Consistently, both AtKCO3 and AtKCO3::GFP are detected as homodimers upon velocity gradient centrifugation, an assembly state that would not allow for activity. We conclude that if AtKCO3 ever functions as a K(+) channel, active tetramers are held by particularly weak interactions, are formed only in unknown specific conditions and may require partner proteins.

In this paper we compare the sequences of a segment of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase gene, isolated from eleven different strains belonging to four species of the fungal order Mucorales, Parasitella parasitica, Absidia glauca, Mucor mucedo (Mucoraceae) and Blakeslea trispora (Choanephoraceae). The segment was obtained by polynucleotide-chain-reaction amplification with primers derived from conservative regions of the gene. For the species M. mucedo and P. parasitica we have obtained evidence for two different types of HMG-CoA reductase genes by hybridization of genomic DNA with the amplified fragment and by cloning and sequencing of two different fragments. The different genes from one species show a sequence similarity of around 80% at the protein sequence level, whereas sequences of the same type from different species show similarity ranging between 91-96%. The highest similarity was found between the genes of type 1 from B. trispora and M. mucedo, although these species belong to different families. Southern-blot analysis of A. glauca DNA and B. trispora DNA revealed a second copy of the genes.

The assembly, sorting signals, and turnover of the tonoplast potassium channel AtTPK1 of Arabidopsis (Arabidopsis thaliana) were studied. We used transgenic Arabidopsis expressing a TPK1-green fluorescent protein (GFP) fusion or protoplasts transiently transformed with chimeric constructs based on domain exchange between TPK1 and TPK4, the only TPK family member not located at the tonoplast. The results show that TPK1-GFP is a dimer and that the newly synthesized polypeptides transiently interact with a thus-far unidentified 20-kD polypeptide. A subset of the TPK1-TPK4 chimeras were unable to assemble correctly and these remained located in the endoplasmic reticulum where they interacted with the binding protein chaperone. Therefore, TPK1 must assemble correctly to pass endoplasmic reticulum quality control. Substitution of the cytosolic C terminus of TPK4 with the corresponding domain of TPK1 was sufficient to allow tonoplast delivery, indicating that this domain contains tonoplast sorting information. Pulse-chase labeling indicated that TPK1-GFP has a half-life of at least 24 h. Turnover of the fusion protein involves internalization into the vacuole where the GFP domain is released. This indicates a possible mechanism for the turnover of tonoplast proteins.

The Arabidopsis thaliana genome contains five genes that encode two pore K + (TPK) channels. The most abundantly expressed isoform of this family, TPK1, is expressed at the tonoplast where it mediates K + -selective currents between cytoplasmic and vacuolar compartments. TPK1 open probability depends on both cytoplasmic Ca 2+ and cytoplasmic pH but not on the tonoplast membrane voltage. The channel shows intrinsic rectification and can be blocked by Ba 2+ , tetraethylammonium, and quinine. TPK1 current was found in all shoot cell types and shows all of the hallmarks of the previously described vacuolar K (VK) tonoplast channel characterized in guard cells. Characterization of TPK1 loss-of-function mutants and TPK1-overexpressing plants shows that TPK1 has a role in intracellular K + homeostasis affecting seedling growth at high and low ambient K + levels. In stomata, TPK1 function is consistent with vacuolar K + release, and removal of this channel leads to slower stomatal closure kinetics. During germination, TPK1 contributes to the radicle development through vacuolar K + deposition to provide expansion growth or in the redistribution of essential minerals.

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 Ca2+- 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, Ca2+ 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 Ca2+, 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, Ca2+-activated, K+-selective ion channel in the vacuolar membrane that does not mediate SV-type ionic currents.

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.

Potassium ion (K+) is the most abundant cation inside prokaryotic and eukaryotic cells and the K+ transport system in biological membranes is essential for maintenance of membrane potential and diverse cellular processes in all organisms studied thus far. Bacterial K+ uptake systems have been investigated extensively. In E. coli, three kinds of K+ transporters have been identified as major K+ transport uptake systems. E. coli Kdp is a P-type ATPase which has been classified as a high-affinity K+ transport system. The Kup transporter in E. coli is a low affinity type of K+ uptake transporter and the Trk system is the third K+ transport system. In fact, an E. coli mutant, lacking the three K+ uptake systems, Trk, Kup, and Kdp, could not grow on K+-limiting medium. In Arabidopsis plants, the K+ channels proteins exist [1]. More than ten genes encoding the homologous channels are dispersed in all of the genome and these homologous channels are expressed in Eucalyptus. We isolated the gene encoding the novel K+ channel from Arabidopsis and characterized the function using E. coli expression system.

Potassium (K+) channels play multiple roles in higher plants, and have been characterized electrophysiologically in various subcellular membranes. The K+ channel AtKCO1 from Arabidopsis thaliana is the prototype of a new family of plant K+ channels. In a previous study the protein has been functionally characterized after heterologous expression in Baculovirus-infected insect cells. In order to obtain further information on the physiological function of AtKCO1, the gene expression pattern and subcellular localization of the protein in plants were investigated. The regulatory function of the 5' region of the AtKCO1 gene was examined in transgenic A. thaliana plants carrying beta-glucuronidase (GUS) fusion constructs. Our analysis demonstrates that the AtKCO1 promoter is active in various tissues and cell types, and the highest GUS activity could be detected in mitotically active tissues of the plant. Promoter activity was strongly dependent on the presence of a 5' leader intron. The same overall structure was identified in two genes encoding AtKCO1-like K+ channels from Solanum tuberosum (StKCO1alpha and StKCO1beta). To investigate the subcellular localization of AtKCO1, the channel protein, as well as a fusion protein of AtKCO1 with green fluorescence protein (GFP), were expressed in transgenic tobacco BY2 cells. In sucrose density gradients, both proteins co-fractionate with tonoplast markers (Nt-TIPa, vATPase). In fluorescence images from transgenic AtKCO1-GFP BY2 cells fluorescence was exclusively detected in the tonoplast. Thus AtKCO1 is the first cloned K+ channel demonstrated to be a vacuolar K+ channel.

Parasitella parasitica (synonyms: P. parasitica and Mucor parasiticus) is a heterothallic facultative mycoparasite which belongs phylogenetically to the Mucor group within the zygomycetes. It is a facultative parasite of several but not all mucoraceous fungi. The interactions between host and parasite present several peculiarities. (1) Infection of the host organism Absidia glauca especially is strongly mating type-specific. (+) strains of P. parasitica will infect exclusively (-) strains of A. glauca whereas (-) strains of the parasite are required for the infection of (+) type hosts. We propose the hypothesis that host/parasite recognition is mediated by the same gamone system as is required for communication between sexual partners within mucorales (trisporic acid). (2) P. parasitica is a fusion biotroph. Infection is accompanied by a limited cytoplasmic continuum between the two partners, which allows invasion of the host by nuclei of the parasite. P. parasitica is not very aggressive. The host, A. glauca survives the infection well and forms normal sporangiospores even close to infection sites. We were able to demonstrate by genetic and molecular techniques that genetic markers of the parasite are transferred into the host’s nuclei at high rates. Absidia and Parasitella form a naturally occurring parasexual system with high efficiency. (3) Sexually compatible pairs of P. parasitica are not able to form zygospores. In order to reproduce sexually at least one of the partners has to grow parasitically. Measurements of trisporic acid levels in cocultures of P. parasitica with A. glauca led us to assume that interaction with a host organism is required for the cooperative synthesis of trisporic acid at a level high enough to induce sexual differentiation.

We have purified the NADP-dependent 4-dihydromethyltrisporate dehydrogenase from the zygomycete Mucor mucedo. The enzyme is involved in the biosynthesis of trisporic acid, the sexual hormone of zygomycetes, which induces the first steps of zygophore development. Protein was obtained from the (-) mating type of M. mucedo after induction with trisporic acid, and purified by gel filtration and affinity chromatography steps. On SDS-PAGE a band with an apparent molecular mass of 33 kDa was ascribed to the enzyme. After transferring onto PVDF membranes the protein was digested with endoprotease Lys-C, and several peptides were sequenced. Oligonucleotides derived from protein sequence data were used for PCR amplification of genomic M. mucedo DNA. The PCR fragment was used as probe for isolation of the corresponding cDNA and complete genomic DNA clones. Comparison of protein and DNA sequence data showed that the cloned fragment corresponded to the purified protein. Search for similarity with protein sequences of the Swiss-Prot database revealed a relationship to enzymes belonging to the aldo/keto reductase superfamily. Southern-blot analysis of genomic DNA with the labelled cloned fragment detected a single-copy gene in both mating types of M. mucedo. PCR with genomic DNA from other zygomycetes gave rise to several fragments. Hybridization analysis with the cloned M. mucedo fragment showed that a fragment of similar length cross-hybridized in Blakeslea trispora (Choanephoraceae) as well as in Parasitella parasitica and Absidia glauca (Mucoraceae). The promoter region of the gene contains DNA elements with similarity to a cAMP-regulated gene of Dictyostelium discoideum.

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