Plant Ion Channels Research Articles

Differential gene expression of two outward-rectifying shaker-like potassium channels OsSKOR and OsGORK in rice

Shaker-like potassium channels are highly conserved voltage-dependent ion channels in plants. Rice, a monocot model plant, has similar numbers of shaker-like potassium channels as Arabidopsis. Although several inward-rectifying potassium channels have been identified, no outward-rectifying potassium channels have yet been reported for rice. Here, we identified two outward-rectifying shaker-like potassium channels in rice, with high amino acid sequence similarities to Arabidopsis stelar K+ outward rectifier (SKOR) and guard cell outward rectifying K+ channel (GORK). To characterize these channels we monitored their expression patterns in several tissues. qRTPCR and promoter-GUS analysis showed that OsSKOR is expressed in root vascular tissues, flower, and seed scutellum. OsGORK was expressed to some degree in most tissues, such as leaf blade, node, leaf sheath and root, but at high levels in flowers. This is the first report characterizing rice outward-rectifying potassium channels and contributes to understanding the regulation of potassium homeostasis in monocot plants.

United in diversity: mechanosensitive ion channels in plants.

Mechanosensitive (MS) ion channels are a common mechanism for perceiving and responding to mechanical force. This class of mechanoreceptors is capable of transducing membrane tension directly into ion flux. In plant systems, MS ion channels have been proposed to play a wide array of roles, from the perception of touch and gravity to the osmotic homeostasis of intracellular organelles. Three families of plant MS ion channels have been identified: the MscS-like (MSL), Mid1-complementing activity (MCA), and two-pore potassium (TPK) families. Channels from these families vary widely in structure and function, localize to multiple cellular compartments, and conduct chloride, calcium, and/or potassium ions. However, they are still likely to represent only a fraction of the MS ion channel diversity in plant systems.

Alternative Splicing-Mediated Targeting of the Arabidopsis GLUTAMATE RECEPTOR3.5 to Mitochondria Affects Organelle Morphology

Since the discovery of 20 genes encoding for putative ionotropic glutamate receptors in the Arabidopsis (Arabidopsis thaliana) genome, there has been considerable interest in uncovering their physiological functions. For many of these receptors, neither their channel formation and/or physiological roles nor their localization within the plant cells is known. Here, we provide, to our knowledge, new information about in vivo protein localization and give insight into the biological roles of the so-far uncharacterized Arabidopsis GLUTAMATE RECEPTOR3.5 (AtGLR3.5), a member of subfamily 3 of plant glutamate receptors. Using the pGREAT vector designed for the expression of fusion proteins in plants, we show that a splicing variant of AtGLR3.5 targets the inner mitochondrial membrane, while the other variant localizes to chloroplasts. Mitochondria of knockout or silenced plants showed a strikingly altered ultrastructure, lack of cristae, and swelling. Furthermore, using a genetically encoded mitochondria-targeted calcium probe, we measured a slightly reduced mitochondrial calcium uptake capacity in the knockout mutant. These observations indicate a functional expression of AtGLR3.5 in this organelle. Furthermore, AtGLR3.5-less mutant plants undergo anticipated senescence. Our data thus represent, to our knowledge, the first evidence of splicing-regulated organellar targeting of a plant ion channel and identify the first cation channel in plant mitochondria from a molecular point of view.

The phosphoinositide PI(3,5)P₂ mediates activation of mammalian but not plant TPC proteins: functional expression of endolysosomal channels in yeast and plant cells.

Two-pore channel proteins (TPC) encode intracellular ion channels in both animals and plants. In mammalian cells, the two isoforms (TPC1 and TPC2) localize to the endo-lysosomal compartment, whereas the plant TPC1 protein is targeted to the membrane surrounding the large lytic vacuole. Although it is well established that plant TPC1 channels activate in a voltage- and calcium-dependent manner in vitro, there is still debate on their activation under physiological conditions. Likewise, the mode of animal TPC activation is heavily disputed between two camps favoring as activator either nicotinic acid adenine dinucleotide phosphate (NAADP) or the phosphoinositide PI(3,5)P₂. Here, we investigated TPC current responses to either of these second messengers by whole-vacuole patch-clamp experiments on isolated vacuoles of Arabidopsis thaliana. After expression in mesophyll protoplasts from Arabidopsis tpc1 knock-out plants, we detected the Arabidopsis TPC1-EGFP and human TPC2-EGFP fusion proteins at the membrane of the large central vacuole. Bath (cytosolic) application of either NAADP or PI(3,5)P₂ did not affect the voltage- and calcium-dependent characteristics of AtTPC1-EGFP. By contrast, PI(3,5)P₂ elicited large sodium currents in hTPC2-EGFP-containing vacuoles, while NAADP had no such effect. Analogous results were obtained when PI(3,5)P₂ was applied to hTPC2 expressed in baker's yeast giant vacuoles. Our results underscore the fundamental differences in the mode of current activation and ion selectivity between animal and plant TPC proteins and corroborate the PI(3,5)P₂-mediated activation and Na(+) selectivity of mammalian TPC2.

Ion Channels in Plants

Since the first recordings of single potassium channel activities in the plasma membrane of guard cells more than 25 years ago, patch-clamp studies discovered a variety of ion channels in all cell types and plant species under inspection. Their properties differed in a cell type- and cell membrane-dependent manner. Guard cells, for which the existence of plant potassium channels was initially documented, advanced to a versatile model system for studying plant ion channel structure, function, and physiology. Interestingly, one of the first identified potassium-channel genes encoding the Shaker-type channel KAT1 was shown to be highly expressed in guard cells. KAT1-type channels from Arabidopsis thaliana and its homologs from other species were found to encode the K(+)-selective inward rectifiers that had already been recorded in early patch-clamp studies with guard cells. Within the genome era, additional Arabidopsis Shaker-type channels appeared. All nine members of the Arabidopsis Shaker family are localized at the plasma membrane, where they either operate as inward rectifiers, outward rectifiers, weak voltage-dependent channels, or electrically silent, but modulatory subunits. The vacuole membrane, in contrast, harbors a set of two-pore K(+) channels. Just very recently, two plant anion channel families of the SLAC/SLAH and ALMT/QUAC type were identified. SLAC1/SLAH3 and QUAC1 are expressed in guard cells and mediate Slow- and Rapid-type anion currents, respectively, that are involved in volume and turgor regulation. Anion channels in guard cells and other plant cells are key targets within often complex signaling networks. Here, the present knowledge is reviewed for the plant ion channel biology. Special emphasis is drawn to the molecular mechanisms of channel regulation, in the context of model systems and in the light of evolution.

Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action

The plant hormone abscisic acid (ABA) is produced in response to abiotic stresses and mediates stomatal closure in response to drought via recently identified ABA receptors (pyrabactin resistance/regulatory component of ABA receptor; PYR/RCAR). SLAC1 encodes a central guard cell S-type anion channel that mediates ABA-induced stomatal closure. Coexpression of the calcium-dependent protein kinase 21 (CPK21), CPK23, or the Open Stomata 1 kinase (OST1) activates SLAC1 anion currents. However, reconstitution of ABA activation of any plant ion channel has not yet been attained. Whether the known core ABA signaling components are sufficient for ABA activation of SLAC1 anion channels or whether additional components are required remains unknown. The Ca(2+)-dependent protein kinase CPK6 is known to function in vivo in ABA-induced stomatal closure. Here we show that CPK6 robustly activates SLAC1-mediated currents and phosphorylates the SLAC1 N terminus. A phosphorylation site (S59) in SLAC1, crucial for CPK6 activation, was identified. The group A PP2Cs ABI1, ABI2, and PP2CA down-regulated CPK6-mediated SLAC1 activity in oocytes. Unexpectedly, ABI1 directly dephosphorylated the N terminus of SLAC1, indicating an alternate branched early ABA signaling core in which ABI1 targets SLAC1 directly (down-regulation). Furthermore, here we have successfully reconstituted ABA-induced activation of SLAC1 channels in oocytes using the ABA receptor pyrabactin resistant 1 (PYR1) and PP2C phosphatases with two alternate signaling cores including either CPK6 or OST1. Point mutations in ABI1 disrupting PYR1-ABI1 interaction abolished ABA signal transduction. Moreover, by addition of CPK6, a functional ABA signal transduction core from ABA receptors to ion channel activation was reconstituted without a SnRK2 kinase.

Uniquely evolved plant ion channels

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Biologically Closed Electrical Circuits and Voltage Gated Ion Channels in Plants

The Venus flytrap is a marvel of plant electrical, mechanical and biochemical engineering. The rapid closure of the Venus flytrap upper leaf in about 0.1 s is one of the fastest movements in the plant kingdom. We found earlier that the electrical stimulus between a midrib and a lobe closes the Venus flytrap upper leaf without mechanical stimulation of trigger hairs. The Venus flytrap can accumulate small subthreshold charges, and when the threshold value is reached, the trap closes. Thigmonastic movements in the sensitive plant Mimosa pudica L., associated with fast responses to environmental stimuli, appear to be regulated through electrical and chemical signal transductions. The thigmonastic responses of Mimosa pudica can be considered in three stages: stimulus perception, electrical signal transmission, and induction of mechanical, hydrodynamical and biochemical responses. We investigated the mechanical movements of the pinnae and petioles in Mimosa pudica induced by the electrical stimulation of a pulvinus, petiole, secondary pulvinus, or pinna by low electrical voltage and charge. Both voltage and electrical charge are responsible for the electro stimulated closing of a leaf. The mechanism behind closing the leaf in Mimosa pudica is discussed. The hydroelastic curvature mechanism closely describes the kinetics of Mimosa pudica leaf movements.

Biological roles of cyclic-nucleotide-gated ion channels in plants: What we know and don’t know about this 20 member ion channel familyThis paper is one of a selection published in a Special Issue comprising papers presented at the 50th Annual Meeting of the Canadian Society of Plant Physiologists (CSPP) held at the University of Ottawa, Ontario, in June 2008.

Cyclic-nucleotide-gated ion channels (CNGCs) are nonselective cation channels that were first identified in vertebrate retinal photoreceptors and olfactory sensory neurons. The role of CNGCs in animals is well documented, and they are believed to localize to the plasma membrane as heterotetramers. So far, only six CNGC genes (CNGA1–4, CNGB1, and CNGB3) have been identified in mammalian genomes. In contrast, the Arabidopsis thaliana (L.) Heynh. genome encodes 20 putative CNGC subunits that are thought to assemble in the same tetrameric form as animal subunits do. Although the study of Arabidopsis CNGCs is still in its infancy, current research revealed their ability to transport cations that play a role in mediating various biotic and abiotic stresses and developmental processes. This review discusses the current knowledge of plant CNGCs and focuses on functional aspects, with references to heterologous expression studies and reverse genetics analyses. In addition, structural aspects of these channels are discussed.

Plant ion channels: gene families, physiology, and functional genomics analyses.

Distinct potassium, anion, and calcium channels in the plasma membrane and vacuolar membrane of plant cells have been identified and characterized by patch clamping. Primarily owing to advances in Arabidopsis genetics and genomics, and yeast functional complementation, many of the corresponding genes have been identified. Recent advances in our understanding of ion channel genes that mediate signal transduction and ion transport are discussed here. Some plant ion channels, for example, ALMT and SLAC anion channel subunits, are unique. The majority of plant ion channel families exhibit homology to animal genes; such families include both hyperpolarization- and depolarization-activated Shaker-type potassium channels, CLC chloride transporters/channels, cyclic nucleotide-gated channels, and ionotropic glutamate receptor homologs. These plant ion channels offer unique opportunities to analyze the structural mechanisms and functions of ion channels. Here we review gene families of selected plant ion channel classes and discuss unique structure-function aspects and their physiological roles in plant cell signaling and transport.

Phosphatidylinositol (4,5)bisphosphate inhibits K+-efflux channel activity in NT1 tobacco cultured cells.

In the animal world, the regulation of ion channels by phosphoinositides (PIs) has been investigated extensively, demonstrating a wide range of channels controlled by phosphatidylinositol (4,5)bisphosphate (PtdInsP2). To understand PI regulation of plant ion channels, we examined the in planta effect of PtdInsP2 on the K+-efflux channel of tobacco (Nicotiana tabacum), NtORK (outward-rectifying K channel). We applied a patch clamp in the whole-cell configuration (with fixed "cytosolic" Ca2+ concentration and pH) to protoplasts isolated from cultured tobacco cells with genetically manipulated plasma membrane levels of PtdInsP2 and cellular inositol (1,4,5)trisphosphate: "Low PIs" had depressed levels of these PIs, and "High PIs" had elevated levels relative to controls. In all of these cells, K channel activity, reflected in the net, steady-state outward K+ currents (IK), was inversely related to the plasma membrane PtdInsP2 level. Consistent with this, short-term manipulations decreasing PtdInsP2 levels in the High PIs, such as pretreatment with the phytohormone abscisic acid (25 microM) or neutralizing the bath solution from pH 5.6 to pH 7, increased IK (i.e. NtORK activity). Moreover, increasing PtdInsP2 levels in controls or in abscisic acid-treated high-PI cells, using the specific PI-phospholipase C inhibitor U73122 (2.5-4 microM), decreased NtORK activity. In all cases, IK decreases stemmed largely from decreased maximum attainable NtORK channel conductance and partly from shifted voltage dependence of channel gating to more positive potentials, making it more difficult to activate the channels. These results are consistent with NtORK inhibition by the negatively charged PtdInsP2 in the internal plasma membrane leaflet. Such effects are likely to underlie PI signaling in intact plant cells.

The plant innate immunity response in stomatal guard cells invokes G-protein-dependent ion channel regulation.

Stomata in the epidermis of terrestrial plants are important for CO2 absorption and transpirational water loss, and are also potential points of entry for pathogens. Stomatal opening and closure are controlled by distinct mechanisms. Arabidopsis stomata have been shown to close in response to bacteria and pathogen-associated molecular patterns (PAMPs) as part of PAMP-triggered immunity (PTI). Here we show that flg22, a PAMP derived from bacterial flagellin, also inhibits light-induced stomatal opening. Consistent with our observations on stomatal opening, flg22 inhibits the inward K+ channels (K+ (in) currents) of guard cells that mediate K+ uptake during stomatal opening. Similar to previously documented K+ current changes triggered by exogenous elevation of H(2)O(2) and nitric oxide (NO), with prolonged duration of flg22 exposure the outward K+ channels (K+ (out) currents) of guard cells are also inhibited. In null mutants of the flg22 receptor, FLS2, flg22 regulation of stomatal opening, K+ (in) currents, and K+ (out) currents is eliminated. flg22 also fails to elicit these responses in null mutants of the sole canonical G-protein alpha subunit, GPA1. The bacterial toxin, coronatine, produced by several pathogenic strains of Pseudomonas syringae, reverses the inhibitory effects of flg22 on both K+ (in) currents and stomatal opening, indicating interplay between plant and pathogen in the regulation of plant ion channels. Thus, the PAMP-triggered stomatal response involves K+ channel regulation, and this regulation is dependent on signaling via cognate PAMP receptors and a heterotrimeric G-protein. These new findings provide insights into the largely elusive signaling process underlying PTI-associated guard cell responses.

Nitric oxide in plant growth, development and stress physiology. Plant Cell Monographs, Volume 6.

Nitric oxide and its role in biological systems has had an increased prominence in the scientific literature since the 1980s, and really came to light as a signalling molecule in plants in the late 1990s. As discussed in the ‘Preface’ of this book, the number of publications concerning NO in plants has increased dramatically since that time, with little sign of this rise easing off. Therefore, a book that brings together a variety of aspects of NO biology in plants is timely. The book starts with chapters on NO synthesis, from mitochondria and from nitrogen assimilation, and then moves on to a chapter on NO signalling, which is an appropriate way to organize a book such as this. One of the main downstream pathways through which NO signals in animals is through the activation of guanylyl cyclase and the production of cGMP, but NO can also covalently modify proteins through a process referred to as S-nitrosylation. This process is the subject of the next chapter of the book, and highlights how NO can have profound effects on a multitude of proteins. Two subsequent chapters on NO in seed germination are followed by chapters on the role of NO in plant hormone signalling, including discussions on auxin, abscisic acid (ABA), lipid signalling, cytokinins and polyamine signalling. This is followed by a chapter on NO control of plant ion channels. The next few chapters are concerned with the role of NO in physiological events. This includes NO in nitrogen-fixing symbiosis, abioitc stress, the hypersensitive response and how it might function under oxygen deprivation. The book ends with a chapter that has a title indicating that it discusses fluorometric detection of NO, but in fact this chapter has short summaries about many techniques to determine the presence of NO, with a more in-depth discussion on the use of the compound diaminofluorescein (DAF) as a fluorometric probe. The greatest strength of this book is the fact that chapters have been contributed from researchers and groups that have a major prominence in the field, and therefore it has been written by those immersed in this research. Most of the major groups working on NO in plants are represented. However, despite the fact that it is timely to publish such a book there are some major issues to be resolved in NO biology in plants. Firstly, the final chapter of this book discusses how NO can be measured, and highlights several matters that need to be resolved in this area in the future. For most of the methods discussed the authors state problems, not least with the use of DAF and fluorescence, one of the most widely used techniques. Secondly, although a sequence for a nitric oxide synthase (NOS) protein from plants has been published, more recent literature brings into doubt exactly what this protein does. In fairness to the editors, they do have a ‘Note added in Proof’ discussing this in the ‘Preface’, but several of the chapters have not had the chance to be similarly up-dated. Therefore, in several places in the book discussion about the sources of NO in plants will need to be read with care. The book is very well presented, with a good use of diagrams, many with the added advantage of the use of colour. All the chapters are very well referenced and this will give any reader a fantastic place to start a more in-depth reading of the literature. However, the index is rather poor, and quickly trying to find pages for subjects known to be discussed in the book is not easy. Final-year undergraduates undertaking research or literature projects on NO will find this book of value, as would any postgraduates working in the area, and in fact for anyone working on NO in plants it ought to be a book that they should definitely read. Furthermore, as NO has now been implicated in so many functions and responses in plants, for those that are not directly involved in NO research but work on growth, development and stress biology, this is a text that they should be aware of. Overall, this is a book on an exciting topic with what appears to be an ever-increasing growth in interest. It is well written by experts in the field, and an excellent place to begin a reasonably in-depth and yet broad reading of the role of nitric oxide in plants.

In planta AKT2 subunits constitute a pH- and Ca2+-sensitive inward rectifying K+ channel

Heterologous expression of plant genes in yeast and animal cells represents a common approach to study plant ion channels. When expressed in Xenopus oocytes and COS cells the Arabidopsis Shaker-like K+ channel, AKT2 forms a weakly voltage-dependent channel, blocked by Ca2+ and protons. Channels with these characteristics, however, were not found in AKT2-expressing Arabidopsis cell types. To understand this phenomenon, we employed Agrobacterium-mediated transient transformation to functionally characterise Arabidopsis thaliana channels in Nicotiana benthamiana mesophyll cells. In this expression system we used AtTPK4 as a control for voltage-independent A. thaliana channels. Agrobacteria harbouring GFP-tagged constructs with the coding sequences of AKT2 and AtTPK4 were infiltrated into intact tobacco leaves. With quantitative RT-PCR analyses channel transcripts of AKT2 and AtTPK4 were determined in transformed leaves. These results were confirmed by Western blots with V5 epitope-tagged AKT2 and AtTPK4 proteins, showing that the channel protein was indeed synthesised. For functional analysis of these channels, mesophyll protoplasts were isolated from infiltrated leaf sections. Patch-clamp studies revealed that AKT2 channels in mesophyll protoplasts retained Ca2+ and pH sensitivity, characteristics of the heterologously expressed protein, but displayed pronounced differences in voltage-dependence and kinetics. AKT2-transformed mesophyll cells, displayed inward-rectifying, rather than voltage-independent K+ channels, initially characterised in AKT2-expressing animal cells. In contrast, AtTPK4 showed the same electrophysiological characteristics both, in oocytes and plant cells. Our data suggest that heterologous systems do not always possess all regulatory components for functional expression of plant channels. Therefore, transient expression of plant proteins in planta provides an additional research tool for rapid biophysical analysis of plant ion channels.

The Potassium Channel KAT1 Is Activated by Plant and Animal 14-3-3 Proteins

14-3-3 proteins modulate the plant inward rectifier K+ channel KAT1 heterologously expressed in Xenopus oocytes. Injection of recombinant plant 14-3-3 proteins into oocytes shifted the activation curve of KAT1 by +11 mV and increased the tau(on). KAT1 was also modulated by 14-3-3 proteins of Xenopus oocytes. Titration of the endogenous 14-3-3 proteins by injection of the peptide Raf 621p resulted in a strong decrease in KAT1 current (approximately 70% at -150 mV). The mutation K56E performed on plant protein 14-3-3 in a highly conserved recognition site prevented channel activation. Because the maximal conductance of KAT1 was unaffected by 14-3-3, we can exclude that they act by increasing the number of channels, thus ruling out any effect of these proteins on channel trafficking and/or insertion into the oocyte membrane. 14-3-3 proteins also increased KAT1 current in inside-out patches, suggesting a direct interaction with the channel. Direct interaction was confirmed by overlay experiments with radioactive 14-3-3 on oocyte membranes expressing KAT1.

CDPKs CPK6 and CPK3 Function in ABA Regulation of Guard Cell S-Type Anion- and Ca2+- Permeable Channels and Stomatal Closure

Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca2+ in guard cell ion channel regulation. However, genetic mutants in Ca2+ sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca2+-independent ABA signaling mechanisms suffice for a full response remains unclear. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central signal transduction responses in plants. However, no Arabidopsis CDPK gene disruption mutant phenotype has been reported to date, likely due to overlapping redundancies in CDPKs. Two Arabidopsis guard cell–expressed CDPK genes, CPK3 and CPK6, showed gene disruption phenotypes. ABA and Ca2+ activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca2+-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells. Furthermore, ABA- and Ca2+-induced stomatal closing were partially impaired in these cpk3cpk6 mutant alleles. However, rapid-type anion channel current activity was not affected, consistent with the partial stomatal closing response in double mutants via a proposed branched signaling network. Imposed Ca2+ oscillation experiments revealed that Ca2+-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca2+-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca2+-induced stomatal closing. Our findings show important functions of the CPK6 and CPK3 CDPKs in guard cell ion channel regulation and provide genetic evidence for calcium sensors that transduce stomatal ABA signaling.

Diacidic Motif Is Required for Efficient Transport of the K+ Channel KAT1 to the Plasma Membrane

For a number of mammalian ion channels, trafficking to the plasma membrane was found to be controlled by intrinsic sequence motifs. Among these sequences are diacidic motifs that function as endoplasmic reticulum (ER) export signals. So far it is unclear if similar motifs also exist in plant ion channels. In this study we analyzed the function of four diacidic DXE/DXD motifs of the plant K(+) channel KAT1. Mutation of the first diacidic DXE motif resulted in a strong reduction of the KAT1 conductance in both guard cell protoplasts and HEK293 cells (human embryonic kidney cells). Confocal fluorescence microscopy of guard cells expressing the mutated KAT1 fused to green fluorescent protein revealed localization of the mutated channel only in intracellular structures around the nucleus. These structures could be identified as part of the ER via coexpression of KAT1 fused to yellow fluorescent protein with an ER-retained protein (HDEL) fused to cyan fluorescent protein. Block of vesicle formation from the ER by overexpression of the small GTP-binding protein Sar1 fixed in its GDP-bound form led to retention of wild-type KAT1 in similar parts of the ER. Mutation of the three other diacidic motifs had no effect. Together, the results demonstrate that one diacidic motif of KAT1 is essential for ER export of the functional channel in both guard cell protoplasts and HEK293 cells. This suggests that trafficking of plant plasma membrane ion channels is controlled via a conserved mechanism.

A procedure for localisation and electrophysiological characterisation of ion channels heterologously expressed in a plant context

BackgroundIn silico analyses based on sequence similarities with animal channels have identified a large number of plant genes likely to encode ion channels. The attempts made to characterise such putative plant channels at the functional level have most often relied on electrophysiological analyses in classical expression systems, such as Xenopus oocytes or mammalian cells. In a number of cases, these expression systems have failed so far to provide functional data and one can speculate that using a plant expression system instead of an animal one might provide a more efficient way towards functional characterisation of plant channels, and a more realistic context to investigate regulation of plant channels.ResultsWith the aim of developing a plant expression system readily amenable to electrophysiological analyses, we optimised experimental conditions for preparation and transformation of tobacco mesophyll protoplasts and engineered expression plasmids, that were designed to allow subcellular localisation and functional characterisation of ion channels eventually in presence of their putative (possibly over-expressed) regulatory partners. Two inward K+ channels from the Shaker family were functionally expressed in this system: not only the compliant KAT1 but also the recalcitrant AKT1 channel, which remains electrically silent when expressed in Xenopus oocytes or in mammalian cells.ConclusionThe level of endogenous currents in control protoplasts seems compatible with the use of the described experimental procedures for the characterisation of plant ion channels, by studying for instance their subcellular localisation, functional properties, structure-function relationships, interacting partners and regulation, very likely in a more realistic context than the classically used animal systems.

Modulation of plant ion channels by oxidizing and reducing agents

Ion channels are proteins forming hydrophilic pathways through the membranes of all living organisms. They play important roles in the electrogenic transport of ions and metabolites. Because of biophysical properties such as high selectivity for the permeant ion, high turnover rate, and modulation by physico-chemical parameters (e.g., membrane potential, calcium concentration), they are involved in several physiological processes in plant cells (e.g., maintenance of the turgor pressure, stomatal movements, and nutrient absorption by the roots). As plants cannot move, plant metabolism must be flexible and dynamic, to cope with environmental changes, to compete with other living species and to prevent pathogen invasion. An example of this flexibility and dynamic behavior is represented by their handling of the so-called reactive oxygen species, inevitable by-products of aerobic metabolism. Plants cope with these species on one side avoiding their toxic effects, on the other utilizing them as signalling molecules and as a means of defence against pathogens. In this review, we present the state-of-the-art of the modulation of plant ion channels by oxidizing and reducing agents.

Potassium and carrot embryogenesis: Are K+ channels necessary for development?

The expression pattern of the KDC1 gene, coding for an inwardly-rectifying K(+) channel of Daucus carota , is described in several embryo stages and seedling tissues. Relative quantitative RT-PCR experiments indicated that, during (somatic) embryonic development, the KDC1 transcript appears as early as the globular stage and that the transcript level remains constant throughout the successive heart and torpedo stages. Thereafter, the KDC1 transcript is preferentially expressed in plant roots, but is also present in other tissues, and in particular, in the shoot apical meristem. In situ hybridisation experiments showed that in embryos KDC1 mRNA is detectable preferentially in protoderm cells with a stage dependent expression pattern. At later times, the hybridisation signal is particularly evident in root hairs, root epidermis and endodermis, but is also observed in single cell layers corresponding to L1 of the shoot apical meristem and leaf primordia. Promoter studies with the beta -glucuronidase reporter gene confirm preferential expression of KDC1 in embryo protoderm cells and in plant root epidermis and root hairs. Western blot analysis of embryonic proteins and immunolocalisation experiments on somatic embryos sections revealed the presence of KDC1 during embryo development. Consistent with these observations, patch-clamp experiments performed on protoplasts isolated from embryos at the torpedo stage demonstrated the presence of functional inward rectifying K(+) channels. This is the first report on the expression of a plant ion channel during embryo development.