Sabine Zimmermann

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 ∼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.

Functional characterisation of LKT1, a K+ uptake channel from tomato root hairs, and comparison with the closely related potato inwardly rectifying K+ channel SKT1 after expression in Xenopus oocytes

Abstract. A cDNA encoding a novel inwardly rectifying potassium (K+in) channel, LKT1, was cloned from a root-hair-specific cDNA library of tomato (Lycopersicon esculentum Mill.). The LKT1 mRNA was shown to be most strongly expressed in root hairs by Northern blot analysis. The LKT1 channel is a member of the AKT family of K+in channels previously identified in Arabidopsis thaliana (L.) Heynh. and potato (Solanum tuberosum L.). Moreover, LKT1 is closely related (97% identical amino acids) to potato SKT1. An electrophysiological comparison of the two channels should therefore assist the identification of possible molecular bases for functional differences. For this comparison, both channels were functionally expressed and electrophysiologically characterised within the same expression system, i.e. Xenopus laevis oocytes. Voltage-clamp measurements identified LKT1 as a K+-selective inward rectifier which activates with slow kinetics upon hyperpolarising voltage pulses to potentials more negative than −50 mV. The activation potential of LKT1 is shifted towards positive potentials with respect to SKT1 which might be due to single amino acid exchanges in the rim of the channels pore region or in the S4 domain. Like SKT1, LKT1 reversibly activated upon shifting the external pH from 6.6 to 5.5, which indicates a physiological role for pH-dependent regulation of AKT-type K+in channels. The pharmacological inhibitor Cs+, applied externally, inhibited K+in currents mediated by LKT1 and SKT1 half-maximally with a concentration (IC50) of 21 μM and 17 μM, respectively. In conclusion, LKT1 may serve as a low-affinity influx pathway for K+ into root hair cells. Comparison of homologous K+in rectifiers from different plant species expressed in the same heterologous system allows conclusions to be drawn in respect to structure-function relationships.

Ion channels in plant signaling

Abstract Plant ion channel activities are rapidly modulated in response to several environmental and endogenous stimuli such as light, pathogen attack and phytohormones. Electrophysiological as well as pharmacological studies provide strong evidence that ion channels are essential for the induction of specific cellular responses, implicating their tight linkage to signal transduction cascades. Ion channels propagate signals by modulating the membrane potential or by directly affecting cellular ion composition. In addition, they may also be effectors at the end of signaling cascades, as examplified by ion channels which determine the solute content of stomatal guard cells. Plant channels are themselves subject to regulation by a variety of cellular factors, including calcium, pH and cyclic nucleotides. In addition, they appear to be regulated by (de)-phosphorylation events as well as by direct interactions with cytoskeletal and other cellular proteins. This review summarizes current knowledge on the role of ion chan nels in plant signaling.

Biotrophic transportome in mutualistic plant–fungal interactions

Understanding the mechanisms that underlie nutrient use efficiency and carbon allocation along with mycorrhizal interactions is critical for managing croplands and forests soundly. Indeed, nutrient availability, uptake and exchange in biotrophic interactions drive plant growth and modulate biomass allocation. These parameters are crucial for plant yield, a major issue in the context of high biomass production. Transport processes across the polarized membrane interfaces are of major importance in the functioning of the established mycorrhizal association as the symbiotic relationship is based on a ‘fair trade’ between the fungus and the host plant. Nutrient and/or metabolite uptake and exchanges, at biotrophic interfaces, are controlled by membrane transporters whose regulation patterns are essential for determining the outcome of plant–fungus interactions and adapting to changes in soil nutrient quantity and/or quality. In the present review, we summarize the current state of the art regarding transport systems in the two major forms of mycorrhiza, namely ecto- and arbuscular mycorrhiza.

Association of plant K+in channels is mediated by conserved C‐termini and does not affect subunit assembly

Inward rectifying potassium (K+ in) channels play an important role in turgor regulation and ion uptake in higher plants. Here, we report a previously unrecognized feature of these proteins: K+ in channel C‐terminal polypeptides mediate channel protein interactions. Using a C‐terminal fragment of potato guard cell K+ in channel KST1 in a yeast two‐hybrid screen two novel putative K+ in channel proteins (SKT2 and SKT3) were identified by interaction of their C‐termini which contained a conserved domain (KHA). Interactions were confirmed by Western blot‐related assays utilizing K+ in channel C‐termini fused to green fluorescence protein. Although deletion of the KHA‐domain abolished these interactions, K+ in currents were still detectable by patch‐clamp measurements of insect cells expressing these KST1 mutants, indicating that formation of a functional channel does not depend on this C‐terminal domain.

Calcium channel antagonists induce direct inhibition of the outward rectifying potassium channel in tobacco protoplasts

Verapamil, nifedipine and bepridil, three antagonists of L‐type calcium channels in animal cells, were shown to induce direct inhibition of outward rectifying potassium current in Nicotiana tabacumcv. Xanthi protoplasts with IC50 of 5 μM, 5 μM and 1 μM, respectively. In the outside‐out configuration, verapamil reduced the open probability of the ion channel responsible for the outward rectifying potassium conductance. Verapamil also blocked the outward rectifying potassium conductance in protoplasts from the N. tabacum cv. Bright Yellow cell line. Thus, studies using these molecules to demonstrate the involvement of calcium channels in plant physiological responses should be regarded with caution.

Plant ion channels: from molecular structures to physiological functions

Progress in identification of plant ion channels and development of electrophysiological analyses in heterologous expression systems and in planta, in combination with reverse genetic approaches, are providing the possibility of associating molecular entities with physiological functions. Recently, the first attempts to determine in vivo functions using knockout mutants demonstrated the roles of root ion channels. The search for proteins interacting with such channels leads to an even more complex view of the concerted action in protein networks.

A novel fungal family of oligopeptide transporters identified by functional metatranscriptomics of soil eukaryotes

Functional environmental genomics has the potential to identify novel biological functions that the systematic sequencing of microbial genomes or environmental DNA may fail to uncover. We targeted the functions expressed by soil eukaryotes using a metatranscriptomic approach based on the use of soil-extracted polyadenylated messenger RNA to construct environmental complementary DNA expression libraries. Functional complementation of a yeast mutant defective in di/tripeptide uptake identified a novel family of oligopeptide transporters expressed by fungi. This family has a patchy distribution in the Basidiomycota and Ascomycota and is present in the genome of a Saccharomyces cerevisiae wine strain. High throughput phenotyping of yeast mutants expressing two environmental transporters showed that they both displayed broad substrate specificity and could transport more than 60–80 dipeptides. When expressed in Xenopus oocytes one environmental transporter induced currents upon dipeptide addition, suggesting proton-coupled co-transport of dipeptides. This transporter was also able to transport specifically cysteine. Deletion of the two copies of the corresponding gene family members in the genome of the wine yeast strain severely reduced the number of dipeptides that it could assimilate. These results demonstrate that these genes are functional and can be used by fungi to efficiently scavenge the numerous, low concentration, oligopeptides continuously generated in soils by proteolysis.

ATP-Dependent Regulation of an Anion Channel at the Plasma Membrane of Protoplasts from Epidermal Cells of Arabidopsis Hypocotyls.

Although Arabidopsis is the object of many genetic and molecular biology investigations, relatively few studies deal with regulation of its transmembrane ion exchanges. To clarify the role of ion transport in plant development, organ-and tissue-specific ion channels must be studied. We identified a voltage-dependent anion channel in epidermal cells of Arabidopsis hypocotyls, thus providing a new example of the occurrence of voltage-dependent anion channels in a specific plant cell type distinct from the stomatal guard cell. The Arabidopsis hypocotyl anion channel is able to function under two modes characterized by different voltage dependences and different kinetic behaviors. This switch between a fast and a slow mode is controlled by ATP. In the presence of intracellular ATP (fast mode), the channels are closed at resting potentials, and whole-cell currents activate upon depolarization. After activation, the anion current deactivates rapidly and more and more completely at potentials negative to the peak. In the absence of ATP, the current switches from this fast mode to a mode characterized by a slow and incomplete deactivation at resting potentials. In addition, the whole-cell currents can be correlated with the activity of single channels. In the outside-out configuration, the presence of ATP modulates the mean lifetimes of the open and closed states of the channel at hyperpolarized potentials, thus controlling its open probability. The fact that ATP-dependent voltage regulation was observed in both whole-cell and outside-out configurations suggests that a single type of anion channel can switch between two modes with distinct functional properties.

Two differentially regulated phosphate transporters from the symbiotic fungus Hebeloma cylindrosporum and phosphorus acquisition by ectomycorrhizal Pinus pinaster.

Ectomycorrhizal symbiosis markedly improves plant phosphate uptake, but the molecular mechanisms underlying this benefit are still poorly understood. We identified two ESTs in a cDNA library prepared from the ectomycorrhizal basidiomycete Hebeloma cylindrosporum with significant similarities to phosphate transporters from the endomycorrhizal fungus Glomus versiforme and from non-mycorrhizal fungi. The full-length cDNAs corresponding to these two ESTs complemented a yeast phosphate transport mutant (Deltapho84). Measurements of (33)P-phosphate influx into yeast expressing either cDNA demonstrated that the encoded proteins, named HcPT1 and HcPT2, were able to mediate Pi:H(+) symport with different affinities for Pi (K(m) values of 55 and 4 mum, respectively). Real-time RT-PCR showed that Pi starvation increased the levels of HcPT1 transcripts in H. cylindrosporum hyphae grown in pure culture. Transcript levels of HcPT2 were less dependent on Pi availability. The two transporters were expressed in H. cylindrosporum associated with its natural host plant, Pinus pinaster, grown under low or high P conditions. The presence of ectomycorrhizae increased net Pi uptake rates into intact Pinus pinaster roots at low or high soil P levels. The expression patterns of HcPT1 and HcPT2 indicate that the two fungal phosphate transporters may be involved in uptake of phosphate from the soil solution under the two soil P availability conditions used.