Joachim Scholz-Starke

Malate transport by the vacuolar AtALMT6 channel in guard cells is subject to multiple regulation

Gas exchange in plants is controlled by guard cells, specialized cells acting as turgor pressure-driven valves. Malate is one of the major anions accumulated inside the vacuole during stomatal opening counteracting the positive charge of potassium. AtALMT6, a member of the aluminum-activated malate transporter family, is expressed in guard cells of leaves and stems as well as in flower organs of Arabidopsis thaliana. An AtALMT6-GFP fusion protein was targeted to the vacuolar membrane both in transient and stable expression systems. Patch-clamp experiments on vacuoles isolated from AtALMT6-GFP over-expressing Arabidopsis plants revealed large inward-rectifying malate currents only in the presence of micromolar cytosolic calcium concentrations. Further analyses showed that vacuolar pH and cytosolic malate regulate the threshold of activation of AtALMT6-mediated currents. The interplay of these two factors determines the AtALMT6 function as a malate influx or efflux channel depending on the tonoplast potential. Guard cell vacuoles isolated from Atalmt6 knock-out plants displayed reduced malate currents compared with wild-type vacuoles. This reduction, however, was not accompanied by phenotypic differences in the stomatal movements in knock-out plants, probably because of functional redundancy of malate transporters in guard cell vacuoles.

Identification of mutations allowing Natural Resistance Associated Macrophage Proteins (NRAMP) to discriminate against cadmium

Each essential transition metal plays a specific role in metabolic processes and has to be selectively transported. Living organisms need to discriminate between essential and non-essential metals such as cadmium (Cd(2+) ), which is highly toxic. However, transporters of the natural resistance-associated macrophage protein (NRAMP) family, which are involved in metal uptake and homeostasis, generally display poor selectivity towards divalent metal cations. In the present study we used a unique combination of yeast-based selection, electrophysiology on Xenopus oocytes and plant phenotyping to identify and characterize mutations that allow plant and mammalian NRAMP transporters to discriminate between their metal substrates. We took advantage of the increased Cd(2+) sensitivity of yeast expressing AtNRAMP4 to select mutations that decrease Cd(2+) sensitivity while maintaining the ability of AtNRAMP4 to transport Fe(2+) in a population of randomly mutagenized AtNRAMP4 cDNAs. The selection identified mutations in three residues. Among the selected mutations, several affect Zn(2+) transport, whereas only one, E401K, impairs Mn(2+) transport by AtNRAMP4. Introduction of the mutation F413I, located in a highly conserved domain, into the mammalian DMT1 transporter indicated that the importance of this residue in metal selectivity is conserved among NRAMP transporters from plant and animal kingdoms. Analyses of overexpressing plants showed that AtNRAMP4 affects the accumulation of metals in roots. Interestingly, the mutations selectively modify Cd(2+) and Zn(2+) accumulation without affecting Fe transport mediated by NRAMP4 in planta. This knowledge may be applicable for limiting Cd(2+) transport by other NRAMP transporters from animals or plants.

Fluorescence combined with excised patch: measuring calcium currents in plant cation channels

Combined application of the patch-clamp technique and fura-2 fluorescence detection enables the study of study calcium fluxes or related increases in cytosolic calcium concentration. Here we used the excised patch configuration, focusing the photomultiplier on the tip of the recording pipette where the fluorescent dye was present (FLEP, fluorescence combined with excised patch). This configuration has several advantages, i.e. a lack of delay in loading the fluorophore, of interference by internal calcium buffers and of photobleaching, due to the quasi-infinite dye reservoir inside the pipette. Upon voltage stimulation of tonoplast patches, sustained and robust fluorescence signals indicated permeation of calcium through the slow vacuolar (SV) channel. Both SV currents and fluorescence signal changes were absent in the presence of SV channel inhibitors and in vacuoles from Arabidopsis tpc1 knockout plants that lack SV channel activity. The fractional calcium currents of this non-selective cation channel were voltage-dependent, and were approximately 10% of the total SV currents at elevated positive potentials. Interestingly, calcium permeation could be recorded as the same time as oppositely directed potassium fluxes. These events would have been impossible to detect using patch-clamp measurements alone. Thus, we propose use of the FLEP technique for the study of divalent ion-selective channels or transporters that may be difficult to access using conventional electrophysiological approaches.

Redox-dependent modulation of the carrot SV channel by cytosolic pH

Currents mediated by a slow vacuolar (SV) channel were recorded and characterized in vacuoles from cultured carrot cells. The carrot channel shows the typical functional characteristics reported for channels of the SV category previously identified in other plants, i.e., slow voltage‐dependent activation kinetics, current activation favoured by cytosolic calcium and permeability to different monovalent cations. The carrot channel is strongly activated by cytosolic reducing agents (such as dithiothreitol, DTT, and glutathione, GSH) and has a peculiar dependence on cytosolic pH, which, in turn, is affected by the concentration of cytosolic reducing agents. Specifically, in 1 mM DTT or GSH the channel displayed a maximum conductance at neutral pH. The normalized conductance did not depend significantly on DTT concentration at acidic pH, while at alkaline pH the attenuation of the normalized conductance declines with increasing DTT concentration. Our results suggest two pH‐titratable groups within the carrot SV channel, one of these depending on cysteine residues exposed to the cytosolic side of the vacuole.

On the Interaction of Neomycin with the Slow Vacuolar Channel of Arabidopsis thaliana

This study investigates the interaction of the aminoglycoside antibiotic neomycin with the slow vacuolar (SV) channel in vacuoles from Arabidopsis thaliana mesophyll cells. Patch-clamp experiments in the excised patch configuration revealed a complex pattern of neomycin effects on the channel: applied at concentrations in the submicromolar to millimolar range neomycin (a) blocked macroscopic SV currents in a voltage- and concentration-dependent manner, (b) slowed down activation and deactivation kinetics of the channel, and most interestingly, (c) at concentrations above 10 μM, neomycin shifted the SV activation threshold towards negative membrane potentials, causing a two-phasic activation at high concentrations. Single channel experiments showed that neomycin causes these macroscopic effects by combining a decrease of the single channel conductance with a concomitant increase of the channels open probability. Our results clearly demonstrate that the SV channel can be activated at physiologically relevant tonoplast potentials in the presence of an organic effector molecule. We therefore propose the existence of a cellular equivalent regulating the activity of the SV channel in vivo.

The phosphoinositide PI(3,5)P 2 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.

The Arabidopsis central vacuole as an expression system for intracellular transporters: functional characterization of the Cl-/H+ exchanger CLC-7.

•  Ion transport proteins in intracellular membranes of eukaryotic cells play key roles in many physiological and pathological processes. •  The function of many of these transporters is poorly understood, because their intracellular localization makes them difficult to study. •  Here, we used the large organelle of plant cells, the central vacuole, as a novel system to study an intracellular transporter from animal cells. •  Our data showed that the lysosomal chloride transporter CLC‐7 from rat constitutes a functional transport protein in the central vacuole of the model plant Arabidopsis thaliana (thale cress). •  This novel approach has the potential to elucidate the transport properties of further, poorly studied intracellular ion channels and transporters.

A perspective on the slow vacuolar channel in vacuoles from higher plant cells

Historically, the vacuole has been an important system for the application of the patch-clamp technique to plant cells, and not only for reasons of simple preparation and the purity of its membrane. The vacuole is an intracellular organelle that can occupy up to 90% of the cellular volume of mature plant cells. For the plant cell, the tonoplast represents an essential crossing point between the cytosol and the vacuolar lumen containing a variety of transport systems. 1 Not only do these allow the storage of ions, metabolites, and xenobiotics, they also contribute to the homeostasis of cytosolic parameters, since the homeostasis of a given ion can be maintained by its release or uptake across the vacuolar membrane. In addition, the vacuole is likely to be involved in cellular signal transduction and can be an excellent model system for the study of calcium release from internal stores, leading to increases in cytosolic calcium. It is clear that the regulation of these processes is based on the permeability properties of the vacuolar membrane. Since its initial characterization, 2 the slowly activating vacuolar (SV) channel has been found in all tissue and plant species investigated so far. The SV channel represents the dominating conductance of the tonoplast at elevated cytosolic calcium concentrations. Interestingly, hypotheses about its physiological role have taken into consideration all the vacuolar functions mentioned above. Recent experimental evidence3 from Arabidopsis thalianastrongly indicates that the structure of the SV channel is determined by the TPC1 gene (two-pore channel 1). This work will briefly summarize the principal characteristics of this channel, concentrating on recent findings and otherwise by referring to previous reviews. 4-7

The signaling lipid phosphatidylinositol‐3,5‐bisphosphate targets plant CLC‐a anion/H+ exchange activity

Phosphatidylinositol‐3,5‐bisphosphate (PI(3,5)P2) is a low‐abundance signaling lipid associated with endo‐lysosomal and vacuolar membranes in eukaryotic cells. Recent studies on Arabidopsis indicated a critical role of PI(3,5)P2 in vacuolar acidification and morphology during ABA‐induced stomatal closure, but the molecular targets in plant cells remained unknown. By using patch‐clamp recordings on Arabidopsis vacuoles, we show here that PI(3,5)P2 does not affect the activity of vacuolar H+‐pyrophosphatase or vacuolar H+‐ATPase. Instead, PI(3,5)P2 at low nanomolar concentrations inhibited an inwardly rectifying conductance, which appeared upon vacuolar acidification elicited by prolonged H+ pumping activity. We provide evidence that this novel conductance is mediated by chloride channel a (CLC‐a), a member of the anion/H+ exchanger family formerly implicated in stomatal movements in Arabidopsis. H+‐dependent currents were absent in clc‐a knock‐out vacuoles, and canonical CLC‐a‐dependent nitrate/H+ antiport was inhibited by low concentrations of PI(3,5)P2. Finally, using the pH indicator probe BCECF, we show that CLC‐a inhibition contributes to vacuolar acidification. These data provide a mechanistic explanation for the essential role of PI(3,5)P2 and advance our knowledge about the regulation of vacuolar ion transport.

The putative K+ channel subunit AtKCO3 forms stable dimers in arabidopsis

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.