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Electrical and calcium signals in plants are one of the basic carriers of information transmitted over a long distance. Together with reactive oxygen species (ROS) waves, electrical and calcium signals can participate in cell-to-cell signaling, conveying information about different stimuli, e.g. abiotic stress, pathogen infection, or mechanical injury. There is no information on the ability of ROS to evoke systemic electrical or calcium signals in the model moss Physcomitrella and on the relationships between these responses. Here, we show that external application of hydrogen peroxide evokes electrical signals in the form of long-distance changes in the membrane potential, which transmit through the plant instantly after stimulation. The responses were calcium dependent, since their generation was inhibited by lanthanum, a calcium channel inhibitor (2 mM) or EDTA, a calcium chelator (0.5 mM). The electrical signals were partially dependent on glutamate receptor ion channels (GLR), since the knockout of GLR genes only slightly reduced the amplitude of the responses. The basal part of the gametophyte, which is rich in protonema cells, was the most sensitive to hydrogen peroxide. The measurements carried out on the protonema expressing fluorescent calcium biosensor GCaMP3 proved that calcium signals propagated slowly (above 5 µm/s) and with a decrement. We also demonstrate upregulation of a stress-related gene which appears in a distant section of the moss 8 minutes after H2O2 treatment. The results help to understand the importance of both types of signals in the transmission of information about the appearance of ROS in the plant cell apoplast.

The two-pore channel (TPC) family is widely conserved in eukaryotes. Many vascular plants, including Arabidopsis and rice, possess a single TPC gene which functions as a slow vacuolar (SV) channel—voltage-dependent cation-permeable channel located in the vacuolar membrane (tonoplast). On the other hand, a liverwort Marchantia polymorpha genome encodes three TPC homologs: MpTPC1 is similar to TPCs in vascular plants (type 1 TPC), while MpTPC2 and MpTPC3 are classified into a distinctive group (type 2 TPC). Phylogenetic analysis suggested that the type 2 TPC emerged before the land colonization in plant evolution and was lost in vascular plants and hornworts. All of the three MpTPCs were shown to be localized at the tonoplast. We generated knockout mutants of tpc1, tpc2, tpc3 and tpc2 tpc3 double mutant by clustered regularly interspaced short palindromic repeats/Cas9 genome editing and performed patch-clamp analyses of isolated vacuoles. The SV channel activity was abolished in the Mptpc1 loss-of-function mutant (Mptpc1-1KO), while Mptpc2-1KO, Mptpc3-1KO and Mptpc2-2/tpc3-2KO double mutant exhibited similar activity to the wild type, indicating that MpTPC1 (type 1) is solely responsible for the SV channel activity. Activators of mammalian TPCs, phosphatidylinositol-3,5-bisphosphate and nicotinic acid adenine dinucleotide phosphate, did not affect the ion channel activity of any MpTPCs. These results indicate that the type 1 TPCs, which are well conserved in all land plant species, encode the SV channel, while the type 2 TPCs likely encode other tonoplast cation channel(s) distinct from the SV channel and animal TPCs.

Background In this review, we summarize data concerning action potentials (APs) – long-distance electrical signals in Characean algae and liverworts. These lineages are key in understanding the mechanisms of plant terrestrialization. Liverworts are postulated to be pioneer land plants, whereas aquatic charophytes are considered the closest relatives to land plants. The drastic change of the habitat was coupled with the adaptation of signalling systems to the new environment. Scope APs fulfil the ‘all-or-nothing’ law, exhibit refractory periods and propagate with a uniform velocity. Their ion mechanism in the algae and liverworts consists of a Ca2+ influx (from external and internal stores) followed by/coincident with a Cl– efflux, which both evoke the membrane potential depolarization, and a K+ efflux leading to repolarization. The molecular identity of ion channels responsible for these fluxes remains unknown. Publication of the Chara braunii and Marchantia polymorpha genomes opened up new possibilities for studying the molecular basis of APs. Here we present the list of genes which can participate in AP electrogenesis. We also point out the differences between these plant species, e.g. the absence of Ca2+-permeable glutamate receptors (GLRs) and Cl–-permeable SLAC1 channel homologues in the Chara genome. Both these channels play a vital role in long-distance signalling in liverworts and vascular plants. Among the common properties of APs in liverworts and higher plants is their duration (dozens of seconds) and the speed of propagation (mm s–1), which are much slower than in the algae (seconds, and dozens of mm s–1, respectively). Conclusions Future studies with combined application of electrophysiological and molecular techniques should unravel the ion channel proteins responsible for AP generation, their regulation and transduction of those signals to physiological responses. This should also help to understand the adaptation of the signalling systems to the land environment and further evolution of APs in vascular plants.

Mycotoxin stress, one of the currently intensively studied, was stimulated in wheat hypocotyl cells through the application of zearalenone, produced by Fusarium fungi. The differences of reactions in the activation of the SV ion channels between stress-tolerant (Parabola) and sensitive (Raweta) wheat cultivars have been studied by the patch-clamp technique. No impact of zearalenone on SV channel activity was observed in Parabola. However, in the sensitive cultivar, mycotoxin (30 µmol L−1) evoked a significant decrease in the unitary conductance of the channels—from 22 ± 0.4 pS (n = 5) in the control to 18 ± 0.5 pS (n = 7) in zearalenone-treated vacuoles (potential clamped at 100 mV). A combined treatment of zearalenone and selenium (5 µmol L−1 of Na2SeO4) abolished this effect totally, indicating the importance of this element in the defense mechanism of cells under the stress of mycotoxins. The role of differences in the membrane structure between tolerant and sensitive cultivars induced in stress conditions, as one of the main factors in mechanical stability of channels, has been discussed.

The mode of transmission of signals between plant cells is an important aspect of plant physiology. The main role in the generation of long-distance signals is played by changes in the membrane potential and cytoplasm calcium concentration, but the relationship between these responses evoked by the same stimuli in the same plant remains unknown. As one of the first plants that colonized land, the moss Physcomitrella patens is a suitable model organism for studying the evolution of signaling pathways in plants. Here, by the application of glutamate as a stimulus, we demonstrated that electrical but not calcium signals can be true carriers of information in long-distance signaling in Physcomitrella. The generation of electrical signals in a form of propagating transient depolarization seems to be dependent on the opening of calcium channels since the responses were reduced or totally blocked by calcium channel inhibitors. While the microelectrode measurements demonstrated the transmission of electric signals between leaf cells and juvenile cells (protonema), the fluorescence imaging of cytoplasmic calcium changes indicated that calcium response occurs only locally—at the site of glutamate application, and only in protonema cells. This study indicates different involvement of glutamate-induced electrical and calcium signals in cell-to-cell communication in these evolutionarily old terrestrial plants.

Potassium-permeable slow activating vacuolar channels (SV) and chloride-permeable channels in the vacuole of the liverwort Marchantia polymorpha were characterized in respect to calcium dependence, selectivity, and pharmacology. The patch-clamp method was used in the study of ion channel activity in the vacuoles from the liverwort Marchantia polymorpha. The whole-vacuole recordings allowed simultaneous observation of two types of currents—predominant slow activated currents recorded at positive voltages and fast activated currents recorded at negative voltages. Single-channel recordings carried out in the gradient of KCl indicated that slow activated currents were carried by potassium-permeable slowly activating vacuolar channels (SV) and fast activated currents—by chloride-permeable channels. Both types of the channels were dependent in an opposite way on calcium, since elimination of this ion from the cytoplasmic side caused inhibition of SV channels, but the open probability of chloride-permeable channels even increased. The dependence of the activity of both channels on different types of ion channel inhibitors was studied. SV channels exhibited different sensitivity to potassium channel inhibitors. These channels were insensitive to 3 mM Ba2+, but were blocked by 3 mM tetraethyl ammonium (TEA). Moreover, the activity of the channels was modified in a different way by calcium channel inhibitors. 200 µM Gd3+ was an effective blocker, but 50 µM ruthenium red evoked bursts of the channel activity resulting in an increase in the open probability. Different effectiveness of anion channel inhibitors was observed in chloride-permeable channels. After the application of 100 µM Zn2+, a decrease in the open probability was recorded but the channels were still active. 50 µM DIDS was more effective, as it completely blocked the channels.

Liverworts are pioneer plants that colonized lands. They had to cope with frequent sea water flooding causing salt stress. The role of vacuoles and in particular slow-activating (SV) channels in the salt stress tolerance was addressed in the present study. A patch-clamp method was used to study sodium fluxes through the tonoplast of the liverwort Conocephalum conicum. The whole-vacuole measurements carried out in a symmetrical Na+ concentration allowed recording of slowly activated outward currents typical for SV channels. In a Na+ gradient promoting an efflux of Na+ from the vacuole, the outward rectifying properties of SV channels were reduced and inward Na+ currents with different inactivation rates were recorded. Single channel analysis proved that a decrease in cytoplasmic Na+ concentration evoked an increase in the open probability of the channels and shifted the activation voltages towards negative values. The number of SV channels recorded at negative voltages was dependent on the vacuolar calcium and decreased at the high concentration of this ion in the vacuole. In some of the tested patches, the channels exhibited a flickering type of activity and two different conductance levels. The role of SV channels in Na+ accumulation during salt stress and its removal after periods of flooding is discussed in the present paper.

Inhibitors of human two-pore channels (TPC1 and TPC2), i.e., verapamil, tetrandrine, and NED-19, are promising medicines used in treatment of serious diseases. In the present study, the impact of these substances on action potentials (APs) and vacuolar channel activity was examined in the aquatic characean algae Nitellopsis obtusa and in the terrestrial liverwort Marchantia polymorpha. In both plant species, verapamil (20–300 µM) caused reduction of AP amplitudes, indicating impaired Ca2+ transport. In N. obtusa, it depolarized the AP excitation threshold and resting potential and prolonged AP duration. In isolated vacuoles of M. polymorpha, verapamil caused a reduction of the open probability of slow vacuolar SV/TPC channels but had almost no effect on K+ channels in the tonoplast of N. obtusa. In both species, tetrandrine (20–100 µM) evoked a pleiotropic effect: reduction of resting potential and AP amplitudes and prolongation of AP repolarization phases, especially in M. polymorpha, but it did not alter vacuolar SV/TPC activity. NED-19 (75 µM) caused both specific and unspecific effects on N. obtusa APs. In M. polymorpha, NED-19 increased the duration of repolarization. However, no inhibition of SV/TPC channels was observed in Marchantia vacuoles, but an increase in open probability and channel flickering. The results indicate an effect on Ca2+ -permeable channels governing plant excitation.

Microelectrode measurements carried out on leaf cells from Physcomitrella patens revealed that a sudden temperature drop and application of menthol evoked two types of different-shaped membrane potential changes. Cold stimulation evoked spike-type responses. Menthol depolarized the cell membrane with different rates. When it reached above 1 mV s-1 , the full response was recorded. Characteristic for the full responses was also a few-minute plateau of the membrane potential recorded after depolarization. The influence of inhibitors of calcium channels (5 mM Gd3+ ), potassium channels (5 mM Ba2+ ), chloride channels (200 μM Zn2+ , 50 μM niflumic acid) and proton pumps (10 μM DES), an activator of calcium release from intracellular stores (Sr2+ ), calcium chelation (by 400 μM EGTA) and phytohormones (50 μM auxin, 50 μM abscisic acid (ABA), 500 μM salicylic acid) on cold- and menthol-evoked responses was tested. Both responses are different in respect to the ion mechanism: cold-evoked depolarizations were influenced by Ba2+ and DES; in turn, menthol-evoked potential changes were most effectively blocked by Zn2+ . Moreover, the effectiveness of menthol in generation of full responses was reduced after administration of auxin or ABA, i.e. phytohormones known for their participation in responses to cold and regulation of proton pumps. The effects of DES indicated that one of the main conditions for generation of menthol-evoked responses is inhibition of the proton pump activity. Our results indicate that perception of cold and menthol by plants proceeds in different ways due to the differences in ionic mechanism and hormone dependence of cold- and menthol-evoked responses.

Main conclusion In this work, for the first time the activity of nitrate-permeable channels in the tonoplast of the moss Physcomitrella patens was recorded. The channels allowed nitrate flow in one direction—from the cytoplasm to the vacuole. Selectivity of nitrate over chloride of the channels was proved. The activity of the channels was dependent on cytoplasmic calcium, magnesium, and pH. A patch-clamp study carried out on the vacuolar membrane of the moss Physcomitrella patens has revealed that inhibition of cation-selective channels leads to disclosure of channels permeable to NO3−. These channels were inwardly rectifying and allowed anions to flow from the cytoplasm to the vacuole. After a decrease in the cytoplasmic NO3− concentration, the current density recorded in the whole-vacuole configuration and amplitude of the currents flowing through single channels were reduced. Application of the NO3− gradient caused a shift in the reversal potential (Erev) towards ENO3−, indicating NO3− permeability. Research of the selectivity of the channels to Cl− and NO3− was also done; it indicated that Cl− is less permeable than NO3− (PNO3/PCl = 3.08). Measurements with different concentrations of cytoplasmic Ca2+ and Mg2+ revealed that the channel was activated by different concentrations of these ions—100 µM Ca2+ and 10 mM Mg2+. Calcium dependence of the channels was also modulated by a redox agent—DTT (dithiothreitol), which added on the cytoplasmic side, caused a reduction in the threshold of channel activation with cytoplasmic Ca2+. The NO3− permeable channel was also pH dependent. A decrease in the cytoplasmic pH reduced the open probability of the channel; in turn, an increase in the vacuolar pH did not decrease ion channel activity but lowered its conductance. Electronic supplementary material The online version of this article (doi:10.1007/s00425-015-2250-3) contains supplementary material, which is available to authorized users.

Amphotericin B (AmB) is an antifungal polyene for which the most accepted mode of action is formation of protein-like ion channels in the cell membrane. Patch-clamp research on Candida albicans protoplasts carried out in the outside-out configuration showed that application of 0.05 and 0.1 μM AmB caused a decrease in seal resistance. Such a phenomenon can be correlated with a decrease in membrane tightness. AmB applied at a 0.05 μM concentration also caused a decrease in the number of active TOK1 (two-pore outward rectifiers) potassium channels, but did not significantly change their open probability. The results indicate that in C. albicans protoplast AmB causes a decrease in cell membrane integrity by interaction with its lipid phase but not with ion channels. Fluorescence microscopy techniques showed that AmB treatment, in clinical concentrations, had no effect on the percentage of PI-positive protoplasts. AmB treatment in the concentrations tested did not cause a rapid reduction of the number of C. albicans protoplasts. However, there was a significant loss of replication competency and numerous morphological and physiological disorders, including cytoplasm shrinking, abnormal morphology of the nucleus and mitochondria, a sudden decrease in the MTT reduction level and oxidative stress. Our results show that the induction of yeast cell death by AmB, at therapeutic doses, is a multistage and long-term process involving multiple intracellular pathways.

Patch-clamp studies carried out on the tonoplast of the moss Physcomitrella patens point to existence of two types of cation-selective ion channels: slowly activated (SV channels), and fast-activated potassium-selective channels. Slowly and instantaneously saturating currents were observed in the whole-vacuole recordings made in the symmetrical KCl concentration and in the presence of Ca(2+) on both sides of the tonoplast. The reversal potential obtained at the KCl gradient (10 mM on the cytoplasmic side and 100 mM in the vacuole lumen) was close to the reversal potential for K(+) (E K), indicating K(+) selectivity. Recordings in cytoplasm-out patches revealed two distinct channel populations differing in conductance: 91.6 ± 0.9 pS (n = 14) at -80 mV and 44.7 ± 0.7 pS (n = 14) at +80 mV. When NaCl was used instead of KCl, clear slow vacuolar SV channel activity was observed both in whole-vacuole and cytoplasm-out membrane patches. There were no instantaneously saturating currents, which points to impermeability of fast-activated potassium channels to Na(+) and K(+) selectivity. In the symmetrical concentration of NaCl on both sides of the tonoplast, currents have been measured exclusively at positive voltages indicating Na(+) influx to the vacuole. Recordings with different concentrations of cytoplasmic and vacuolar Ca(2+) revealed that SV channel activity was regulated by both cytoplasmic and vacuolar calcium. While cytoplasmic Ca(2+) activated SV channels, vacuolar Ca(2+) inhibited their activity. Dependence of fast-activated potassium channels on the cytoplasmic Ca(2+) was also determined. These channels were active even without Ca(2+) (2 mM EGTA in the cytosol and the vacuole lumen), although their open probability significantly increased at 0.1 μM Ca(2+) on the cytoplasmic side. Apart from monovalent cations (K(+) and Na(+)), SV channels were permeable to divalent cations (Ca(2+) and Mg(2+)). Both monovalent and divalent cations passed through the channels in the same direction-from the cytoplasm to the vacuole. The identity of the vacuolar ion channels in Physcomitrella and ion channels already characterised in different plants is discussed.

Glass microelectrodes were inserted into Physcomitrella patens gametophyte leaves and action potentials (APs) were recorded in response to sudden illumination as well as after darkening, i.e., when the dark-induced membrane depolarization crossed a threshold. Application of 5 mM La(3+) (a calcium channel inhibitor), 10 mM TEA(+) (a potassium channel inhibitor) and increased free Ca(2+) resulted in a loss of excitability. Lack of Ca(2+) in the external medium did not prevent APs from occurring. It was concluded that during light- dark-induced excitation of Physcomitrella patens, APs might rely upon calcium influxes from the intracellular compartments. APs were not blocked by the proton pump inhibitors (DES, DCCD), although the resting potential (RP) diminished significantly.

Intracellular microelectrode measurements revealed that a resting potential (RP), an action potential (AP) and a calcium component of AP (named voltage transient, VT) can be influenced by glutamic acid (Glu) and aminoacetic acid (glycine, Gly) in the liverwort Conocephalum conicum. In the continuous presence of 5mM Glu or 5mM Gly, the RP hyperpolarized constantly and the plants became desensitized to the excitatory amino acids (Glu or Gly). Under such circumstances, the amplitudes of APs evoked by stimuli other than Glu or Gly grew, as did their calcium components (VTs). The sudden application of 1-15 mM Glu or Gly to a thallus not yet desensitized resulted in an excitation, i.e. a single AP or AP series. Aspartate (Asp) could not substitute for Glu in any way. Simultaneous action of both amino acids acted synergically to trigger APs. The same phenomenon was observed when glycine solution was enriched with N-methyl-D-aspartic acid (NMDA). Gly-induced APs were totally hindered by 1mM D-amino-5-phosphonopentanoic acid (AP5)--an inhibitor of ionotropic glutamate receptors of the NMDA kind. Glu-induced APs could be totally suppressed by 1mM AP5 as well as by 1mM 6,7-dinitroquinoxaline-2,3-dione (DNQX)--an inhibitor of AMPA/KA receptors. DNQX also completely blocked the calcium component of Glu-evoked APs. After DNQX treatment, the only response to Glu was a membrane potential hyperpolarization (like the Glu response in a desensitized plant). It was concluded that the Glu-induced depolarization and hyperpolarization are separate phenomena. The stimulatory effects of both Glu and Gly on liverwort excitability may be the consequences of an activation of a variety of ionotropic Glu receptor subtypes.