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In response to both biotic and abiotic stresses, vascular plants transmit long-distance Ca2+ and electrical signals from localized stress sites to distant tissues through their vasculature. Various models have been proposed for the mechanisms underlying the long-distance signaling, primarily centered around the presence of vascular bundles. We here demonstrate that the non-vascular liverwort Marchantia polymorpha possesses a mechanism for propagating Ca2+ waves and electrical signals in response to wounding. The propagation velocity of these signals was approximately 1-2 mm s-1, equivalent to that observed in vascular plants. Both Ca2+ waves and electrical signals were inhibited by La3+ as well as tetraethylammonium chloride, suggesting the crucial importance of both Ca2+ channel(s) and K+ channel(s) in wound-induced membrane depolarization as well as the subsequent long-distance signal propagation. Simultaneous recordings of Ca2+ and electrical signals indicated a tight coupling between the dynamics of these two signaling modalities. Furthermore, molecular genetic studies revealed that a GLUTAMATE RECEPTOR-LIKE (GLR) channel plays a central role in the propagation of both Ca2+ waves and electrical signals. Conversely, none of the three two-pore channels were implicated in either signal propagation. These findings shed light on the evolutionary conservation of rapid long-distance Ca2+ wave and electrical signal propagation involving GLRs in land plants, even in the absence of vascular tissue., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Electrical and calcium signals in plants are some of the basic carriers of information that are 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 nor on the relationships between these responses. Here, we show that the external application of hydrogen peroxide (H2O2) 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), and EDTA, a calcium chelator (0.5 mM). The electrical signals were partially dependent on glutamate receptor (GLR) ion channels since knocking-out the 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 H2O2. The measurements carried out on the protonema expressing fluorescent calcium biosensor GCaMP3 proved that calcium signals propagated slowly (>5 µm/s) and showed a decrement. We also demonstrate upregulation of a stress-related gene that appears in a distant section of the moss 8 min after the H2O2 treatment. The results help understand the importance of both types of signals in the transmission of information about the appearance of ROS in the plant cell apoplast., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

An unsolved problem of contemporary plant electrophysiology is the identity of Ca2+ channels responsible for the initiation of the action potential. We took a pharmacological approach and applied several Ca2+ channel blockers (verapamil, tetrandrine, and NED-19) on a Characean (Nitellopsis obtusa ) algae model system. The impact of the selected pharmaceuticals on the parameters of excitation transients of a single cell was analysed employing the two-electrode voltage clamp technique. It was revealed that tetrandrine exerted no effect, while both verapamil and NED-19 prolonged activation and inactivation durations of the excitatory Cl- current. NED-19 also significantly depolarised the excitation threshold membrane potential and shifted Ca2+ current reversal potential. Thus, NED-19 most specifically targeted Ca2+ channels. A viability assay paired with observations of cytoplasmic streaming revealed that verapamil affected not only Ca2+ channels but also exhibited non-specific effects, which eventually lead to cell death. Since many potential Ca2+ channel blockers exert additional undesirable non-specific effects, our study underlines the necessity to search for new more specific modulators of plant Ca2+ transport systems.

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 Ca 2+ 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 Ca 2+ -permeable channels governing plant excitation.

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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 Gd 3+ ), potassium channels (5 mM Ba 2+ ), chloride channels (200 μM Zn 2+ , 50 μM niflumic acid) and proton pumps (10 μM DES), an activator of calcium release from intracellular stores (Sr 2+ ), 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 Ba 2+ and DES; in turn, menthol-evoked potential changes were most effectively blocked by Zn 2+ . 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., (© 2019 Scandinavian Plant Physiology Society.)

Main Conclusion: 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 Ba 2+ , 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 Gd 3+ 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 Zn 2+ , 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.

Carotenoid pigments play numerous important physiological functions in human organism. Very special is a role of lutein and zeaxanthin in the retina of an eye and in particular in its central part, the macula lutea. In the retina, carotenoids can be directly present in the lipid phase of the membranes or remain bound to the protein-pigment complexes. In this work we address a problem of binding of carotenoids to proteins and possible role of such structures in pigment transport to lipid membranes. Interaction of three carotenoids, beta-carotene, lutein and zeaxanthin with two proteins: bovine serum albumin and glutathione S-transferase (GST) was investigated with application of molecular spectroscopy techniques: UV-Vis absorption, circular dichroism and Fourier transform infrared spectroscopy (FTIR). Interaction of pigment-protein complexes with model lipid bilayers formed with egg yolk phosphatidylcholine was investigated with application of FTIR, Raman imaging of liposomes and electrophysiological technique, in the planar lipid bilayer models. The results show that in all the cases of protein and pigment studied, carotenoids bind to protein and that the complexes formed can interact with membranes. This means that protein-carotenoid complexes are capable of playing physiological role in pigment transport to biomembranes., (Copyright © 2015. Published by Elsevier Inc.)

Our previous study has shown that the liverwort Conocephalum conicum generates action potentials (APs) in response to both temperature drop and menthol, which are also activators of the TRPM8 (transient receptor potential melastatin 8) receptor in animals. Not only similarities but also differences between electrical reactions to menthol and cooling observed in the liverwort aroused our interest in the action of menthol at the molecular level. Patch-clamp investigations have shown that menthol causes a reduction of current flowing through slow vacuolar (SV) channels to 29 ± 10% of the initial value (n = 9); simultaneously, it does not influence magnitudes of currents passing through a single SV channel. This may point to an unspecific interaction between menthol and the lipid phase of the membrane. An influence of menthol on lipid organization in membranes was investigated in two-component monomolecular layers formed with menthol and dipalmitoylphosphatidylcholine (DPPC) at the argon-water interface. Analyses of the mean molecular area parameters vs the molar fraction of the menthol component have shown over-additivity (approximately 20 Å(2) ) in the region of high molar fractions of menthol. Infrared absorption spectroscopy studies have shown that menthol, most probably, induces breaking of a hydrogen bond network formed by ester carbonyl groups and water bridges in the lipid membrane and binds to the polar head group region of DPPC. We conclude that the disruption in the lipid phase of the membrane influences ion channels and/or pumps and subsequently causes generation of APs in excitable plants such as C. conicum., (© 2014 Scandinavian Plant Physiology Society.)

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 Ca(2+) and Mg(2+) revealed that the channel was activated by different concentrations of these ions-100 µM Ca(2+) and 10 mM Mg(2+). 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 Ca(2+). 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.

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.

Background: A plant is considered carnivorous if it receives any noticeable benefit from catching small animals. The morphological and physiological adaptations to carnivorous existence is most complex in plants, thanks to which carnivorous plants have been cited by Darwin as 'the most wonderful plants in the world'. When considering the range of these adaptations, one realizes that the carnivory is a result of a multitude of different features., Scope: This review discusses a selection of relevant articles, culled from a wide array of research topics on plant carnivory, and focuses in particular on physiological processes associated with active trapping and digestion of prey. Carnivory offers the plants special advantages in habitats where nutrient supply is scarce. Counterbalancing costs are the investments in synthesis and the maintenance of trapping organs and hydrolysing enzymes. With the progress in genetic, molecular and microscopic techniques, we are well on the way to a full appreciation of various aspects of plant carnivory., Conclusions: Sufficiently complex to be of scientific interest and finite enough to allow conclusive appraisal, carnivorous plants can be viewed as unique models for the examination of rapid organ movements, plant excitability, enzyme secretion, nutrient absorption, food-web relationships, phylogenetic and intergeneric relationships or structural and mineral investment in carnivory.

The mechanism of cold perception by plants is still poorly understood. It was found that temperature drop evokes changes in the activity of ion pumps and channels, which leads to plasma membrane depolarization. The nature of the primary step of its action (alteration in membrane composition, transient influx of Ca2+ etc.,) has not been elicited yet. Our electrophysiological experiments conducted on the liverwort Conocephalum conicum showed that its cells respond not only to sudden cooling but also to menthol, generating depolarization of the plasma membrane and action potentials (APs). Similar results are well documented in mammals; cold or “cooling compounds” including menthol cause activation of thermosenstitive channel TRPM8 permeable to Ca2+ and generation of AP series. TRP receptors are detected, among others, in green and brown algae. Possible existence of TRPM8-like channel-receptor in Conocephalum conicum is discussed here.

In animals, cooling substances such as menthol are perceived as cold sensation because they bind to the same receptor TRPM8 (transient receptor potential melastatin) that activates upon temperature drops. We investigated the effect of menthol on the plant membrane potential to search for analogies between animal and plant perception systems. The study was conducted on the liverwort Conocephalum conicum- a non-vascular plant generating action potentials (APs) in response to different stimuli including cold. (+)Menthol, (-)menthol and (+/-)menthol induced one or more APs, depending on the concentration. In contrast to animal reactions to menthol, threshold concentrations of these isomers were the same (1 mM). The presence of menthol in medium shortened cold-induced APs, whereas low temperature prolonged the repolarization phase of AP evoked by menthol. Cells of C. conicum with anion and potassium channels blocked by anthracene-9-carboxylic acid (A9C) and tetraethylammonium chloride (TEACl) generate short spike-like voltage transients (VTs) in response to cold and light stimulation. Membrane potential changes evoked by menthol in A9C- and TEACl-treated plants differed significantly from VTs - lasted much longer and frequently occurred in series. 5 mM LaCl(3) , 1 mM EGTA (ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid) (0 Ca(2+) ) but not 0.2 mM verapamil blocked the putative calcium component of AP induced by menthol. Similar inhibitory effect was observed after the application of proton pump inhibitors: 0.05 mM N,N-dicyclohexylcarbodiimide (DCCD), 0.05 mM diethylstilbestrol (DES) or 0.01 mM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP). Our results indicate that cold and menthol act independently, activating different membrane transporters in C. conicum cells., (Copyright © Physiologia Plantarum 2011.)

Currents flowing through slow vacuolar SV channels of rape (Brassica napus) growing on media supplemented with Cd²+ (400 μM), and/or SeO₄(²⁻) (2μM) were examined. The aim of the study was to investigate the role of Cd²+ in modulation of SV channel activity and to determine whether Se reverses the effect of cadmium. Vacuoles were isolated using a quick surgical method to avoid application of any cell wall-degrading enzymes. Vacuoles of rape exhibited typical SV channel activity with slow activation at positive potentials and strong rectification into the vacuolar lumen. Single-channel conductance in cytoplasm-side-out tonoplast patches ranged between 68.8±1.9 pS in the control, 80.1±2.5pS, in Cd²+, 74.2±2.4 pS in Cd²+/selenate, and 80.1±1.8 pS in selenate-pretreated plants. The lack of a clear tendency was likely an effect of equilibration of the pipette solution (without Cd²+/SeO₄(²⁻) with that of the luminal side of the vesicles. In the vacuole-attached configuration, in which natural vacuolar solution was not exchanged, there was a significant reduction in single-channel conductance in the Cd²+ (40.3±2.8 pS), Cd²+/selenate (47.1±2.8 pS) and selenate-pretreated (42.3±1.4 pS) plants, compared to the control (60.2±1.7 pS). The reduction in single-channel conductance only partially explained the significant decline in the densities of ion current flowing through the vacuolar membrane in the whole-vacuole configuration in the plants growing on Cd²+ and Cd²+/selenate media. Thus, Cd²+ accumulation in the vacuole reduced the number of active SV channels from 0.28±0.05 μm⁻² in the control to 0.021±0.005 and 0.039±0.004 μm⁻² in Cd²+ and Cd²+/selenate-pretreated plants, respectively., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Genistein (4',5,7-trixydroxyflavone) is a member of the family of plant flavonoids that widely occurs in crop plants. It is involved in a wide spectrum of pharmacological activities, and is suggested to have anti-cancer dietary properties. Cell membranes are one of the targets of anti-cancer drugs. In the present study, we used the liverwort Conocephalum conicum as a model plant in an electrophysiological study. Intracellular microelectrode measurements were carried out to examine the effects of genistein alone and in combination with verapamil on resting and action potentials. The application of isoflavone genistein resulted in a statistically significant elevation in action potential amplitudes. An increase of 13-62% compared with the control was noted. An increase was also found in the membrane resting potentials in genistein-treated plants. Verapamil, the known calcium channel inhibitor, caused a gradual decline of AP amplitudes, whereas preincubation of Conocephalum thalli with genistein prevented inhibition of APs by verapamil. It is concluded that genistein strongly affects the membranes, and the effect of genistein in canceling the activity of verapamil is discussed.

Polar carotenoid pigment zeaxanthin (beta,beta-carotene-3,3'-diol) incorporated into planar lipid membranes formed with diphytanoyl phosphatidylcholine increases the specific electric resistance of the membrane from ca. 4 to 13 x 10(7) Omega cm2 (at 5 mol% zeaxanthin with respect to lipid). Such an observation is consistent with the well known effect of polar carotenoids in decreasing fluidity and structural stabilization of lipid bilayers. Zeaxanthin incorporated into the lipid membrane at 1 mol% has very small effect on the overall membrane resistance but facilitates equilibration of the transmembrane proton gradient, as demonstrated with the application of the H+-sensitive antimony electrodes. Relatively low changes in the electrical potential suggest that the equilibration process may be associated with a symport/antiport activity or with a transmembrane transfer of the molecules of acid. UV-Vis linear dichroism analysis of multibilayer formed with the same lipid-carotenoid system shows that the transition dipole moment of the pigment molecules forms a mean angle of 21 degrees with respect to the axis normal to the plane of the membrane. This means that zeaxanthin spans the membrane and tends to have its two hydroxyl groups anchored in the opposite polar zones of the membrane. Detailed FTIR analysis of beta-carotene and zeaxanthin indicates that the polyene chain of carotenoids is able to form weak hydrogen bonds with water molecules. Possible molecular mechanisms responsible for proton transport by polyenes are discussed, including direct involvement of the polyene chain in proton transfer and indirect effect of the pigment on physical properties of the membrane.

Currents passing through slowly activating vacuolar channels (SV) in isolated vacuoles from winter (Górczański) and spring (Młochowski) varieties of rape (Brassica napus) were examined using the patch-clamp technique. Eight-week-long vernalization at 5/2 degrees C (day/night) was applied to obtain the generative stage of winter rape. SV channels of vacuoles isolated from vegetative (rosette) and generative leaves of both varieties were examined in order to investigate a possible role of these ion channels in rape flowering. Single SV channel conductance measured in a vacuole-attached configuration (natural cell sap) ranged from 60 to 83 pS. Lower values were observed in the generative leaves of both varieties. Unitary conductance measured in excised cytoplasm-out membrane patches did not differ significantly among the experimental variants, with the exception of spring generative vacuoles, where it was significantly lower. There was also no difference in SV current densities measured in the whole-vacuole configuration. Gibberellic acid (GA(3)) (2mg/l) caused lowering of macroscopic SV currents by 20%, and had no significant effect on the single channel conductance. We conclude that SV channels play a role in rape vernalization and flowering owing to their multifactor regulation abilities rather than structural changes.

As a liverwort Conocephalum conicum belongs to the oldest terrestrial plants1 and is phylogenetically located between green algae and higher plants. Recent patch-clamp recordings on Conocephalum vacuoles2,3 demonstrate ion channels very similar to higher plants and clearly different from vacuolar ion channels described in green algae. Here we summarize the features of a vacuolar cation channel and a vacuolar anion channel that both are common in terrestrial plants but are not detected in green algae, and we speculate about the molecular identity of these channels in the liverwort Conocephalum.

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.

Annexins are calcium-dependent phospholipid-binding proteins existing both in animal and plant cells. Mammalian and especially human annexins were examined for many years, and their functions in these organisms are already well known, but it is not the case for plant annexins. On the basis of existing literature and experimental evidence, it can be proposed that plant annexins may have a role in stress response. Annexin At1 of Arabidopsis thaliana (AnnAt1) is one of eight proteins of this family in A. thaliana. In its sequence many potentially functional domains are found, owing to that this protein can play an important role in stress response of the organism. Considering literature data and our own experiments one can postulate that AnnAt1 has weak peroxidase activity and form oligomers in hydrogen peroxide-dependent manner. This can be important in response to oxidative stress. Also we found that this protein forms ion channel in pH-dependent manner. This phenomenon may have particular significance in maintaining calcium homeostasis in the cell and calcium signaling, therefore AnnAt1 may play different roles in regulating stress response of plant. This is extremely important because plants during growth and development have to cope different stress factors like drought, deficiency or excess of mineral compounds in the soil, as well as low or high temperatures.

A pH sensitive fluorescence probe piranine trisulfonate, entrapped inside small unilamellar liposomes formed with egg yolk phosphatidylcholine, was applied to investigate effect of polyene antibiotic amphotericin B (AmB) on proton transport across lipid membranes. Time dependencies of fluorescence-monitored pH changes inside lipid vesicles, upon sudden acidification of the liposome suspension, were analyzed in terms of two-exponential kinetics. It appears that addition of AmB at 3 mol%, with respect to lipid, considerably increases the rate constant of the fast component of proton transport (a change from (60 to 149) x 10(-3)s(-1)) and decreases the rate constant of the slow component (a change from (11 to 5) x 10(-3)s(-1)). Incorporation of 0.1 mol% AmB results in the decrease of both parameters (to (33 and 2) x 10(-3)s(-1), respectively). The increase in the rate of proton transfer across the lipid membrane is interpreted as related to the formation of membrane channels by AmB, at higher concentration of the drug or nonspecific destabilization of the membrane structure. At low concentrations, at which formation of molecular structures of AmB is not possible, the antibiotic molecules are oriented horizontally with respect to the plane of the membrane and act in making the membrane more compact and less permeable to ions. The presence of sterols (cholesterol, ergosterol and cholesterol dimer) in the lipid phase, in the concentration 3 mol% and lower, decreased the rate constants of proton transfer across the membranes but did not influence significantly the effect of AmB on the ion transport. The presence of AmB in the bathing solutions of tonoplast membranes isolated from Conocephalum conicum at the concentrations range 1 x 10(-7) to 3.6 x 10(-5) does not influence considerably the ion current, as monitored by means of the patch-clamp technique.

Glass microelectrodes were inserted into mesophyll cells of intact leaves from higher plants: Arabidopsis thaliana, Helianthus annuus and Vicia faba var minor, and transient membrane potential changes were recorded in response to a sudden temperature drop. The cold-induced potential changes were unaffected by an anion channel inhibitor (anthracene-9-carboxylic acid) and potassium channel inhibitor (tetraethyl ammonium ion). Verapamil, a calcium channel inhibitor, caused significant suppression of the cold-induced potential changes. In the presence of calmoduline antagonists (trifluoperazine and N-6-aminohexyl-5-chloro-1-naphtalenesulphonamide), their amplitudes decreased and their durations were prolonged. Neomycin, which suppresses phospholipase C, also caused substantial inhibition of the amplitudes of the cold-induced potential changes. It is concluded that cold-evoked membrane potential changes are due to calcium influxes from both the apoplast and internal stores.

Electrical activity (action and variation potentials) and ethylene emission were measured in thermally stimulated Vicia faba minor seedlings. It was determined that variation potential with or without super-imposed action potentials was generated and propagated basipetally in response to scorching of the upper leaf. In stimulated plants the level of ethylene production measured in lower, non stimulated leaf was significantly higher than that in the control plants and the difference correlated with the amplitude of the electrical response. Neither variation nor action potential was recorded when ethylene was injected to the chamber covering the experimental leaf. The level of ethylene emission showed clear circadian rhythm when measured at photoperiod 16:8 (LD) or at constant light (LL). It is concluded that the sequence of ion fluxes registered as an electrical response of a plant to the thermal stimulus is a signal evoking an enhancement of ethylene emission.

Intracellular microelectrode measurements revealed that the liverwort Conocephalum conicum generates all-or-none action potentials (APs) in response to a sudden temperature drop. In plants with anion and potassium conductance blocked, dose-dependent voltage transients (VTs) were evoked by cold stimuli. These VTs did not propagate. When the external concentration of Ca(2+) was decreased or calcium channel inhibitors (La(3+), Gd(3+), verapamil, Mg(2+), Mn(2+)) were used, inhibition of VTs was observed. Amplitudes of both APs and VTs grew when Sr(2+) ions, known to release calcium from internal stores, were added to the medium. Neomycin, which suppresses phospholipase C and indirectly affects inositol triphosphate formation, caused substantial inhibition of both APs and VTs. It is concluded that a temperature drop elucidated membrane potential changes due to calcium influx both from external and internal stores.

The relationship between evoked electrical activity and stem movements in three-week old sunflowers was demonstrated. Electrical potential changes (recorded by Ag/AgCl extracellular electrodes) and time-lapse images (from a top view camera) were recorded and analyzed. A heat stimulus applied to the tip of one of the second pair of leaves evoked a variation potential, transmitted basipetally along one side of the stem. After stimulation, disturbances of circumnutations occurred. They included: changes in the period, disorders in the elliptical shape, and, in some cases, reversion of direction (of movement). We suggest that asymmetrically propagated variation potential induces asymmetric stem shrinking and bending, which strongly disturbs circumnutations. Our results confirm the involvement of electrical potential changes in the mechanism of stem nutations.

A diaheliotropic response of sunflower, Helianthus annuus, following a short pulse of low intensity blue light to a small area of leaf surface was examined with the application of the very low-force-measurements technique (the order of magnitude of 10(-5) N). One leaf from a pair was illuminated with a low intensity blue-light-pulse and the force was recorded, generated by the stem of the plant tending to bend. A very low phototropic effect in response to blue light alone was observed which could be considerably enhanced by the application of background illumination with red light. Microelectrode measurements of the membrane potential of the mesophyll cells of the sunflower leaf showed hyperpolarization in response to a blue light pulse, observed very clearly under application of the red light background illumination. The hyperpolarization of the membrane potential was accompanied by acidification of extracellular compartments as monitored with a miniature pH-sensitive electrode, placed in the epidermis of the stem. The relatively short lag period between the hyperpolarization of the membrane potential and the decrease in pH suggests that the hyperpolarization is a direct effect of the blue light-induced proton extrusion. The acidification correlates with the light response, which suggests that acidification-induced stem wall loosening is responsible for the blue light-induced bending. The examined mechanisms are discussed in terms of sun tracking by a sunflower.

Electrophysiological methods, such as intracellular potential recording, ion activity measurements, patch-clamp were described. Special attention was paid to their modifications allowing application to plant cells.

Evoking of action potentials (APs) in the trap of Dionaea muscipula Ellis at intervals shorter than 20 s caused a gradual decrease in the amplitude of the APs. At longer intervals the amplitude was constant. The calcium ionophore A23187 (1 μM) caused a considerable decrease of AP amplitude. Pretreatment of a segment of the Dionaea trap with cyclopiazonic acid (CPA), which is a specific inhibitor of the Ca 2+ -ATPase in the sarcoplasmic seticulum of animal cells and in ER vesicles isolated from plant cells, only slightly affected the amplitude when APs were evoked every 10 min; however, it caused a considerable decrease in the amplitude when the stimulation was repeated every 2 min. Assuming that APs increase the concentration of cytosolic Ca 2+ and the amplitude of AP depends on the gradient of Ca 2+ across the plasma membrane, the effect of CPA on the AP amplitude indicates that CPA inhibits the sequestration of Ca 2+ in Dionaea cells.