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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.

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.

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.

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.

Under electrical stimulation of Lupinus stem phenomena of accommodation and anode break excitation appear. Their characteristic is the same as in the axon or nerve. Only their duration is about 103-104 longer in plants. The constant characterizing the rate of accommodation was calculated. A limiting threshold value was found beyond which excitation occurs, irrespective of the rate of stimulus rise (voltage gradient). The accommodation rate is approximately constant, whereas the range of accommodation varies and is dependent on the difference between the rheobase value and the limiting threshold value. Hence plants with a low rheobase are characterized by a wider range of accommodation. It is suggested that the changes in potential (including AP) recorded on the stem surface are connected with changes of the potential on cell membranes (Sibaoka, 1962).

Action potentials (APs) in plants are involved in fast leaf or trap closure as well as elongation, respiration, photosynthesis, and fertilization regulation. Here, spontaneous APs (SAPs) in relation to endogenous stem movement named circumnutation (CN) have been investigated in Helianthus annuus in different light conditions in freely circumnutating and immobilized plants. Extracellular electrical measurements and time-lapse photography were carried out simultaneously. The parameters of CN (trajectory length, period, and direction) and the number and transmission direction of SAPs were analysed. In continuous light (25–40 μmol m−2 s−1), all plants circumnutating vigorously in a regular elliptical manner and no SAPs were observed. In light/dark conditions, the plants circumnutated in a daily pattern, most SAPs were observed in the dark and freely circumnutated sunflowers had two times more SAPs (10 SAPs/24 h/plant) than the immobilized plants (5 SAPs/24 h/plant). In continuous very low light (5 μmol m−2 s−1), the plants circumnutated weakly and irregularly and SAPs appeared without the circadian pattern. 3–5 SAPs/24/plant occurred in the freely circumnutating and immobilized plants. In light/dark and continuous very low light conditions, an ultradian rhythm of SAPs was observed and the mean spacing between SAPs was approx. 121–271 min in the freely circumnutating and immobilized plants. Under all light conditions, more SAPs were transmitted basipetally than acropetally. One-hour lasting series of 3–4 min spaced SAPs locally propagated were observed as well in very low light. Basipetal and acropetal SAPs passing along the stem motor region accompany irregularity, changes in the CN trajectory direction, and stem torsion. These results demonstrate that APs and CN changes play a role in plant adaptation to light conditions and that there is an ultradian rhythm of SAPs beside ultradian CN rhythm.

Quercetin (3,3′,4′,5,7-pentahydroxyflavone) is claimed to exert many beneficial health effects. With application of 1H NMR (nuclear magnetic resonance) and FTIR (Fourier-transform infrared) techniques, quercetin interaction with liposomes formed with dipalmitoylphosphatidylcholine (DPPC) was analyzed. Patch-clamp technique was employed to study quercetin effects at single channel level of vacuolar membranes in the liverwort Conocephalum conicum. Light and electron microscopy were applied to study quercetin effects on human negroid cervix carcinoma cells (HeLa). Enzymatic measurements along with DPPH (1,1-diphenyl-2-picrylhydrazyl) bioassay were performed to investigate the influence of quercetin on antioxidant enzymes and reactive oxygen species (ROS) production. The inclusion of quercetin to the membrane exerted pronounced ordering effect on the motional freedom of lipids in the head group region as manifested by broadening of the 1H NMR spectral line representing the choline groups. FTIR analysis revealed quercetin incorporation into DPPC liposomes via hydrogen bonding between its own hydroxyl groups and lipid polar head groups in the C–O–P–O–C segment. Both, FTIR and NMR techniques indicated also quercetin spectral effects in the region corresponding to alkyl chains. Patch-clamp experiments showed that quercetin stabilizes tonoplast and promotes a close state of SV channels. Microscopic observations of HeLa cells revealed characteristic changes in ultrastructure and morphology of the examined cells in comparison to control cells. Pretreatment of HeLa cells with quercetin alleviated H2O2-induced cell injury by improving redox balance as indicated by the increase in glutathione content and SOD (superoxide dismutase) levels as well as by the decrease in ROS level. \In conclusion, the incorporation, distribution and the changes of biophysical properties of the membranes are very important for the effectiveness of phenolic compounds as antioxidant and anticancer factors.

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.

Plants are subject to stimuli from the environment on which they strongly depend and in contrast to animals, they are unable to escape harmful influences. Therefore, being able to receive stimuli they have developed adequate responses to them. Such a reaction can occur in the area of a stimulus action or cover the whole plant or its parts. In the latter case, it is a systemic reaction. The plant reaction is expressed by various intensity, rate and kind of response. It is interesting to know the character of the signal informing about a stimulus, the routes of its propagation and the transmission mechanism. Three conceptions of excitation are distinguished: 1) propagation of chemical agents formed at the site of a stimulus action with the flow of the phloem sap or through the atmosphere (in the case of volatile substances) to other plant parts, 2) a very fast transmission by the xylem in the wave of hydraulic pressure formed after a plant damage. From combining the "hydraulic" and "chemical" hypothesis a conception of hydraulic dispersion has been formulated which assumes that chemical substances synthetized after an injury can be transferred very fast with the wave of hydraulic pressure changes in the whole plant, 3) a stimulus evokes the action potential (AP), and its transmission along the whole plant, plant organ or specialized tissue, by local circuits from cell to cell. Strong, damaging stimuli can evoke variation potentials (VPs), the character of which differs from APs. It is postulated that transmission of VP occurs by a hydraulic dispersion and electrical changes seem to be secondary phenomena.

Currents flowing through slow vacuolar SV channels of rape ( Brassica napus ) growing on media supplemented with Cd 2+ (400 μM), and/or SeO 4 2− (2 μM) were examined. The aim of the study was to investigate the role of Cd 2+ 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 ± 2.5 pS, in Cd 2+ -, 74.2 ± 2.4 pS in Cd 2+ /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 Cd 2+ /SeO 4 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 Cd 2+ - (40.3 ± 2.8 pS), Cd 2+ /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 Cd 2+ and Cd 2+ /selenate media. Thus, Cd 2+ 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 Cd 2+ and Cd 2+ /selenate-pretreated plants, respectively.

In plants, an electrical potential and circumnutation disturbances are a part of a response to environmental and internal stimuli. Precise relations between electrical potential changes and circumnutation mechanisms are unclear. We have found recently that glutamate (Glu) injection into Helianthus annuus stem induced a series of action potentials (APs) and a transient decrease in circumnutation activity. A theoretical explanation for this finding is discussed here taking into considerations data about the ion mechanism of AP and circumnutation as well as about the metabolic and signaling pathways of glutamate and their possible interactions.

Reports concerning the function of glutamate (Glu) in the electrical and movement phenomena in plants are scarce. Using the method of extracellular measurement, we recorded electrical potential changes in the stem of 3-week-old Helianthus annuus L. plants after injection of Glu solution. Simultaneously, circumnutation movements of the stem were measured with the use of time-lapse images. Injection of Glu solution at millimolar (200, 50, 5 mM) concentrations in the basal part of the stem evoked a series of action potentials (APs). The APs appeared in the site of injection and in different parts of the stem and were propagated acropetally and/or basipetally along the stem. Glu injection also resulted in a transient, approximately 5-h-long decrease in the stem circumnutation rate. The APs initiated and propagating in the sunflower stem after Glu injection testify the existence of a Glu perception system in vascular plants and suggest its involvement in electrical, long-distance signaling. Our experiments also demonstrated that Glu is a factor affecting circumnutation movements.

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.

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.

Glass microelectrodes were inserted into Dionaea muscipula (Venus flytrap) lobes and the action potentials (APs) were recorded in response to a sudden temperature drop or a direct current (DC) application. The effect of potassium channel inhibitor, tetraethylammonium ion, was the lengthening of the depolarization phase of AP. APs were also affected by the anion channel inhibitor, anthracene-9-carboxylic acid, that made them slower and smaller. Neomycin, which disturbs inositol triphosphate-dependent Ca2+ release, caused the visible inhibition of AP, too. Ruthenium red, which blocks cyclic ADP-ribose-dependent Ca2+ release, totally inhibited DC-triggered APs and induced the decrease in the amplitudes of cold-evoked APs. Lanthanum ions significantly inhibited both cold- and DC-induced membrane potential changes. It was concluded that during excitation Dionaea muscipula relied upon the calcium influxes from both the extra- and intracellular compartments.

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) × 10 −3 s −1 ) and decreases the rate constant of the slow component (a change from (11 to 5) × 10 −3 s −1 ). Incorporation of 0.1 mol% AmB results in the decrease of both parameters (to (33 and 2) × 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 × 10 −7 to 3.6 × 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.

Slowly activating vacuolar channels (SV), were examined in embryogenic and non-embryogenic cultures of winter wheat using a patch-clamp technique. Four different types of cultures were examined: embryogenic and non-embryogenic calli from embryos, embryogenic and non-embryogenic calli from inflorescences. In a cell-attached mode single SV channel events were recorded. Unitary conductance of single SV channels was between 37 pS and 48 pS and did not significantly depend on the kind of the culture, although it was a tendency that SV channels of embryogenic calli possessed lower unitary conductance than those of non-embryogenic. 2,4-D caused significant lowering of unitary conductance from 48±6 pS in the control culture of embryogenic embryos to 28±6 pS in vacuoles treated. The SV channel density was estimated as 0.34 µm−2.

Summary 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 superimposed 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.

Summary Helianthus annuus generates two types of electrical responses to different kinds of stimuli. Non-damaging electrical stimulation evokes action potentials. Damaging, thermal stimuli trigger complex responses: long lasting variation potentials with superimposed action potentials. Electrically evoked action potentials are transmitted along the stem both acro- and basipetally, and on its way, excitation covers nearly all living tissues. Action potentials usually do not enter leaves and cease in petioles. Variation potentials appearing in response to thermal stimulation of one leaf spread preferentially down the leaf trace, probably along conducting bundles. Electrodes inserted into the vicinity of conducting bundles coming from the stimulated leaf register variation potentials, while those inserted on the opposite side of the stem register no change or responses of strongly reduced amplitudes. We point out that electrode arrangement during the monitoring of the transmission of electrical signals in plants is of great importance when physiological responses of excited cells are simultaneously investigated.

A novel experimental set-up and method of recording of electrical potential differences in plants have been developed which enable continuous, 8-channel monitoring of electrical activity over extended periods of time using inserted, extracellular electrodes. The investigations were carried out on 21- to 23-day-old Helianthus annuus plants, and spontaneously-generated action potentials were recorded during monitoring sessions lasting for 3 days and nights. Characteristics of these spontaneous action potentials were elaborated, adopting as parameters their typical form, amplitude, duration, velocity, direction, and distance of propagation and frequency of occurrence in morphologically different parts of the plant, Variability, similarities, and interdependence of the above parameters in individual plants and in a group of 15 plants were determined. A hypothesis concerning propagation of action potentials in plants along specific impulse-propagating ‘columns’ is discussed. The frequency of generated impulses is highest at night and lowest in the day and also displays an apparent 24-h rhythm. Presumably this mechanism is under both endogenous and exogenous control and may be partly dependent on a biological clock.

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 action potentials induced by nondamaging electrical stimuli in 16- to 22-day-old plants of Helianthus annuus were examined. Typical recordings are presented. Mean values of their amplitudes and conduction velocities in the stem, the strength-duration relation, the ‘all-or-none’ law and the refractory periods have been determined. The amplitude and velocity of propagation were essentially identical in the upward and downward direction, but greater in the upper than in the lower half. In ‘electrically active’ plants, the rheobase value is 2 V, the minimum period for stimulation is 1.8 s. and the chronaxie 2.3 s. It is noted that the excitability level between similar plants on the same day and in the same plant on different days is highly variable and undergoes periodic changes.

Isolated vacuoles of the liverwort Conocephalum conicum thallus cells were investigated using the patch-clamp technique. At high cytosolic Ca(2+) activities, slowly activating currents were evoked by positive potentials. The currents were conducted by the SV (slow-vacuolar) channel. When isolation of vacuoles was carried out at high Mg(2+) and low Ca(2+) concentration and the same proportion of the cations was kept in the bath, currents were recorded at negative potentials. Once activated, these currents persisted even after replacing Mg(2+) with K(+) in the bath. Sr(2+) and Ba(2+) were also effective activators of the currents. With a Cl(-) gradient, 10 mM in the bath and 100 mM in the lumen, currents were significantly reduced and the current-voltage characteristics shifted towards the reversal potential of Cl(-), indicating Cl(-) selectivity. Currents almost vanished after substituting Cl(-) with gluconate. They were strongly reduced by anion channel inhibitors 4,4'-diisothicyanatostilbene-2,2'-disulfonic acid (DIDS; 1 mM), anthracene-9-carboxylic acid (A9C; 2 mM) and ethacrinic acid (0.5 mM). Single-channel recordings revealed a 32 pS channel activating at negative voltages. It is concluded that the currents at negative potentials are carried by anion channels suitable for conducting anions from the cytosol to the vacuole. The anion channels were weakly calcium dependent, remaining active at physiological calcium concentration. The channels were almost equally permeable to Cl(-), NO3(-) and SO4(2-), and much less permeable to malate(2-). Anion channels did not respond to ATP addition. cAMP (10 microM) had a weak effect on anion channels. Protein kinase A (0.4 U) added to the medium caused no significant effect on anion channels.

The growth and circumnutation of the stem of three-week old Helianthus annuus in the 16:8 h light:dark photoperiod were monitored using an angular position-sensing transducer and a time lapse photography system. It was found that the rate of growth and circumnutation reached a high level in the dark stage; in the light stage, however, only the growth rate reached the same high level, whereas the circumnutations were weak. These results showed that in the light stage the stem circumnutation was downregulated more strongly than the growth. Short-term stem responses to darkening and illumination were a further display of the relation between growth and circumnutations. Switching off the light caused an increase in the growth and circumnutation rate. In some cases it was accompanied by changes in the rotation direction. On the other hand, switching the light on caused an immediate transient (several-minute long) decrease in the growth rate resulting in stem contraction, and this was accompanied by an almost complete pause of circumnutation. Additionally, under light, there occurred a subsequent decrease in the magnitude, disturbance of circumnutation trajectory and, in some cases, changes in the direction of rotation. The observed stem contraction and disturbance of circumnutation imply the occurrence of turgor changes in sunflower stem, which may be caused by a non-wounding, darkening or illumination stimulus. Our experiments indicate that the disturbances of the growth rate are accompanied by changes in circumnutation parameters but we have also seen that there is no simple quantitative relation between growth rate and circumnutation rate.

Action potentials (APs) belong to long-distance signals in plants. They fulfill the all-or-none law, propagate without decrement and their generation is limited by refractory periods. The ion mechanism of APs was elaborated in giant Characean algae and extended by another model plant — the liverwort Conocephalum conicum. It consists of an increase in cytoplasmic Ca2+ concentration ([Ca2+]cyt) which activates anion channels responsible for Cl− efflux and for membrane depolarization. Repolarization occurs after the opening of potassium channels and K+ efflux. The resting potential is restored by the electrogenic proton pump. A number of ion channels which may play a role in AP were identified by the patch-clamp technique. APs propagate on the principle of local electrical circuits. They cover whole plants, plant organs or definite tissues, mainly phloem, phloem parenchyma and protoxylem. APs mediate between local stimulation and movements in carnivorous Dionaea muscipula, Aldrovanda vesiculosa, and tigmonastic Mimosa pudica. The role of APs in regulation of respiration, photosynthesis, growth, pollination, fertilization and gene expression is well documented. An AP-coupled increase in [Ca2+]cyt seems to play a central role in signal transduction.

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.

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.

Background An endogenous, helical plant organ movement named circumnutation is ubiquitous in the plant kingdom. Plant shoots, stems, tendrils, leaves, and roots commonly circumnutate but their appearance is still poorly described. To support such investigations, novel software Circumnutation Tracker (CT) for spatial-temporal analysis of circumnutation has been developed. Results CT works on time-lapse video and collected circumnutation parameters: period, length, rate, shape, angle, and clockwise- and counterclockwise directions. The CT combines a filtering algorithm with a graph-based method to describe the parameters of circumnutation. The parameters of circumnutation of Helianthus annuus hypocotyls and the relationship between cotyledon arrangement and circumnutation geometry are presented here to demonstrate the CT options. Conclusions We have established that CT facilitates and accelerates analysis of circumnutation. In combination with the physiological, molecular, and genetic methods, this software may be a powerful tool also for investigations of gravitropism, biological clock, and membrane transport, i.e. processes involved in the mechanism of circumnutation.

The characteristics of the potential changes caused by excitation of the thallus of Conocephalum conicum L. were investigated and compared with the basic criteria of an action potential. Electrical stimulation (square DC pulses) was applied, and extraand intracellular potentials were recorded. The all-or-nothing law, strength-duration relation, refractory periods, and general characteristics of the changes of transmembrane potential difference were examined. The electrical phenomena occurring during the excitation of Conocephalum fulfil the basic criteria of an action potential. The results contribute additional evidence for the hypothesis that action potentials occur both in plants and in animals.

Calculations of the resistance r and capacity c of cell membrances and the resistancer 1 of cell interiors of a community of cells in Marchantia polymorpha L. thalli are presented. These parameters of a multicellular system were determined by the adaptation of methods employed for the calculation of the resistance and capacity of single cells. The obtained results indicate that such a procedure is justified. A generally accepted resistance-capacity model of the cell was used as a basis for the determination of r, c, and r1 (representing membrane resistance, membrane capacity, and resistance of cell interior, respectively). The calculations were based on measurements of impedance and phase shift within the frequency range of 5 Hz-1000 Hz. Stainless steel plates were employed as the measuring electrodes; polarization resistance and capacity were determined by separate measurements. The calculations confirmed the assumption that the parameters r, c, and r1 were constant within the investigated frequency range. The calculations of resistance and capacity for 25 plants were constant within the investigated frequency range. The calculations of resistance and capacity for 25 plants were carried out by four different methods and they yielded results of the order of : r = 0.45 kΩ± 0.15 kΩ, r1= 1.0 kΩ± 0.45 kΩ, c = 11 μF ± 3.5 μF. Circular diagrams of impedance also confirmed the validity of the accepted model within the frequency range of 25–300 Hz.

Simultaneous measurements were taken of the electrical activity and the rate of respiration of thalli of Conocephalum conicum L. stimulated electrically and mechanically (by cutting). The measurements of the rate of respiration employed a modified Warburg apparatus for O2 consumption and an infra-red gas analyzer with computer recording and data processing for CO2 evolution. The action potential, produced by either a cut (a damaging stimulus) or an electrical stimulus (a non-damaging stimulus), caused a transient rise in the rate of respiration. The course of changes in the rate of respiration depends on the character of the excitation and the area of the thallus covered by it. If stimulation does not produce excitation, the increase in the rate of respiration does not take place, regardless of the magnitude and type of the stimulus applied.

The aim of the present study was to characterise bioelectrical changes in the membrane potential of Marchantia polymorpha gametophyte cells after light/dark transitions and to determine the role of the proton pump and energy status of the M. polymorpha cells in generation of these changes. Darkening caused persistent depolarisation of the resting potential (RP) and generation of short-lasting potential changes that were not uniform among different thalli. In some plants (18%), the changes evoked by darkening were typical action potentials (APsdark), whereas in 69% of the plants, the changes had a form of action potential-like responses (APsdark-like) consisting of a transient depolarisation followed by a plateau phase, whose magnitude and duration were inconstant. The illumination of the M. polymorpha always evoked action potentials (APslight) if the thallus was illuminated with light intensity of at least 120 µmol photons m−2 s−1 after 30-min darkening. To analyse the involvement of H+-ATPase in formation of the illumination/darkening-induced electrical responses in M. polymorpha, the proton pump regulators were used. The proton pump inhibitor (20 µM FCCP) significantly diminished the RP and inhibited dark-induced APdark and/or APdark-like responses and illumination-induced APslight. After application of DCMU (20 µM), the RP was strongly depolarised and no response to light/dark was observed. Fusicoccin (20 µM), i.e., an activator of the proton pump, strongly hyperpolarised the membrane potential and blocked dark-induced APdark/APdark-like responses and illumination-induced APslight.

Action potentials generated spontaneously (SAPs) and evoked by electrical stimulation (APs) in tomato plants (Solanum lycopersicum L.) cv. Micro-Tom ABA-deficient mutants (sitiens—MTsit) and its wild type (MTwt) were characterized by continuous monitoring of electrical activity for 66 h and by application of an electrical stimulation supplied extracellularly. MTsit generated SAPs which spread along the stem, including petioles and roots with an amplitude of 44.6 ± 4.4 mV, half-time (t½) of 33.1 ± 2.9 s and velocity of 5.4 ± 1.0 cm min−1. Amplitude and velocity were 43 and 108 % higher in MTsit than in MTwt, respectively. The largest number of SAPs was registered in the early morning in both genotypes. MTsit was less responsive to electrical stimuli. The excitation threshold and the refractory period were greater in MTsit than in MTwt. After current application, APs were generated in the MTwt with 21.2 ± 2.4 mV amplitude and propagated with 5.6 ± 0.5 cm min−1 velocity. Lower intensity stimuli did not trigger APs in these plants. In MTsit APs were measured with amplitude of 26.8 ± 4.8 mV and propagated with velocity of 8.5 ± 0.1 cm min−1.