Kazimierz Trebacz

Electrical Signals in Long-Distance Communication in Plants

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.

Transmission route for action potentials and variation potentials in Helianthus annuus L.

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.

Effects of ion channel inhibitors on cold- and electrically-induced action potentials in Dionaea muscipula

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.

Variation and action potentials evoked by thermal stimuli accompany enhancement of ethylene emission in distant non-stimulated leaves ofVicia faba minorseedlings

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.

Simple method to isolate vacuoles and protoplasts for patch-clamp experiments

It was not possible to obtain protoplasts or vacuoles from the thallus of the liverwortConocephalum conicum by applying cell-wall-degrading enzymes. Therefore, a surgical method was developed to isolate protoplasts and vacuoles. A thallus was plasmolyzed and cut. The few protoplasts along the cutting edge that were not destroyed emerged from the edge under deplasmolysis and became thus accessible for a patch pipette. Whereas under slightly hypoosmolar conditions the emerging protoplast remained largely intact, more hypoosmolar conditions gave rise to isolated vacuoles. This method to isolate protoplasts and vacuoles could also be applied to other plant tissues like leaves ofArabidopsis thaliana. Patch-clamp measurements were performed with isolated vacuoles and excised tonoplast patches. A slowly activating vacuolar channel inC. conicum displayed the characteristic features of higher-plant slowly activating vacuolar channels.

Carotenoid binding to proteins: Modeling pigment transport to lipid membranes

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.

Cadmium and selenium modulate slow vacuolar channels in rape (Brassica napus) vacuoles.

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.

Light- and dark-induced action potentials in Physcomitrella patens

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 La3+ (a calcium channel inhibitor), 10 mM TEA+ (a potassium channel inhibitor), and increased free Ca2+ resulted in a loss of excitability. Lack of Ca2+ 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.

The influence of apigenin on membrane and action potential in the liverwort Conocephalum conicum

Apigenin (4′,5,7-trixydroxyflavone) is a member of the family of plant flavonoids considered to prevent a number of human diseases, for instance cancer development. It displays a lot of activities and part of its beneficial effects could come from its affinity to the cellular membranes. In the present study we used the liverwort Conocephalum conicum, a model plant in electrophysiological study. Intracellular microelectrode measurements were carried out to examine the effects of apigenin alone and in combination with verapamil on the resting and action potentials. The application of apigenin caused an increase of action potential amplitudes. An increase even by 110–131% with respect to the control was observed. Little increase was also found in the membrane potentials in apigenin treated plants. Verapamil, the known calcium channel inhibitor, caused gradual decline of AP amplitudes. When apigenin was used simultaneously with verapamil, still high APs were observed. Duration of action potentials amplitudes measuerd at a half of the amplitude decreased in either apigenin or apigenin and verapamil treated plants to 56–62% of the control. It is concluded that apigenin strongly affects the membranes and prevents inhibitory effect of verapamil probably interacting with calcium channel protein.

Slow vacuolar channels of non-embryogenic and embryogenic cultures of winter wheat

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.