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Plant vacuoles occupy up to 90% of mature plant cells. They participate in turgor regulation, cell signaling, storage of ions and other compounds. Ion channels in the tonoplast play an important role in these processes. A number of ion channels have been characterized both at the electrophysiological and molecular level. The most abundant channels are cation-permeable: slow vacuolar channels, SV/TPC, vacuolar potassium channels, VK/TPK, and anion-selective channels belonging to ALMT and CLC groups. SV channels are permeable to both mono- and divalent cations and activate at high cytoplasmic Ca2+ concentrations [Ca2+]cyt (10–100 µM). They rectify from the cytoplasm to the vacuole. SV channels are regulated by phosphorylation, 14-3-3 proteins, redox potential, and heavy metals. VK channels are K+ -selective, operate at physiological [Ca2+]cyt, and are outwardly rectifying. VMAL channels transport mainly malate from the cytosol to the vacuole. CLC-type proteins exist as channels or NO3 -/H+ transporters.
Plants are able to generate electrical responses to various mechanical, electrical, chemical, or light stimuli causing the disturbance of the balance between cation and anion fluxes through the plasma membrane. The aim of the research was to determine the impact of H2O2 (11–20 mM) on activation of ion channels in the plasma membrane of the liverwort Conocephalum conicum. The study was conducted using intracellular microelectrodes. Application of hydrogen peroxide in concentrations higher than 11 mM resulted in vanishing series of action potentials (APs), leading to permanent depolarization of the membrane potential. Potassium channel inhibitor (TEA, 10 mM) and anion channel inhibitor (A9C, 2 mM) caused the suppression of AP series evoked by H2O2.
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.
The object of our patch-clamp study was the liverwort Marchantia polymorpha. Basic solutions were symmetrical in the bath and in the pipette and contained 100 mM KCl and 0.5 mM CaCl2. Whole vacuole recordings mainly showed slowly activating outward rectifying currents, typical for SV (slowvacuolar) channels. The unitary conductance registered from isolated patches was 74.7±4.0 pS at 100 mV. Replacement of K+ with Na+ caused reduction of the unitary conductance to 59.8±6.87 pS. Tenfold decrease of the KCl shifted the reversal potential close to EK and caused reduction of the SV unitary conductance to 31.5±5.5 pS. Moreover, the gradient of KCl revealed the current at the negative potential; the unitary conductance at −100 mV was 43.3±0.5 pS, whereas the gradient of Na+ did not evoke such an effect. This may suggest that inwardly rectifying K+ channels exist in the tonoplast of the liverwort. Supported by NCN grant 2013/09/B/NZ1/01052
In specialised sensitive plants such as Mimosa, Dionaea, and Aldrovanda, rapid organ movement is observed and the excitationturgor loss mechanism is the basis for rapid leaf or trap closure. In non-specialised ubiquitous plants, slow movement named circumnutation is common and it is driven by turgor. We examine whether transmembrane potential changes such as oscillations and long distance signal action and variation potentials are involved in this movement. Video camera recordings combined with extracellular measurement of electrical potential changes are applied. Additionally, intracellular microelectrodes and patchclamp measurements are engaged. Novel software Circumnutation Tracker has been developed to track organ movement and standardisation of circumnutation parameters. Helianthus annuus and Arabidopsis thaliana are studied and a model of the circumnutation mechanism is proposed.
Apigenin, quercetin and genistein are members of the family of plant flavonoids suspected to prevent a number of human diseases, for instance cancer development. They display a number of activities, and part of their beneficial effects may be due to their affinity to cellular membranes. In this study, we used Conocephalum conicum, a well-elaborated model of liverworts. Intracellular microelectrode measurements were carried out to examine the effects of flavonoids in combination with neomycin on the resting and action potentials. Neomycin triggered gradual decline of action potential amplitudes through a membrane potential decrease (membrane potential became less negative) and a decrease of the action potential peak value. Additionally, duration of action potential amplitudes measured at half of the amplitude increased in neomycintreated plants. However, the simultaneous use of quercetin or genistein (but not apigenin) with neomycin hindered neomycin-specific actions. Hence, the membrane resting potential and action potential amplitudes regained neomycin-free values. It may be concluded that application of at least some flavonoids (namely quercetin and genistein) exerts strong influence on electrical membrane responses in C. conicum.
Black Lipid Membrane (BLM) technique was used to examine effects of BSA (Bovine serum albumin) on properties on the model membrane. BLMs were formed with EYPC (L-αPhospatydylocholine from eggs yolk) in n-dekane. BLM studied were performed in phosphate buffer (pH 7.4). Various concentrations of BSA (between 0.01 mg/ml and 2.5×10-3 mg/ml) were used in order to optimize experimental conditions. BSA concentrations above 0.01 mg/ml proved too high as they caused collapse of the lipid membrane. Supplementation of the protein resulted in change of resistance of the lipid membrane. The experiment indicates that the protein incorporates into the lipid membrane.
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