Electrophysiology of pumpkin seeds: Memristors in vivo.

Leon Chua, the discoverer of a memristor, theoretically predicted that voltage gated ion channels can be memristors. We recently found memristors in different plants such as the Venus flytrap, Mimosa pudica, Aloe vera, apple fruits, and in potato tubers. There are no publications in literature about the existence of memristors in seeds. The goal of this work was to discover if pumpkin seeds might have memristors. We selected Cucurbita pepo L., cv. Cinderella, Cucurbita maxima L. cv Warty Goblin, and Cucurbita maxima L., cv. Jarrahdale seeds for this analysis. In these seeds, we found the presence of resistors with memory. The analysis was based on cyclic voltammetry where a memristor should manifest itself as a nonlinear two-terminal electrical element, which exhibits a pinched hysteresis loop on a current-voltage plane for any bipolar cyclic voltage input signal. Dry dormant pumpkin seeds have very high electrical resistance without memristive properties. The electrostimulation by bipolar sinusoidal or triangular periodic waves induces electrical responses in imbibed pumpkin seeds with fingerprints of memristors. Tetraethylammonium chloride, an inhibitor of voltage gated K(+) channels, transforms a memristor to a resistor in pumpkin seeds. NPPB (5-Nitro-2-(3-phenylpropylamino)benzoic acid) inhibits the memristive properties of imbibed pumpkin seeds. The discovery of memristors in pumpkin seeds creates a new direction in the understanding of electrophysiological phenomena in seeds.

An analytical model of memristors in plants.

The memristor, a resistor with memory, was postulated by Chua in 1971 and the first solid-state memristor was built in 2008. Recently, we found memristors in vivo in plants. Here we propose a simple analytical model of 2 types of memristors that can be found within plants. The electrostimulation of plants by bipolar periodic waves induces electrical responses in the Aloe vera and Mimosa pudica with fingerprints of memristors. Memristive properties of the Aloe vera and Mimosa pudica are linked to the properties of voltage gated K(+) ion channels. The potassium channel blocker TEACl transform plant memristors to conventional resistors. The analytical model of a memristor with a capacitor connected in parallel exhibits different characteristic behavior at low and high frequency of applied voltage, which is the same as experimental data obtained by cyclic voltammetry in vivo.

Memory elements in the electrical network of Mimosa pudica L.

The fourth basic circuit element, a memristor, is a resistor with memory that was postulated by Chua in 1971. Here we found that memristors exist in vivo. The electrostimulation of the Mimosa pudica by bipolar sinusoidal or triangle periodic waves induce electrical responses with fingerprints of memristors. Uncouplers carbonylcyanide-3-chlorophenylhydrazone and carbonylcyanide-4-trifluoromethoxy-phenyl hydrazone decrease the amplitude of electrical responses at low and high frequencies of bipolar sinusoidal or triangle periodic electrostimulating waves. Memristive behavior of an electrical network in the Mimosa pudica is linked to the properties of voltage gated ion channels: the channel blocker TEACl reduces the electric response to a conventional resistor. Our results demonstrate that a voltage gated K(+) channel in the excitable tissue of plants has properties of a memristor. The discovery of memristors in plants creates a new direction in the modeling and understanding of electrical phenomena in plants.

Morphing structures of the Dionaea muscipula Ellis during the trap opening and closing.

The Venus flytrap is a marvelous plant that has intrigued scientists since the times of Charles Darwin. This carnivorous plant is capable of very fast movements to catch a prey. We found that the maximal speed of the trap closing in the Dionaea muscipula Ellis is about 130,000 times faster than the maximal speed of the trap opening. The mechanism and kinetics of this movement was debated for a long time. Here, the most recent Hydroelastic Curvature Model is applied to the analysis of this movement during closing and opening of the trap with or without a prey. Equations describing the trap movement were derived and verified with experimental data. Chloroform and ether, both anesthetic agents, induce action potentials and close the trap without the mechanical stimulation of trigger hairs. We tested this by dropping 10 µL of ether on the midrib inside the trap without touching any of the mechanosensitive trigger hairs. The trap closed slowly in 10 s. This is at least 20 times slower than the closing of the trap mechanically or electrically. The similar effect can be induced by placing 10 µL of chloroform on the midrib inside the trap, however, the lobes closing time in this case is as fast as closing after mechanical stimulation of the trigger hairs.

Memristors in plants.

We investigated electrical circuitry of the Venus flytrap, Mimosa pudica and Aloe vera. The goal was to discover if these plants might have a new electrical component--a resistor with memory. This element was postulated recently and the researchers were looking for its presence in different systems. The analysis was based on cyclic current-voltage characteristic where the resistor with memory should manifest itself. We found that the electrostimulation of plants by bipolar sinusoidal or triangle periodic waves induces electrical responses in the Venus flytrap, Mimosa pudica and Aloe vera with fingerprints of memristors. Tetraethylammonium chloride, an inhibitor of voltage gated K(+) channels, transforms a memristor to a resistor in plant tissue. Our results demonstrate that a voltage gated K(+) channel in the excitable tissue of plants has properties of a memristor. This study can be a starting point for understanding mechanisms of memory, learning, circadian rhythms, and biological clocks.

Memristors in the electrical network of Aloe vera L.

A memristor is a resistor with memory, which is a non-linear passive two-terminal electrical element relating magnetic flux linkage and electrical charge. Here we found that memristors exist in vivo. The electrostimulation of the Aloe vera by bipolar sinusoidal or triangle periodic waves induce electrical responses with fingerprints of memristors. Uncouplers carbonylcyanide-3-chlorophenylhydrazone and carbonylcyanide-4-trifluoromethoxy-phenyl hydrazone decrease the amplitude of electrical responses at low and high frequencies of bipolar periodic sinusoidal or triangle electrostimulating waves. Memristive behavior of an electrical network in the Aloe vera is linked to the properties of voltage gated ion channels: the K(+) channel blocker TEACl reduces the electric response to a conventional resistor. Our results demonstrate that a voltage gated K(+) channel in the excitable tissue of plants has properties of a memristor. The discovery of memristors in plants creates a new direction in the modeling and understanding of electrical phenomena in plants.

Memristors in the Venus flytrap.

A memristor is a nonlinear element because its current-voltage characteristic is similar to that of a Lissajous pattern for nonlinear systems. We investigated the possible presence of memristors in the electrical circuitry of the Venus flytrap's upper and lower leaves. The electrostimulation of this plant by bipolar sinusoidal or triangle periodic waves induces electrical responses in the upper and lower leaves of the Venus flytrap with fingerprints of memristors. The analysis was based on cyclic voltammetric characteristics where the memristor, a resistor with memory, should manifest itself. Tetraethylammonium chloride, an inhibitor of voltage gated K(+) channels, or NPPB, a blocker of voltage gated Cl(-) and K(+) channels, transform a memristor to a resistor in plant tissue. Uncouplers carbonylcyanide-3-chlorophenylhydrazone (CCCP) and carbonylcyanide-4-trifluoromethoxy-phenyl hydrazone (FCCP) decrease the amplitude of electrical responses at low and high frequencies of bipolar periodic electrostimulating waves. Our results demonstrate that voltage gated K(+) channels in the Venus flytrap have properties of memristors of type 1 and type 2. The discovery of memristors in plants creates a new direction in the modeling and understanding of electrical phenomena in plants.

Morphing structures and signal transduction in Mimosa pudica L. induced by localized thermal stress.

Leaf movements in Mimosa pudica, are in response to thermal stress, touch, and light or darkness, appear to be regulated by electrical, hydrodynamical, and chemical signal transduction. The pulvinus of the M. pudica shows elastic properties. We have found that the movements of the petiole, or pinnules, are accompanied by a change of the pulvinus morphing structures. After brief flaming of a pinna, the volume of the lower part of the pulvinus decreases and the volume of the upper part increases due to the redistribution of electrolytes between these parts of the pulvinus; as a result of these changes the petiole falls. During the relaxation of the petiole, the process goes in the opposite direction. Ion and water channel blockers, uncouplers as well as anesthetic agents diethyl ether or chloroform decrease the speed of alert wave propagation along the plant. Brief flaming of a pinna induces bidirectional propagation of electrical signal in pulvini. Transduction of electrical signals along a pulvinus induces generation of an action potential in perpendicular direction between extensor and flexor sides of a pulvinus. Inhibition of signal transduction and mechanical responses in M. pudica by volatile anesthetic agents chloroform or by blockers of voltage gated ion channels shows that the generation and propagation of electrical signals is a primary effect responsible for turgor change and propagation of an excitation. There is an electrical coupling in a pulvinus similar to the electrical synapse in the animal nerves.

Electrotonic and action potentials in the Venus flytrap.

The electrical phenomena and morphing structures in the Venus flytrap have attracted researchers since the nineteenth century. We have observed that mechanical stimulation of trigger hairs on the lobes of the Venus flytrap induces electrotonic potentials in the lower leaf. Electrostimulation of electrical circuits in the Venus flytrap can induce electrotonic potentials propagating along the upper and lower leaves. The instantaneous increase or decrease in voltage of stimulating potential generates a nonlinear electrical response in plant tissues. Any electrostimulation that is not instantaneous, such as sinusoidal or triangular functions, results in linear responses in the form of small electrotonic potentials. The amplitude and sign of electrotonic potentials depend on the polarity and the amplitude of the applied voltage. Electrical stimulation of the lower leaf induces electrical signals, which resemble action potentials, in the trap between the lobes and the midrib. The trap closes if the stimulating voltage is above the threshold level of 4.4V. Electrical responses in the Venus flytrap were analyzed and reproduced in the discrete electrical circuit. The information gained from this study can be used to elucidate the coupling of intracellular and intercellular communications in the form of electrical signals within plants.

Venus flytrap biomechanics: forces in the Dionaea muscipula trap.

Biomechanics of morphing structures in the Venus flytrap has attracted the attention of scientists during the last 140 years. The trap closes in a tenth of a second if a prey touches a trigger hair twice. The driving force of the closing process is most likely due to the elastic curvature energy stored and locked in the leaves, which is caused by a pressure differential between the upper and lower layers of the leaf. The trap strikes, holds and compresses the prey. We have developed new methods for measuring all these forces involved in the hunting cycle. We made precise calibration of the piezoelectric sensor and performed direct measurements of the average impact force of the trap closing using a high speed video camera for the determination of time constants. The new equation for the average impact force was derived. The impact average force between rims of two lobes in the Venus flytrap was found equal to 149 mN and the corresponding pressure between the rims was about 41 kPa. Direct measurements of the constriction force in the trap of Dionaea muscipula was performed during gelatin digestion. This force increases in the process of digestion from zero to 450 mN with maximal constriction pressure created by the lobes reaching to 9 kPa. The insects and different small prey have little chance to escape after the snap of the trap. The prey would need to overpower the "escaping" force which is very strong and can reach up to 4N.

Parking problem and negative cooperativity of binding of myosin subfragment 1 to F-actin.

Previously we provided evidence that myosin subfragment 1 (S1) can bind either one (state 1) or two actin monomers (state 2) in solution and in muscle fiber. Here we present results of the kinetics study of binding of S1 to F-actin labeled with fluorescent dye pyrene. A transition from state 1 to state 2 depends on probability that the second actin is free, which is high when molar ratio of S1/actin (R) is less than 0.5, and it decreases dramatically when R>2.0 due to the parking problem. The kinetics data obtained at different molar ratios were well fitted by two binding states model. The sequential binding of myosin head initially with one actin monomer and then with the second actin monomer in F-actin can play a key role in force generation by actin-myosin and their directed movement.

Modulation of the pHLIP transmembrane helix insertion pathway.

The membrane-associated folding/unfolding of pH (low) insertion peptide (pHLIP) provides an opportunity to study how sequence variations influence the kinetics and pathway of peptide insertion into bilayers. Here, we present the results of steady-state and kinetics investigations of several pHLIP variants with different numbers of charged residues, with attached polar cargoes at the peptide's membrane-inserting end, and with three single-Trp variants placed at the beginning, middle, and end of the transmembrane helix. Each pHLIP variant exhibits a pH-dependent interaction with a lipid bilayer. Although the number of protonatable residues at the inserting end does not affect the ultimate formation of helical structure across a membrane, it correlates with the time for peptide insertion, the number of intermediate states on the folding pathway, and the rates of unfolding and exit. The presence of polar cargoes at the peptide's inserting end leads to the appearance of intermediate states on the insertion pathway. Cargo polarity correlates with a decrease of the insertion rate. We conclude that the existence of intermediate states on the folding and unfolding pathways is not mandatory and, in the simple case of a polypeptide with a noncharged and nonpolar inserting end, the folding and unfolding appears as an all-or-none transition. We propose a model for membrane-associated insertion/folding and exit/unfolding and discuss the importance of these observations for the design of new delivery agents for direct translocation of polar therapeutic and diagnostic cargo molecules across cellular membranes.

Circadian rhythms in biologically closed electrical circuits of plants.

The circadian clock regulates a wide range of electrophysiological and developmental processes in plants. Here, we discuss the direct influence of a circadian clock on biologically closed electrochemical circuits in vivo. The biologically closed electrochemical circuits in the leaves of C. miniata (Kaffir lily), Aloe vera and Mimosa pudica, which regulate their physiology, were analyzed using the charge stimulation method. Plants are able to memorize daytime and nighttime. Even at continuous light or darkness, plants recognize nighttime or daytime and change the input resistance. The circadian clock can be maintained endogenously and has electrochemical oscillators, which can activate ion channels in biologically closed electrochemical circuits. The activation of voltage gated channels depends on the applied voltage, electrical charge, and the speed of transmission of electrical energy from the electrostimulator to plants.

Energetics and forces of the Dionaea muscipula trap closing.

The Venus flytrap is the most famous carnivorous plant. The electrical stimulus between a midrib and a lobe closes the Venus flytrap upper leaf in 0.3s without mechanical stimulation of trigger hairs. Here we present results for direct measurements of the closing force of the trap of Dionaea muscipula Ellis after mechanical or electrical stimulation of the trap using the piezoelectric thin film or Fuji Prescale indicating sensor film. The closing force was 0.14N and the corresponding pressure between rims of two lobes was 38 kPa. We evaluated theoretically using the Hydroelastic Curvature Model and compared with experimental data velocity, acceleration and kinetic energy from the time dependencies of distance between rims of lobes during the trap closing. The Charge Stimulation Method was used for trap electrostimulation between the midrib and lobes. From the dependence of voltage between two Ag/AgCl electrodes in the midrib and one of the lobes, we estimated electrical charge, current, resistance, electrical energy and electrical power dependencies on time during electrostimulation of the trap.

Circadian rhythms in electrical circuits of Clivia miniata.

The biological clock regulates a wide range of physiological processes in plants. Here we show circadian variation of the Clivia miniata responses to electrical stimulation. The biologically closed electrochemical circuits in the leaves of C. miniata (Kaffir lily), which regulate its physiology, were analyzed in vivo using the charge stimulation method. The electrostimulation was provided with different voltages and electrical charges. Resistance between Ag/AgCl electrodes in the leaf of C. miniata was higher at night than during the day or the following day in the darkness. The biologically closed electrical circuits with voltage gated ion channels in C. miniata are activated the next day, even in the darkness. C. miniata memorizes daytime and nighttime. At continuous light, C. miniata recognizes nighttime and increases the input resistance to the nighttime value even under light. These results show that the circadian clock can be maintained endogenously and has electrochemical oscillators, which can activate voltage gated ion channels in biologically closed electrochemical circuits. The activation of voltage gated channels depends on the applied voltage, electrical charge and speed of transmission of electrical energy from the electrostimulator to the C. miniata leaves. We present the equivalent electrical circuits in C. miniata and its circadian variation to explain the experimental data.

Circadian variations in biologically closed electrochemical circuits in Aloe vera and Mimosa pudica.

The circadian clock regulates a wide range of electrophysiological and developmental processes in plants. This paper presents, for the first time, the direct influence of a circadian clock on biologically closed electrochemical circuits in vivo. Here we show circadian variation of the plant responses to electrical stimulation. The biologically closed electrochemical circuits in the leaves of Aloe vera and Mimosa pudica, which regulate their physiology, were analyzed using the charge stimulation method. The electrostimulation was provided with different timing and different voltages. Resistance between Ag/AgCl electrodes in the leaf of Aloe vera was higher during the day than at night. Discharge of the capacitor in Aloe vera at night was faster than during the day. Discharge of the capacitor in a pulvinus of Mimosa pudica was faster during the day. The biologically closed electrical circuits with voltage gated ion channels in Mimosa pudica are also activated the next day, even in the darkness. These results show that the circadian clock can be maintained endogenously and has electrochemical oscillators, which can activate ion channels in biologically closed electrochemical circuits. We present the equivalent electrical circuits in both plants and their circadian variation to explain the experimental data.

Modeling ion channels in the gigaseal.

The ability to form gigaseals is essential for patch-clamp electrophysiology; however, ion channels located in the seal can produce measureable currents. To explore the expected properties of channels in the seal (i.e., rim channels), we created a mathematical model. The seal was a two-dimensional cable filled with saline and bounded on one side by membrane (with resistance and capacitance) and on the other side by glass (nonconductive and noncapacitive). We included ion depletion/accumulation around the channels. The channels were ohmic with a conductance that increased with the concentration of permeant ions. The aqueous seal thickness was set nominally to 1 nm. Imaging with fluorescent dyes in the pipette showed that the hydrophilic dye Alexa 488 is impermeant, but lipophilic FM1-43 labels the seal. The model showed that to obtain high-resistance seals, the conductivity of the seal media has to be <10% that of the bath. Stimulus voltages decreased with distance down the seal. In agreement with results in the literature, channels in the seal can produce currents similar to those in the pipette-spanning dome. The transition times of currents are slower due to membrane capacitance. If channel densities are uniform, patch currents are dominated by channels in the dome.

Anisotropy and nonlinear properties of electrochemical circuits in leaves of Aloe vera L.

Plant tissue has biologically closed electrical circuits and electric fields that regulate its physiology. The biologically closed electrochemical circuits in the leaves of Aloe vera were analyzed using the charge stimulation method with Ag/AgCl electrodes inserted along a leaf at 1-2 cm distance. The electrostimulation was provided with different timing and different voltages. Strong electrical anisotropy of the leaves was found. In the direction across the leaf the electrical circuits remained passive and linear, while along the leaf the response remained linear only at small voltages not exceeding 1 V. At higher potentials the circuits became strongly non-linear pointing to the opening of voltage gated ion channels in the plant tissues. Changing the polarity of electrodes located along conductive bundles led to a strong rectification effect and to different kinetics of capacitor discharge. Equivalent electrical circuit models of the leaf were proposed to explain the experimental data.

Gating behaviour of sodium currents in adult mouse muscle recorded with an improved two-electrode voltage clamp.

Muscle contraction is triggered by the spread of an action potential along the fibre. The ionic current to generate the action potential is conducted through voltage-activated sodium channels, and mutations of these channels are known to cause several human muscle disorders. Mouse models have been created by introducing point mutations into the sodium channel gene. This achievement has created the need for a high-fidelity technique to record sodium currents from intact mouse muscle fibres. We have optimized a two-electrode voltage clamp, using sharp microelectrodes to preserve the myoplasmic contents. The voltage-dependent behaviour of sodium channel activation, inactivation and slow-inactivation were characterized. The voltage range for these gating behaviours was remarkably hyperpolarized, in comparison to studies in artificial expression systems. These results provide normative data for sodium channels natively expressed in mouse muscle and illustrate the need to modify model simulations of muscle excitability to account for the hyperpolarized shift.

Complete hunting cycle of Dionaea muscipula: consecutive steps and their electrical properties.

The total hunting cycle of the Venus flytrap consists of five stages: 1. Open state→2. Closed state→3. Locked state→4. Constriction and digestion→5. Semi-open state→1. Open state. The opening of the trap after digestion consists of two steps: opening of the lobes, and changing of their curvature from concave to convex shape. Uncouplers carbonylcyanide-4-trifluoromethoxyphenyl hydrazone (FCCP) and carbonylcyanide-3-chlorophenylhydrazone (CCCP) inhibit the trap from opening for two weeks and antracene-9-carboxylic acid inhibits the trap from constricting. Different stages of the hunting cycle have different electrical characteristics. The biologically closed electrochemical circuits in the Venus flytrap are analyzed using the charged capacitor method. If the initial voltage applied to the Venus flytrap is 0.5V or greater, changing the polarity of the electrodes between the midrib and one of the lobes results in a rectification effect and in different kinetics of discharge capacitance. These effects can be caused by the fast transport of ions through ion channels. The electrical properties of the Venus flytrap were investigated and equivalent electrical circuits within the upper leaf were proposed to explain the experimental data.