Electrical Memory in Venus Flytrap

ARTICLE IN PRESS BIOJEC-06329; No of Pages 6 Bioelectrochemistry xxx (2009) xxx–xxx

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Bioelectrochemistry

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Electrical memory in Venus ﬂytrap

Alexander G. Volkov a,⁎, Holly Carrell a, Andrew Baldwin a, Vladislav S. Markin b a Department of Chemistry and Biochemistry, Oakwood University, Huntsville, AL 35896, USA b Department of Neurology, University of Texas, Southwestern Medical Center, Dallas, TX 75390-9068, USA article info abstract

Article history: Electrical signaling, memory and rapid closure of the carnivorous plant Dionaea muscipula Ellis (Venus Received 5 October 2008 flytrap) have been attracting the attention of researchers since the XIX century. The electrical stimulus Received in revised form 5 March 2009 between a midrib and a lobe closes the Venus flytrap upper leaf in 0.3 s without mechanical stimulation of Accepted 8 March 2009 trigger hairs. Here we developed a new method for direct measurements of the exact electrical charge Available online xxxx utilized by the D. muscipula Ellis to facilitate the trap closing and investigated electrical short memory in the Venus flytrap. As soon as the 8 µC charge for a small trap or a 9 µC charge for a large trap is transmitted Keywords: Plant memory between a lobe and midrib from the external capacitor, the trap starts to close at room temperature. At Bioelectrochemistry temperatures 28–36 °C a smaller electrical charge of 4.1 µC is required to close the trap of the D. muscipula. Electrophysiology The cumulative character of electrical stimuli points to the existence of short-term electrical memory in the Electrical signaling Venus flytrap. We also found sensory memory in the Venus flytrap. When one sustained mechanical stimulus Venus flytrap was applied to only one trigger hair, the trap closed in a few seconds. Dionaea muscipula Ellis © 2009 Elsevier B.V. All rights reserved.

1. Introduction inhibitors of voltage gated channels [3,4,27,41]. The two mechanical stimuli required for the trap closing should be applied within an Electrical signaling and memory play fundamental roles in plant interval from 0.75 s to 40 s. responses [1–8]. Examples of memory and learning have been ob- The inducement of non-excitability after excitation (refractory served in plants, including: storage and recall functions in seedlings period) and the summation of subthreshold irritations were devel- [9], chromatin remodelling in plant development [10,11], transgenera- oped in the vegetative and animal kingdoms in protoplasmic tion memory of stress [12–14], immunological memory of tobacco structures prior to morphological differentiation of nervous tissues. plants [15–16] and mountain birches [17], vernalization and epige- These protoplasmic structures merged into the organs of a nervous netic memory of winter [18–20], induced resistance and susceptibility system and adjusted the interfacing of the organism with the to herbivores [21], memory response in ABA-entrained plants [13], environment. Some neuromotoric components include acetylcholine phototropically and gravitotropically induced memory in maize neurotransmitters, cellular messenger calmodulin, cellular motors [22,23], ozone sensitivity of grapevine as a memory effect in a actin and myosin, voltage-gated channels, and sensors for touch, light, perennial crop plant [24], memory of stimulus [3,4,25,26], systematic gravity and temperature [1,2,42]. Although this nerve-like cellular acquired resistance in plants exposed to a pathogen [16], and electrical equipment has not reached the same level of great complexity as in memory in the Venus flytrap [3,4]. animal nerves, a simple neural network has been formed within the Rapid closure of the carnivorous plant Dionaea muscipula Ellis plasma membrane of a phloem or plasmodesmata enabling it to (Venus flytrap) has been attracting the attention of researchers and as communicate ef ficiently over long distances [1,2,5–7,43,44].The a result its mechanism has been widely investigated [3,4,27–37]. reason why plants have developed pathways for electrical signal When an insect touches the trigger hairs (Fig. 1), these mechan- transmission most probably lies in the necessity to respond rapidly to osensors trigger a receptor potential [38], which generate an electrical environmental stress factors [2,45,46]. Different environmental signal that acts as an action potential. Two stimuli generate two action stimuli evoke specific responses in living cells, which have the capa- potentials, which activate the trap closing at room temperature in a city to transmit a signal to the responding region. In contrast to fraction of a second [27]. At high temperatures of 28–36 °C, only one chemical signals such as hormones, electrical signals are able to mechanical stimulus is required for the trap closing [28,39–40]. transmit information rapidly over long distances. Electrical potentials Propagation of action potentials and the trap closing can be blocked by have been measured at the tissue and whole plant levels [8,43–47]. We found that Venus flytrap has a short term electrical memory [3,4]. Using our new charge injection method, it was evident that the application of an electrical stimulus between the midrib (positive fl ⁎ Corresponding author. Tel./fax: +1 256 726 7113. potential) and the lobe (negative potential) causes Venus ytrap to E-mail address: [email protected] (A.G. Volkov). close the trap without any mechanical stimulation [27]. The average

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of D. muscipula (mean 13.63 µC, median 14.00 µC, std. dev. 1.51 µC, n=41) causes trap closure and induces an electrical signal propagat- ing between the lobes and the midrib [3,37]. Not all of the applied “initial” charge was accumulated during 20–40 s and new series of experiments is necessary to determine the exact electrical charge accumulated by the closing trap. The electrical signal in the lobes was not an action potential, because its amplitude depended on the applied voltage from the charged capacitor. Charge induced closing of a trap plant can be repeated 2–3 times on the same Venus flytrap plant after complete reopening of the trap. The Venus flytrap can accumulate small charges, and when the threshold value is reached, the trap closes [3,37]. A summation of stimuli is demonstrated through the repetitive application of smaller charges [3]. Previous work by Brown and Sharp [39] indicated that strong electrical shock between lower and upper leaves can cause the Venus flytrap to close, but in their article, the amplitude and polarity of applied voltage, charge, and electrical current were not reported. The trap did not close when we applied the same electrostimulation between the upper and lower leaves as we applied between a midrib Fig. 1. Location of Ag/AgCl electrodes in the Dionaea muscipula. The three trigger hairs and a lobe, even when the injected charge was increased from 14 µC to in each lobe are seen. 750 µC [3]. It is probable that the electroshock induced by Brown and Sharp [39] had a very high voltage or electrical current. In the present work we investigated electrical memory in the stimulation pulse voltage sufficient for rapid closure of the Venus Venus flytrap and analyzed the exact amount of submitted electrical flytrap was 1.5 V (standard deviation is 0.01 V, n=50) for 1 s. The charge accumulated in the trap. inverted polarity pulse with negative voltage applied to the midrib did not close the plant [27]. Applying impulses in the same voltage range 2. Materials and methods with inverted polarity did not open the trap, even with pulses of up to 100 s [3,27]. It was found that energy for trap closure is generated by 2.1. Images ATP hydrolysis [48]. ATP is used for a fast transport of protons. The amount of ATP drops from 950 µM per midrib before mechanical Digital video recorders Sony DCR-HC36 and Canon ZR300 were stimulation to 650 µM per midrib after stimulation and closure [48]. used to monitor the Venus flytraps and to collect digital images, which However, it is not clear if electrical stimulation triggers the closing were analyzed frame by frame. process, or contributes energy to the closing action. The action potential delivers the electrical signal to the midrib, 2.2. Electrodes which can activate the trap closing. To check this hypothesis, we measured the effects of transmitted electrical charge from the charged All measurements were conducted in the laboratory at a 21 °C and capacitors between the lobe and the midrib of Venus flytrap. some experiments at 30 °C inside a Faraday cage mounted on a Application of a single electrical charge initially stored on the vibration-stabilized table. Ag/AgCl electrodes were prepared from capacitor when it is connected to the electrodes inserted in the trap Teflon coated silver wires. Following insertion of the electrodes into

Fig. 2. Experimental setup.

Please cite this article as: A.G. Volkov, et al., Electrical memory in Venus ﬂytrap, Bioelectrochemistry (2009), doi:10.1016/j.bioelechem.2009.03.005 ARTICLE IN PRESS

A.G. Volkov et al. / Bioelectrochemistry xxx (2009) xxx–xxx 3 lobes and a midrib, the traps closed. We allowed plants to rest until 2.5. Continuous single-touch stimulus the traps were completely open. Using a small wooden probe, one trigger hair was gently held down 2.3. Data acquisition until the trap closed. The wooden probe was removed just before the leaves closed. NI-PXI-4071 digital multimeter, NI-PXI-5124 oscilloscope (National Instruments), data acquisition boards NI-PXI-6115 or NI-PCI-6115 2.6. Plants (National Instruments) interfaced through a NI SCB-68 shielded connector block to 0.1 mm thick nonpolarizable reversible Ag/AgCl Three hundred bulbs of D. muscipula (Venus flytrap) were pur- electrodes were used to record the digital data (Fig. 2). The results chased for this experimental work from Fly-Trap Farm (Supply, North were reproduced on a workstation with data acquisition board NI Carolina) and grown in well drained peat moss in plastic pots at 21 °C 6052E DAQ with input impedance of 100 GΩ interfaced through a NI with a 12:12 h light:dark photoperiod. The peat moss was treated with SC-2040 Simultaneous Sample and Hold. The system integrates distilled water. Humidity averaged 45–50%. Irradiance was 77 µE/m2 s. standard low-pass anti-aliasing filters at one half of the sampling All experiments were performed on healthy adult specimens. frequency. Measuring signals were recorded as ASCII files using Lab- View (National Instruments) software with low pass filters. The NI-PXI- 3. Results 4071 high-resolution digital multimeter delivers fast voltage measurements from 10 nV to 1000 V, current measurements from 1 pA to 3 A, Touching of one sensitive hair twice or two different hairs with an and resistance measurements from 10 µΩ to 5 GΩ. interval up to 40 s induces the trap closing. We found that one sustained mechanical stimulus applied to only one trigger hair can 2.4. Plant electrostimulation close the trap of D. muscipula in a few seconds (Fig. 3). Prolonged pressing of the trigger hair generates two electrical signals similar The Charge-Injection Method [3] has been used to precisely estimate to action potentials reported in reference [27] within an interval the amount of electrical energy necessary to cause trap closure. A double of about 2 s, which stimulate the trap closing. The Venus flytrap can pole, double throw (DPDT) switch was used to connect the known capture insects by any of these three ways of mechanical stimulation capacitor to the 1.5 V battery during charging, and then to the plant of trigger hairs. Insects can touch a few trigger hairs for just 1 or 2 s. In during plant stimulation. Since the charge of capacitor Q connected to biology, this effect is referred to as molecular or sensory memory. It the voltage source U is Q=CU, we can precisely regulate the amount of was shown that sustained membrane depolarization induced a charge using different capacitors and applying various voltages. By “molecular” memory phenomenon and has profound implication on changing switch position, we can instantaneously connect the charged the biophysical properties of voltage-gated ion channels [49]. capacitor to the plant and induce an evoked response. We used different Figs. 4 and 5 illustrate the short term memory in the Venus flytrap. capacitors with a capacitance ranging from 1.0 to 9.4 µF. Recently we found that transmission of an electrical charge between

Fig. 3. Closing of the Dionaea muscipula trap by pressing of only one trigger hair at 21 °C. These results were reproduced 14 times on different Venus ﬂytrap plants. Reproducibility of the movement of the trap is ±33 ms.

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give the possibility of finding the exact electrical charge utilized by the Venus flytrap to facilitate trap closing. We measured voltage between two electrodes #1 and #2 (Fig. 1) inserted in the midrib and one of the lobes connected to a charged capacitor (Fig. 2). Charge was estimated as the voltage U between two Ag/AgCl electrodes multiplied by a capacitance C of the capacitor: Q=UC. The trap closing charge was estimated as the difference between the initial charge of a capacitor and the final charge of a capacitor, when the trap begins to close. In the experiments, when a few small charges were applied to close the trap, the closing charge was estimated as the sum of all charges transmitted from a capacitor. This shows that repeated application of smaller charges demonstrates a summation of stimuli (Figs. 4 and 5). We studied the capacitor discharge on small traps with a midrib length of 1.0 cm and large traps with a midrib length of 3.5 cm. Fig. 4 shows that as soon as 8.05 µC charge (mean 8.05 µC, median 9.00 µC, std. dev. 0.06 µC, n=34) for small trap or 9.01 µC charge (mean 9.01 µC, median 8.00 µC, std. dev. 0.27 µC, n=34) for large trap is transmitted between a lobe and midrib from the capacitor, the trap begins to close at room temperature. At temperatures of 28–36 °C a smaller electrical charge of 4.1 µC (mean 4.1 µC, median 4.1 µC, std. dev. 0.24 µC, n=34) is required to close the trap of the D. muscipula (Fig. 5). Probably, there is a phase transition at temperature between 25 °C and 28 °C and due to this reason only one mechanical stimulus or a small electrical charge is required to close the trap. Line 1 on Fig. 6a and b shows electrical discharge in the D. muscipula trap at 30 °C between electrodes inserted in a lobe and midrib and connected to a charged capacitor. Two other Ag/AgCl

Fig. 4. Closing of the Dionaea muscipula trap at 21 °C by electrical charge Q injected between a lobe and a midrib: (a) small trap with the midrib length of 1.0 cm and (b) large trap with the midrib length of 3.5 cm. Electrical discharge was measured using NI-PXI-4071 digital multimeter. electrodes #1 and #2 (Fig. 1) in a lobe and the midrib causes closure of the trap [3,27]. If we apply two or more injections of electrical charges within a period of less than 40 s, the Venus ﬂytrap upper leaf closes as soon as a 14 µC charge is applied. The capacitor slowly discharges with time, so although a 14 µC charge was applied, not all of this charge was accumulated during 20–40 s to assist in closing the trap. It is important to measure the exact amount of electrical charge used by the Venus ﬂytrap to close. Figs. 4 and 5 show a new series of experiments, which

Fig. 6. Electrical discharge in the Dionaea muscipula trap at 30 °C between electrodes located in a lobe and a midrib connected to charged capacitor and NI-PXI-4071 digital Fig. 5. Closing of the Dionaea muscipula trap at 30 °C by electrical charge Q injected multimeter (1) or between electrodes located in another lobe and a midrib connected between a lobe and a midrib. The length of the midrib was 3.5 cm. Electrical discharge to NI-PXI-6115 data acquisition system (2). Panels (a) and (b) show both curves but at was measured using NI-PXI-4071 digital multimeter. These results were reproduced 16 different time scales. The length of the midrib was 3.5 cm. These results were times on different Venus ﬂytrap plants. reproduced 16 times on different Venus ﬂytrap plants.

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A.G. Volkov et al. / Bioelectrochemistry xxx (2009) xxx–xxx 5 electrodes were located in another lobe and midrib, and connected to a data acquisition system. Electrical discharge at two electrodes connected to the capacitor takes place within many seconds (Fig. 6, line 1), but also induces a fast electrical signal between another lobe and midrib, which completely disappears in 70 ms (Fig. 6, line 2). It merely demonstrates that injecting charge into one leaf elicits also a short electrical signal in another leaf. The upper leaf of the Venus ﬂytrap has very dense network of plasmodesmata and vascular bundles with highly conductive plasma membrane.

4. Discussion

4.1. Plant memory

The general classification of memory in plants is based on the duration of memory retention, and identifies three distinct types of memory: sensory memory, short term memory and long term memory. Sensory memory corresponds approximately to the initial 0.2–3.0 s after an item is perceived. Some information in sensory memory can be transferred then to short-term memory. Short-term memory allows one to recall something from several second to as long as a minute without rehearsal. The storage in sensory memory and short-term memory generally has a strictly limited capacity and duration, which means information is available for a certain period of time, but is not retained indefinitely. Long-term memory can store much larger quantities of information for potentially unlimited duration up to a whole life span of the plant. The Venus flytrap can accumulate small subthreshold charges, and when the threshold value is reached, the trap closes. The cumulative character of electrical stimuli points to the existence of short-term electrical memory in the Venus flytrap.

4.2. Biologically closed electrical circuits Fig. 8. Scheme of a trap closure.

If a capacitor of capacitance C is discharged through a parallel Fig. 7 shows that the experimental time dependencies at different resistor R, the dependence of the capacitor voltage Uc on time is temperatures look like they are predicted by Eq. (1) for tbτ/2, but deviate increasingly from Eq. (1) for tNτ/2, because the equivalent ðÞ − t ; ð Þ electrical circuit of the Venus flytrap is more complicated than just a UC t = U0 × exp τ 1 resistor. The capacitive time constant τ depends on the size of the trap and decreases with increasing trap size. where The trap can be closed by applying a voltage (or action potentials) τ ¼ RC ð2Þ or by electrical charge. Probably, both ways of electrostimulation activated the ion pump in the midrib. and U0 is initial voltage. The circuit time constant τ governs the discharging process. With increasing capacitance, the time of the 4.3. Chain of events in the trap closing capacitor discharge increases according to Eqs. (1) and (2). Fig. 8 shows schematically the possible mechanism of a trap closure. When a mechanical stimuli is received by the trigger hairs in an open trap, a receptor potential is generated [35,36]. After the receptor potential, action potentials are generated, which are propagated to the midrib of the plant through the plasmodesmata [27]. Action potential can be inhibited by uncouplers and blockers of fast anion and potassium channels [3,27,33,34]. The Venus flytrap memorizes first electrical signal for short period of time and as soon as second action potential propagates to the midrib in 1 to 40 s, electrical signals activate the ATP hydrolysis [48], starting fast proton transport [50,51] and initiating aquaporin opening [41]. Fast proton transport induces transport of water and a change in turgor [41]. It is common knowledge that the leaves of the Venus flytrap actively employ turgor pressure and hydrodynamic flow for fast movement and catching insects. In these processes the upper and lower surfaces of the leaf behave quite differently. During the trap closing, the loss of turgor by parenchyma lying beneath the upper epidermis, accompanied by the active expansion of the tissues of the lower layers of parenchyma near Fig. 7. Dependence of voltage on a capacitor (2 µF in experiments at 30 °C and 4.7 µF at the under epidermis, closes the trap. The cells on the inner face of the 21 °C) during discharge in the Dionaea muscipula on time. Small traps were used with the midrib length of 1.0 cm and large trap were with the midrib length of 3.5 cm. These trap jettison their cargo of water, shrink, and allow the trap lobe to results were reproduced 16 times on different Venus flytrap plants. fold over. The cells of the lower epidermis expand rapidly, folding the

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