Action potentials induce biomagnetic fields in carnivorous Venus flytrap plants

Upon stimulation, plants elicit electrical signals that can travel within a cellular network analogous to the animal nervous system. It is well-known that in the human brain, voltage changes in certain regions result from concerted electrical activity which, in the form of action potentials (APs), travels within nerve-cell arrays. Electro- and magnetophysiological techniques like electroencephalography, magnetoencephalography, and magnetic resonance imaging are used to record this activity and to diagnose disorders. Here we demonstrate that APs in a multicellular plant system produce measurable magnetic fields. Using atomic optically pumped magnetometers, biomagnetism associated with electrical activity in the carnivorous Venus flytrap, Dionaea muscipula, was recorded. Action potentials were induced by heat stimulation and detected both electrically and magnetically. Furthermore, the thermal properties of ion channels underlying the AP were studied. Beyond proof of principle, our findings pave the way to understanding the molecular basis of biomagnetism in living plants. In the future, magnetometry may be used to study long-distance electrical signaling in a variety of plant species, and to develop noninvasive diagnostics of plant stress and disease.

The bilobed trap of the Dionaea muscipula plant 4,19 (Fig. 1A,B), formed by the modified upper part of the leaf, snaps closed within a fraction of a second when touched. Three trigger hairs that serve as mechanosensors are equally spaced on each lobe. When a prey insect touches a trigger hair, an AP ( Fig. 1C) is generated and travels along both trap lobes 20 . If a second touch-induced AP is fired within 30 s, the viscoelastic energy stored in the open trap is released and the capture organ closes 21,22 , imprisoning the animal food stock for digestion of a nutrient-rich meal. The leaf stalk, or petiole, is not excitable and is electrically insulated from the trap 23 . Because of this, the trap can be isolated functionally intact from the plant by a cut through the petiole. On the isolated trap, stimuli trigger APs and closure just as on the intact plant. For the comfort of electrophysiological studies, one of the trap lobes can be fixed to a support while the other is removed, without affecting the features of AP firing. It has been shown that this simplified experimental flytrap system is well-suited to study the AP under highly reproducible conditions 24 . Other than by touch (mechanical energy), APs in traps can be induced by salt loads (osmotic energy) 25 and temperature changes (thermal energy).
Since mechanical activation of APs can cause unwanted noise in electric and magnetic recordings, we use thermal stimulation in our experiments. The interdisciplinary work presented here encompasses two complementary sets of experiments: the temperature dependence of flytrap electrical activity was studied in a plantphysiology laboratory, while magnetometer measurements of heat-stimulated traps were conducted in a magnetically shielded room.

Heat-induced action potentials
When we heated up the support to which excised open traps were fixed, APs were elicited and the traps closed ( Supplementary Information, Fig. S1 and Movie S1). To study the temperature dependence of heat-induced AP initiation ( Fig. 2A), from a resting temperature of 20 °C, the trap temperature was increased monotonically to 45 °C at a rate of 4 °C/s (Fig. S2). Below 30 °C, no APs were observed; above 30 °C, the probability of AP firing increased and was maximal (100%) above 40 °C. In 60 independent experiments using 10 different traps from 10 different plants, we recorded the temperature at which an AP was first induced. When these data were plotted as temperature-dependent AP-firing probability (Fig. 2B), the curve could be well-fitted by a single Boltzmann equation characterized by a 50% AP-firing probability at 33.8 °C. This behavior indicates that heat activation of the AP is based on a two-state process. The ion channels that carry the classical animal-type AP also occupy two major states: closed and open. In contrast to the animal sodium-based AP, the plant AP depolarization is operated by a calcium-activated anion channel 4 . Thus, we conclude that the temperature "switch" of the Dionaea AP is based on a calcium-dependent process. Following Ca 2+ binding, the anion-channel gates open. Our experiments indicate that at temperatures of T ≲ 34 °C the cellular Ca 2+ level remains below threshold, but at T ≳ 34 °C there is enough chemical energy to open a critical number of anion channels, driving the fast depolarization phase of the AP. The Dionaea AP can be subdivided into 6 well-defined phases ( Fig. 1C): (1) fast depolarization, (2) slow depolarization, (3) fast repolarization, (4) slow repolarization, (5) transient hyperpolarization, and (6) slow recovery of the membrane potential to the pre-AP state. When comparing APs recorded at different temperatures, we found that temperature affects the signal amplitude and duration. Increasing the thermal energy input changed not only the probability for an AP to be fired, but also led to an increased AP amplitude and decreased half-depolarization time (Supplementary Information). These facts indicate that heat-sensitive ion channels trigger and shape the AP: at higher temperatures, thermal energy input causes more closed Ca 2+ -activated anion channels to open and depolarize the membrane potential. Compared to depolarization, fast repolarization (mediated by K + channels) and transient hyperpolarization (caused by depolarization activation of outward-directed protein pumps) were In contrast to the petiole, the trap contains parallel veins of interconnected cells. These veins consist of both dead low-conductivity water pipes (xylem) and living conductive phloem. Here the trap was destained using a modified protocol according to 26 , as described in Methods. (C) Intracellular AP lasting 2 s is subdivided into six phases (numbers), as explained in the text. The depolarization peak is indicated by an asterisk; the dotted line represents 0 mV. Inset: zoom-in on the AP, resolving the first five phases of the AP. www.nature.com/scientificreports/ much less affected by temperature. The recovery time to reach the resting membrane potential was essentially insensitive to temperature changes. Besides lowering the AP firing threshold and changing certain features of the AP, prolonged heat stimulation can induce trap lobes to enter an autonomous AP firing mode (Fig. S1). When increasing the bottom surface temperature of the recording-chamber base from 20 to 46 °C, AP spiking activity sets in after a couple of seconds, reaching a steady AP firing frequency of 3.8 per minute at a stable 46 °C surface temperature. Induction of autonomous APs has also been obtained using flytraps treated with NaCl salt (osmotic energy) 27 .

Biomagnetism
Having established heat stimulation as a reliable noninvasive technique for inducing flytrap APs, we searched for the magnetic field associated with this electrical excitability. Magnetometry experiments were carried out at Physikalisch-Technische Bundesanstalt (PTB) Berlin in the Berlin Magnetically Shielded Room 2 (BMSR-2) facility 28 , using four QuSpin Zero-Field Magnetometers (QZFM). These commercial optically pumped magnetometers (OPMs) employ a glass cell containing alkali vapor to sense changes in the local magnetic-field environment 29,30 . A magnetically shielded environment is required for operation of these magnetometers, and use of a walk-in shielded room allowed for the constant presence of an experimenter to prepare plant samples and carry out measurements. As shown in Fig. 3, an isolated trap lobe was attached to the housing of the primary sensor (denoted A), such that the distance between the plant sample and the center of the atomic sensing volume was approximately 7 mm. Two secondary sensors (B and C) were placed nearby the primary sensor to measure signal fall-off, and an additional background sensor (D) was used to monitor the magnetic environment in the shielded room. Each magnetometer is sensitive to signals along two orthogonal axes. Resistive heaters in the magnetometer housing, which are used to increase the atomic density and improve sensitivity, also served to induce autonomous AP firing via surface heat transfer at 41 °C. To monitor heat-induced APs, we used two silver-tipped copper surface electrodes, inserted in either end of the plant sample 31 . Prior to the measurements, we performed tests to ensure that no spurious magnetic fields were generated by the electrode system (Supplementary Information, Figs. S3 and S4). The magnetic and electric data, together with other auxiliary trigger signals, were sampled simultaneously using the same data-acquisition system located outside the magnetically shielded room.
To better distinguish possible magnetic signals from background noise, we triggered on the electric signals and averaged the magnetic data in a time window around those trigger points. Examples of averaged magnetic data are shown in Fig. 4. A clear magnetic signal with a time scale corresponding to that of the averaged electric signal is visible in the primary-sensor data. For comparison, data from several different experiments were plotted (Fig. 5). To minimize common background noise, we subtracted the magnetic data of sensor D to create a gradiometer with a 48-mm baseline. Signals of up to 0.5 pT are visible in the y-axis gradiometric data, normal to the sample surface. The signal magnitude obtained is comparable to what one observes in surface measurements of nerve impulses in animals 14 .
To quantify the significance of the measured signals, signal-to-noise ratios (SNRs) were calculated from the average y-axis gradiometric time traces as follows. The noise level is defined as the standard deviation of the gradiometric response in a 1.5 s time window (from time t = − 2 s to t = − 0.5 s in Fig. 4A) prior to signal onset. The signal size is defined as the amplitude of the extreme (minimum) field value, with respect to the mean value in the noise window. For the four experiments shown in Fig. 5, the SNRs range from 8 to 20. The corresponding p-values are p < 9 × 10 −16 , indicating that the probability of such signals arising from random noise is negligible. At the sub-Hz signal frequency, the sensitivity of the gradiometer is approximately 100 fT/√Hz (Fig. S6). For both the electric and magnetic signals, the full width at half extremum (maximum or minimum, FWHM) were also calculated, where the extremum is defined with respect to the mean value in the noise window.   13,16,18 , the magnetic signal from the complex multicellular flytrap lobe has a shape similar to that of the electric signal. We see features in the magnetic signal which appear to correspond to the depolarization and repolarization phases of the AP. In electric recordings using surface electrodes, the exact shape and duration of signals are dependent on the placement of electrodes on the measured sample. By contrast, magnetometry records a "true" physical signal from the organism. In this sense, it is comparable to intracellular electrode techniques. Whereas intracellular electrodes are sensitive to electrical activity of single cells, magnetometers can record both local and systemic activity at the multicellular level. In this way, magnetometry might be considered a complementary tool to the emerging technique of gene-based imaging of plant electrical signaling 23,32 .
The physical origin of the measured biomagnetic fields is related to an outstanding question in plant electrophysiology: how electrical signals propagate over long distances through the plant. Essentially this is a scaling problem: while electrical signaling is well-understood in some unicellular plant systems 16 , much less is known about the propagation mechanisms of such signals between cells and along cellular pathways. For the Venus flytrap system, it is known from electrode measurements that APs propagate through the trap at speeds of around 10 m/s 19 . A proposed pathway of long-distance signal propagation between plant cells in the trap is the electrically conductive phloem in the vasculature (Fig. 1B). Given that the typical resistance between two points on a trap is R ≈ 1 MΩ 33 , we can perform a basic calculation to confirm that the magnitude of the magnetic fields we measure is reasonable. We estimate the expected magnetic-field magnitude at the center of the sensing volume to be where I = V/R ≈ 10 nA is the current passing through the trap between the electrodes, and r ≈ 7 mm is the perpendicular distance from the trap surface. Using these values, we find B ≈ 0.3 pT, a magnitude which corresponds well with the y-axis experimental results of sensor A. Although the precise distribution and directionality of current flow in the trap is unknown, we can use the geometry of the trap (Fig. 1A,B) and magnetometry setup (Fig. 3) to further interpret our results. If the x-oriented parallel-cable structure of the vasculature is the primary conduction pathway, magnetic field along the y-direction is expected at the primary sensor A, but not at the secondary sensors B and C. The symmetry of the trap about the x-direction could explain the relative lack of z-axis magnetic signal in our measurements. Thus, our magnetometry results agree with a hypothesis that the vasculature serves as a network for long-distance electromagnetic signaling within the trap.

Discussion
Previously reported detection of plant biomagnetism, which established the existence of measurable magnetic activity in the plant kingdom, was carried out using superconducting-quantum-interference-device (SQUID) magnetometers 1,5,16 . Atomic magnetometers are arguably more attractive for biological applications, since, unlike SQUIDs 34,35 , they are non-cryogenic and can be miniaturized to optimize spatial resolution of measured biological features 14,15,36 . In the future, the SNR of magnetic measurements in plants will benefit from optimizing the low-frequency stability and sensitivity of atomic magnetometers. Just as noninvasive magnetic techniques have become essential tools for medical diagnostics of the human brain and body, this noninvasive technique could also be useful in the future for crop-plant diagnostics-by measuring the electromagnetic response of plants facing such challenges as sudden temperature change, herbivore attack, and chemical exposure.  To produce the destained image of the flytrap lobe shown in Fig. 1B, the trap chlorophyll was removed using methanol at 37 °C for 4 weeks, followed by final clearing in 4% sodium dodecyl sulfate (SDS) for 4 weeks.
For recording of flytrap APs in our heat-stimulation investigations, we used surface electrodes measuring the extracellular potential of a trap. The measuring electrode (blank silver wire; 0.25 mm, WPI, Sarasota, USA) was inserted into the trap, with the electrical connection enhanced by application of a droplet of contact gel (Laboklinika), while the reference electrode was inserted into wet soil or the petiole midrib. Electrical signals were amplified 100-fold and recorded with Patchmaster software (HEKA). Temperature dependence of AP induction was studied by application of a homemade Peltier device powered by a PTC-10 temperature-control system (npi electronic, NJ 08,510, United States). Constant heat was applied using an IKA RET basic hot plate (IKA-Werke GmbH & Co. KG, Staufen, Germany) heated to 46 °C.
Magnetometry. Several types of magnetometry experiments were conducted at PTB: controls, OPM measurements using four QuSpin sensors (three Gen-2: denoted A, B, C; one Gen-1.5: denoted D), and measurements using the multi-channel SQUID array of BMSR-2. See Supplementary Information for further details of the SQUID measurements.
For the OPM measurements of isolated trap lobes, each sample was cleaved from the plant with a razor blade and placed on the primary sensor A for immediate measurement. The sample was either secured to the sensor housing with double-sided adhesive tape (acrylate, thickness 0.5 mm) or placed on a plastic slide (PET, thickness 0.22 mm) on the housing. Electrode, magnetometer, and electric reference signals were recorded at a 500-Hz acquisition rate on a 9-channel analog data-acquisition system with PC control. The raw difference signal from the two surface electrodes was first sent through a voltage preamplifier (Stanford Research Systems, Model SR560), AC-coupled with a gain of 100. It is essential to use a voltage, rather than current, preamplifier to avoid currents in the electrical leads whose magnetic fields may be detected by the magnetometers. Since leakage of electrical signals into magnetic channels is a serious concern, we address the topic in detail in Supplementary Information.

Data availability
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.