Planta (Berl.) 102, 91-114 (1972) 9 by Springer-Verlag 1972
Spontaneous Electrical Activity in Shoots of Ipomoea, Pisum, and Xanthium B A R B A R A GILLESPIE PICKA~D Department of Biology and Center for the Biology of Natural Systems, Washington University, St. Louis, Mo. Received August 17, 1971
Summary. Extracellular recordings from light-grown shoots of Ipomoea and Xanthium contain trains of spontaneous fluctuations which resemble action potentials. The rise time of the putative action potentials is usually in the range from 1 to 50 ms. Their duration is most frequently between 100 and 400 ms, but they may be either briefer or, in the presence of unusual shoulders, much longer. Their separation interval is typically between 0.1 and 10s. The trains may last from 1 s to over 2 h. Spontaneous fluctuations also occur individually in shoots of Ipomoea, Xanthium, and Pisum. Although the shapes, rise times, and durations of the individual fluctuations fall within the same range as do those of repetitive fluctuations, a greater number of the individual spikes have a relatively slow time course and a higher apparent amplitude. At times, recordings have periods of activity which appear to result from the superposition of numerous individual fluctuations. There is no evidence that any of the fluctuations propagate, but propagation cannot be excluded on the basis of the extracellular recordings. The fluctuations occur in both dark and light. There is no strong evidence for the occurrence of spontaneous fluctuations in roots. Introduction L i t t l e a t t e n t i o n has been given to t h e p o s s i b i l i t y t h a t electrical signals g e n e r a t e d b y cell m e m b r a n e s m a y be of extensive occurrence in higher p l a n t s a n d m a y p l a y a wide v a r i e t y of i m p o r t a n t d e v e l o p m e n t a l a n d physiological roles. This is surprising, considering t h a t a c t i o n p o t e n tials h a v e long been k n o w n to m e d i a t e t h e r a p i d closure of leaves of Dionaea muscipula a n d Mimosa pudica (reviews b y Sibaoka, 1966, 1969) ; i t would be u n u s u a l if such e l a b o r a t e a n d specialized b e h a v i o u r e v o l v e d de novo r a t h e r t h a n b y m o d i f i c a t i o n of less conspicuous b u t m o r e f u n d a m e n t a l a n d u n i v e r s a l processes. The r e l a t i v e l y r e c e n t r e p o r t s b y S i n y u k h i n a n d G o r c h a k o v (1966a, b, 1968) t h a t a t least u n d e r some conditions t h e p h l o e m of higher p l a n t s such as Cucurbita pepo, Helianthus annuus, Phaseolus vulgaris, a n d Fagopyrum sagittaeum can c o n d u c t a c t i o n p o t e n t i a l s s t r e n g t h e n t h e view t h a t n e u r o i d control s y s t e m s m a y exist in m a n y p l a n t s - - e v e n t h o u g h 7 Planta (Berl.),Bd. 102
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i t m u s t be a d d e d t h a t the e x p e r i m e n t s do n o t seem definitive a n d no c o n v i n c i n g f u n c t i o n of the signals has y e t been suggested. Similarly s u p p o r t i v e is t h e r e p o r t b y S i n y u k h i n a n d B r i t i k o v (1967a, b) t h a t a n action p o t e n t i a l courses down the style of Lilium martagon, IncarviUea grandi/lora a n d I. delavayi following pollination, a n d triggers e n h a n c e d r e s p i r a t i o n of o v a r i a n tissue. Still more recently, P i c k a r d (1971) has shown t h a t voltage f l u c t u a t i o n s which h a v e a t least m a n y of the properties of a c t i o n p o t e n t i a l s occur i n response to m e c h a n i c a l s t i m u l a t i o n of the p l u m u l a r hook of y o u n g pea p l a n t s , quite possibly m e d i a t i n g the m o d u l a t i o n of shoot d i a m e t e r which is the e n d result of m e c h a n i c a l stimulation. F u r t h e r , W i l l i a m s a n d P i e k a r d (in press, a) have d e m o n s t r a t e d a role for b o t h receptor p o t e n t i a l s a n d action p o t e n t i a l s i n the p r e y i n g behavior of Drosera. The purpose of the p r e s e n t p a p e r is to report e x p l o r a t o r y extracellular recordings which d e m o n s t r a t e t h e occurrence of u n e x p l a i n e d , s p o n t a n e o u s , r a p i d voltage f l u c t u a t i o n s i n three higher plants. Materials and Methods
a) Plants. Seeds of Ipomoea hederacea L., cv. Scarlett O'I-Iara were obtained from A. H. Hummert Seed Co., St. Louis, Missouri. A voucher specimen of a mature plant (M0 201-9652) is in the herbarium of the Missouri Botanical Garden. The seeds were soaked overnight in aerated deionized water, and those which swelled were planted in sterilized soil in clay pots. Plants were provided with light 22 h per day at an intensity of about 100 ixW ram-2; the light source was a bank of 10 F48T12-VHO-GRO-WS fluorescent tubes (Sylvania, Inc., Mountain View, California) and 10, 60-W incandescent lamps. Day air temperature was 22 ~ and night temperature was 20 ~. Preliminary recordings with plants grown at cooler and warmer temperatures and also with plants grown at lower light intensities yielded results similar to those with standard plants. Because Ipomoea was the plant most intensively studied, all figures illustrate its behaviour; however, except as noted for the fluctuations described in Observations section 1, the patterns of activity seemed similar for Ipomoea, Xanthium and Pisum. Seeds of Xanthium strumarium L. (notomorph pensylvanicum) were collected in November, 1970 on the banks of Sunnen Lake near Potosi, Missouri. A voucher specimen of a mature plant (MO 201-9654) is in the herbarium of the Missouri Botanical Garden. Burs with clipped ends were soaked overnight in rapidly running tap water and planted and grown in the same manner as Ipomoea except that the night length was 6 h. Seedlings of Pisum sativum L. ev. Alaska were grown in either vermiculite or sterilized soil under conditions closely similar to those used for Ipomoea. b) Recording Conditions. During the recording period, plants were maintained at room temperature--usually about 22~ were illuminated with light of constant intensity. I n the shielded room (Section f) incandescent light was about 4 txW mm -2 at plant level. I a the unshielded recording room, mixed fluorescent and incandescent light was about 40 tzW mm -2. It was checked that the three categories of bioeleetric activity reported in the Observations section could occur in complete darkness. Relative humidity was controlled at about 40-60% in the
Electrical Activity in Higher Plants
93
open room. I t was n o t controlled in the shielded room as it was always high enough to prevent rapid drying of wicks (cf. Section d) or excessive electrostatic disturbance. c) Electrodes. Freshly chlorided silver wires served as electrodes. They were inserted into small glass pipettes containing 0.1 M KC1 solution. The tip of each pipette was plugged with the same solution gelled with 1% agar (Bacto-Agar, Difco Laboratories, Detroit, Michigan). Through the tip extended a short length of cotton thread soaked in the saline agar. d) Contacts. The ground (i.e., reference) electrode was placed in a s t a n d a r d salt bridge which was inserted into a glass tube (35 m m long, 5 m m inside diameter) filled with 0.1 M KC1 in 1% agar. The tube was embedded in the moist soil of the pot containing the experimental plant. I n order to m a i n t a i n moisture in the soil, the pot was placed in a dish containing water a few millimeters deep. A second agar-filled tube containing a n identical salt bridge with its electrode was often positioned several centimeters away from the first for use as a n alternate ground. Salt wicks of recording electrodes were applied directly to the surface of the plant. (Before a n experiment on roots, they were excavated with a gentle stream of water.) Small drops of water were added to the wicks from time to time, a n d every few hours saline was added to the shafts of all the pipettes. After each experiment, it was checked t h a t the area of the p l a n t which h a d been in contact with the 0.1 M wick did not show gross signs of damage. On occasion it was checked t h a t the activity could be detected through 0.02 M KC1 wicks. I t was also checked t h a t activity could be detected when 0.1 M wicks were applied to areas of the shoot from which the epidermis h a d been peeled away. 1Noise was sometimes picked up when a salt wick was freshly positioned on shoots of Ipomoea or Xanthium, b u t (presumably as a result of penetration of saline through the cuticle) usually disappeared in a few minutes. W i t h the leaves of Pisum, noise was slower to disappear. I t was in a n y event frequently impossible to record through a freshly positioned wick due to pronounced drift (presumably the shifting of junction potentials as the saline from the wick penetrated the cuticle a n d mingled with extracellular fluid). Vibration of the salt wick a n d p l a n t is a potential cause of variable contact, a n d hence of noise. However, t h e experiments were performed on a 28-kg steel plate set on blocks of cork on a sturdy table also m o u n t e d on blocks of cork; it was routinely checked t h a t vigorously striking the floor or table did not disturb the recording. The plants were shielded from air currents. e) Electrical Apparatus. E q u i p m e n t was fundamentally as described in the Materials and Methods of a preceding paper (Pickard, 1971) : signals from recording electrodes were passed through the same Pico-metric electrometer headstages, the same specially constructed preamplifiers with gain of 100, and recorded on the same B r u s h Recorder s. 1 I t is desirable to clarify the ambiguous statement in the preceding paper t h a t " t h e system underwent considerable drift". This wording was m e a n t to indicate t h a t drift occurred during recording from the plants; the Brush Recorder could not be detected to drift, while the Pico-metric headstages a n d the specially constructed 100 • amplifiers in combination drifted less (usually much less) t h a n 1 tzV s -~. ~)roblems with drifting were minimal in the present experiments with Ipomoea a n d Xanthium, as might be expected from the fact t h a t their surfaces are r a t h e r readily wettable. Experiments with leaves of Pisum, which repel water conspicuously, were troubled b y drifting during the first hour or two (recordings usually continued 5 or 6 h). 7*
94
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1) Electrical Shielding. Some experiments were conducted in an ordinary room but with the plant and electrode assembly as well as the headstages and preamplifiers contained within a screen cage. For these experiments, amplified outputs were routinely passed through simple RC low-pass filters made of 5 cascaded stages; the measured rise time (10-90 % ) of these units was 100 ms, and their cutoff frequency was 2.8 Hz. On some days, relays of equipment in the room adjoining caused sharp spikes in the recording. However, on such days the signals could be identified because 1. the occurrence coincided with an audible snap of the relays or with the turning on of a signal light, 2. the relays caused a very brief disturbance unlike those usually associated with the plants, 3. such disturbances when observed at high recording speed could be seen to decay exponentially. Extensive use was made of a double-walled radio frequency-shielded room (catalog No. 81, serial No. 1~-9662, Ray-Proof Corp., Norwalk, Connecticut). AC power for equipment was supplied within the room by a special line used exclusively for measuring instruments; this line voltage was regulated and filtered as it entered the laboratory and filtered again as it entered the shielded room. Inside the room, the assembly of plant, electrodes, and amplifiers was situated within a closed screen cage. I t was checked that a variety of relays operating outside the shielded room could no~ be detected on recordings made within the room. Within the room, observations were usually carried out without the previously described RC filter. Potentially, electrostatic interference due to the presence of the investigator could be picked up by the plant and electrodes. That this was not a problem when the RC filter was utilized was routinely checked by waving an ungrounded hand within 10-50 mm of the contact points. In general, no indication of this disturbance could be detected unless the motion was vigorous enough to cause air currents. When the RC filter was not in use, it was of course always necessary to keep the door of the screen cage closed; it was routinely checked that movements of the investigator outside the cage did not perturb the recording. g) Test o/Recording Equipment. Before each experiment, the recording system was routinely checked for noise, drift, and other irregularities by recording for about 20 min at an amplification of 50 ~V per 0.8 mm pen deflection with the pipettes which had been prepared for the day's work inserted in a beaker containing 0.1 ~ KCI. Only three electrodes could be checked at once; in general, a fom%h was required, and this was also checked (in combination with two of the three other electrodes) for 20 min. In the absence of sensible noise and drift, recording from plants was commenced. Once the preparation of electrodes had been standardized, it was never necessary to reject an Ag; AgC1 electrode because of irregular behavior. The recording system was often checked again after interesting activity had been observed by shorting the electrodes through 0.1 M KC1 solution. Alternatively, shorting was achieved by placing the wick of the spare reference electrode against the recording wick. Also, an interesting pattern of activity was often observed alternately using the regular reterence electrode and using a spare reference electrode posit'cried for this purpose (see Section d). As a final check that a pattern of activity was associated with a recording site rather than with a recording circuit, the positions were exchanged for the two electrodes which were recording from different sites on the plant. h) Figures. All figures are photographs of the original recordings from expanding cotyledons of Ipomoea. Unless it is indicated that wicks were situated on the petiole, recording was from the lamina. All figured recordings except that of X~ig.5 were obtained in the shielded room. l~iltered recordings are so marked.
Electrical Activity in I-Iigher Plants
95
Observations
1. Repetitive Spiking The most striking voltage fluctuations in Ipomoea and Xanthium were spikes which occurred repetitively. Fig. 1 is a photograph of a recording of a representative train of spikes from Ipomoea. The train of l~ig. 1 and the numerous other such trains which were observed had the following characteristic properties. a) Shape o/ Fluctuation. As best illustrated in Fig. 2, the voltage commonly deviated from the baseline much more rapidly t h a n it returned. I n particular, the initial 10% of the rise was much more abrupt than the final 10% of the return. The rise time (measured between attainment of 10 and 90% amplitude) was typically between 10 and 50 ms, but could be much briefer. Often, there was a conspicuous shoulder on the declining side of the fluctuations (e.g. Fig. 2B). I n the absecnce of unusual shoulders, the duration (indexed as time between attainment of and return to 10 % of the m a x i m u m amplitude) typically ranged from 100 to 400 ms, and could gradually v a r y within this range even in a single train. Durations shorter t h a n 50 ms were sometimes observed, and occasionally, in the presence of unusually big shoulders, durations as great as 5 s were common. Shoulders on the spikes could be so large and regular as to give them the appearance of square waves. Fig. 3 is a photograph of exerpts of an 18.5-min train in which there is a transition between " s q u a r e " waves and the more typically observed waveform. The recording was also notable in t h a t it documented apparent rise times of about 1 ms. Such rapid rise times, while not unique to this train (of. Fig. 4), were observed far less frequently than the slower ones. Another notable feature was the exceptionally long 46-s duration of the 26th fluctuation (so long t h a t it was 6.4 times the length of the traces shown in l~ig. 3). After-potentials were occasionally consistently present. These usually endured much longer t h a n the main fluctuation, and could be of the same or opposite sign. Several times, there were trains of fluctuations which occurred at a regular frequency but which had properties so individualistic t h a t they cannot be categorized with the trains of repetitive spikes. The rarity of such sequences and their individualistic nature makes it u n ~ s e to draw general rules for their behavior, yet it seems equally foolish to ignore such highly structured fluctuations. Therefore, an example of an unusual train is presented in Fig. 4. b) Amplitude. Apparent amplitude typically varied from the beginning to the end of a train. Such transitions were never abrupt, but were only conspicuous in a sequence of several to m a n y fluctuations. The amplitude of the largest fluctuations of the typical train of Fig. 1 (filtered) is
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Fig. 3. Fluctuations with angular shoulders, exerpted from a t r a i n of 1110 s duration containing a b o u t 250 fluctuations. The n u m b e r ahead of each fluctuation tells its position in the sequence. Fluctuations gradually increase in duration, t h e n gTadually decrease until there is no evidence of a shoulder. Some of the fluctuations of this t r a i a showed rise times (10-90%) of a b o u t 1 ms, judged from accelerated recordings. Time ticks indicate 1-s intervals
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Electrical Activity in Higher Plants
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300 ~V; t h e g r e a t e s t a m p l i t u d e ever o b s e r v e d was 11 m V (this p a r t i cular recording was from a c o t y l e d o n of Xanthium). The f l u c t u a t i o n s were p o s i t i v e going (as in Fig. 1) in a b o u t half of t h e cases a n d negativegoing in t h e r e m a i n d e r . Quite t y p i c a l l y , as in Fig. 1, t h e a m p l i t u d e of t h e f l u c t u a t i o n s g r a d u a l l y increased e a r l y in t h e train. L a t e in t h e train, t h e a m p l i t u d e g r a d u a l l y decreased (again, i l l u s t r a t e d in Fig. 1).
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c) Frequency. The frequency of spiking in the train of Fig. 1. is initially about 1 s -1, diminishing so t h a t the last two fluctuations are about 5 s apart. The modal separation of the middle two spikes of all trains was about 2.5 s. However, at one extreme, spikes in a highly regular train were both minimally and maximally separated b y 50 ms while in the other extreme the minimal and maximal separations were 7 and 20 s. Although a drop in frequency was common near the end of a train, acceleration or deceleration of spiking could occur at any point. d) Uni/ormity o/Successive Fluctuations. Within a train, any variations in shape, amplitude and temporal separation generally occurred gradually. This has already been remarked in Sections a und b b u t is such a distinguishing property of the trains t h a t it deserves to be singled out as a special character. I n occasional trains, changes in separation occurred rather erratically, but nevertheless the graduality of any variations in shape and amplitude contributed to an appearance of uniformity. e) Train Duration. The shortest train identified lasted 1.2 s and contained 9 fluctuations (Fig. 2D). There were briefer bursts of fairly regularly spaced, fairly well-matched fluctuations, but these were difficult to classify because the fluctuations tended to be small and because there was so little evidence on which to evaluate the uniformity of the sequences. The train oY Fig. 1 was chosen because of its more or less typical properties; it contains at least 49 fluctuations, and is at least 108 s long. (Because the amplitude of the spikes rises above noise level gradually, it is impossible to be sure when the train begins.) The second longest train lasted 44 rain and contained over 2500 fluctuations. (An exerpt from this train is shown in Fig. 5). Repetitive spiking in the longest train showed no sign of fatigue when observation was terminated after 2 h. l) Site oI Occurrence. Repetitive spiking was observed on both petioles and laminae; the trains chosen for Figs. 1 through 5 were recorded from the laminae of cotyledons of Ipomoea. No such trains were ever observed in recordings from root tissue. g) Biological Origin. I t m a y be questioned whether repetitive spiking results from the activity of the plant or from either imperfections in the recording equipment or electrical interference. Three lines of evidence support the interpretation t h a t the spikes are of biological origin. First, as illustrated in Fig. 5, when the recording electrode is shorted to ground b y transferring the salt wick to the saline gel in which the wick of the reference electrode is embedded, the activity disappears, reappearing when the wick is repositioned on the plant. The activity also disappears or is greatly reduced while the wick of an additional reference pipette is touching the recording wick.
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(which could be turned on and off without producing any signal). I f filtered, these recordings were demonstrated to be stable when the door of then screen cage was opened and a hand was waved close to the salt wicks. (If unfiltered, the pattern of high amplitude trains could be seen to be unchanged b y the introduction of 60-Hz noise when the screen door was opened, but small fluctuations were lost.) Third, it was found t h a t paired pipettes with wicks only 2-5 m m apart tended to display a train synchronously, but although the majority of the recordings were through pipettes with wicks 10-20 m m apart, these showed simultaneous repetitive spiking on only five occasions. (It was carefully checked in all cases t h a t wicks were not connected b y a visible film of water.) I n one of the five trains recorded simultaneously through widely separated wicks, the positive-going spikes of the conspicuous train were of high a m p l i t u d e - - o v e r 10 m V - - a n d the negative-going spikes of the other train were not more than 100 vV high. I n another case, positive-going spikes reached an amplitude of 300 ~V but the negative-going set did not exceed 40 ~V. I n three cases, spikes in both recordings had the same sign and were limited to amplitudes less t h a n 150 ~V. Thus, it appears t h a t in general the fluctuations m a y be detected at some distance from their site of origin, which is not necessarily close to either of the paired wicks, but t h a t this distance has practical limits. More importantly, the scores of cases in which the fluctuations were detected through only one of two recording pipettes with wicks 10-20 m m apart overwhelmingly suggest t h a t the activity is neither a product of disturbances in the grounding system nor of electrical interference, since such noise would in general be seen in each of the neighboring, carefully matched recording circuits. I t seems likely, then, t h a t the trains of fluctuations are of biological origin. h) Apparent Absence in Pisum Leaves. Repetitive fluctuations were not detected in Pisum except from the tendril. Rarely, sequences of fluctuations with fairly regular properties were seen in recordings from leaves, but these were never as conspicuously uniform as the trains of Ipomoea and Xanthium. Whether trains of fluctuations are rare or absent in leaf blades of Pisum, whether they m a y occur but require environmental conditions which were not met, whether they occur in tissue which is effectively insulated from the salt wicks, or whether the rare irregular sequences which were seen in Pisum are functionally comparable with the regular trains of Ipomoea and Xanthium, is unknown.
2. Solitary Fluctuations Fluctuations resembling the individual spikes of the trains described in the preceding section occurred much more often in isolation t h a t in
Electrical Activity in Higher Plants
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sequences. While their shapes and rise times seemed to v a r y in the same range as did those of trains, such independent fluctuations more frequently had a relatively slow time course, and more often their rise was nearly comparable to their return. Fairly frequently, the amplitude was several millivolts; well over half the fluctuations were negative-going. Typical solitary fluctuations are shown in Fig. 6 and 7. I f the wicks of the paired pipetted were placed only 2 or 3 m m apart, solitary fluctuations as well as slow meanderings of the background vottage (see Section 5) often appeared synchronously in the two recordings. As the wicks were moved farther apart, the similarity of the recordings diminished. When wicks were more than 10 m m apart, the slow meanderings tended to be entirely different, but concurrent fluctuations of the type shown in Fig. 7 were occasionally seen. Such paired fluctuations might be the same size, but more frequently were of different sizes. The signs might be the same or opposite. I t sometimes happened t h a t one of the concurrent fluctuations would appear more rounded t h a n the other. I f the pipette wicks were separated more than 20 mm, only very rarely were fluctuations recorded concurrently. In sum, because frequency of concurrent fluctuations increased as wick separation decreased, it appears t h a t fluctuations occurring a short distance from the wick can be detected, but that the probability of picking up a signal decreases as its distance from the wick increases. Because the relative number of differences in shapes and signs of concurrent fluctuations detected with the paired channels increased as frequency of occurence decreased, it seems likely t h a t the electrical properties of the recording p a t h which were responsible for the distortion and inversion were associated not with the electronic equipment but with the tissue of the plant. Solitary fluctuations and arbitrarily grouped fluctuations could be detected in roots as well as in shoots of intact plants. Because such activities disappeared reversibly when electrodes were shorted and occurred in the shidded room, they would seem ~o be biologically generated. However, on the whole, recordings from roots had much less activity than did those from shoots and the activity was invariably of low amplitude. Therefore, it m a y be asked whether the recorded fluctuations might be electrotouically transmitted through the root from active sites in the shoot. I n order to investigate this question, 15-20-mm root tips were excised, the basal ends were inserted in 0.01 M KC1 gelled with 1% agar, and recording was carried out with electrode wicks positioned 2 m m from the apices. Particular care was taken t h a t the ambient humidity remained high enough to prevent wilting and low enough to prevent condensation on the root surface Voltage fluctuations over 50 ~tV were extremely rare; the traces were
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Fig. 6. An unbroken sequence of solitary fluctuations. Relati~rely slowly rising spikes of ~'arious amplitudes are intermingled at random. I n addition to the conspicuous spikes millivolts high, there are numerous smaller ones; a~ the lower limits these are indisg~guishab]e from the background aetivigy. Altl~ough the large signals are all neg~give-going, some tiny ones rise in ~he laosi~ive direction. In the second line, a brief, irregular train of low amplitude pulses may be seen. Ticks mark 1-s inter~rals
105
Electrical Activity in Higher Plants almost as quiet as those obtained with electrodes dipped in saline. As a control, it was checked t h a t solitary fluctuations of normal appearance could be measured from excised cotyledons. While it remains possible t h a t the root can produce fluctuations only when stimulated b y the shoot, it is far more likely t h a t the greater number of the voltage fluctuations which were measured from the surface of roots of intact young plants was transmitted passively from the shoot. While it m a y well be t h a t the isolated roots occasionally produce voltage fluctuations similar to the slower fluctuations of shoots, any activity which might occur would be difficult to study because of its rarity; thus, recording from roots was abandoned. Individual fluctuations were less frequent in recordings from Pisum t h a n in those from Ipomoea and Xanthium. I t is not known whether this reflects genuinely lower levels of activity under the recording conditions or whether it results from some anatomical feature of P i s u m such as the troublesome water-repellent surface which made establishment of good contact a tedious job and which might have diminished the amplitude of fluctuations even when contact was apparently satisfactory.
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Fig. 8A and B. General activity. A The electrode recording from the blade of a cotyledon was rapidly transferred to the ground tube for 142 s and then returned to its original position on the blade; activity is associated only with the plant. B The upper pair of traces were concurrently recorded from a spot on the petiole (electrode 1) and a spot on the blade (electrode 2) of the same cotyledon. The electrodes were rapidly interchanged before recording the lower pair of traces. Activity is associated with a specific region of the plant. A lone spike appears on the second trace from electrode 1; it can be seen simultaneously in the trace from electrode 2 but the differing shapes of the returns suggest either that distortion has occurred or that another fluctuation has been superimposed on one spike. Recorded through a filter. Time ticks, 1 s
3. General Activity Sometimes, as i l l u s t r a t e d in Fig. 8, a recording showed vigorous a c t i v i t y which superficially r e s e m b l e d noise. T h e f l u c t u a t i o n s m i g h t h a v e a m p l i t u d e s as high as a few millivolts, a n d m i g h t continue for a few seconds or for m a n y minutes. T h a t t h e a c t i v i t y is n o t noise in a n y o r d i n a r y sense, b u t originates w i t h i n t h e p l a n t , is i n d i c a t e d b y its d i s a p p e a r a n c e w h e n t h e recording electrode is s h o r t e d a n d r e t u r n w h e n t h e salt wick is r e p o s i t i o n e d a t its p r e v i o u s site on t h e p l a n t (Fig. 8A). I t is also i n d i c a t e d b y t h e a s s o c i a t i o n of a n a c t i v i t y p a t t e r n w i t h a site on t h e p l a n t r a t h e r t h a n w i t h a salt b r i d g e a n d electrode a s s e m b l y , as d e m o n s t r a t e d b y switching p i p e t t e s (Fig. 8B). Moreover, such activ i t y , which occurred even in a closed screen cage w i t h i n t h e r a d i a t i o n
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shielded room, could not be detected when a 10-M ohm resistor was substituted for the electrodes and plant. General activity was sometimes seen simultaneously on the two paired recordings. When paired pipette wicks were 2-5 m m apart, the activity was often imperfectly synchronous, or less frequently might appear to be the same. The activity of one recording might appear more or less imperfectly inverted on the other recording. Pipette wicks which were separated 10-20 m m recorded synchronous activity much less often, and those separated b y greater distances generally did not show a n y similarity. When activity patterns which showed similarity between the two channels were recorded, it was always checked with particular care to ascertain t h a t the fluctuations did not result from misbehavior of the reference electrode. General activity of low amplitude could be detected from roots of intact plants, but was never observed with isolated roots. Therefore, generalized activity, like individual fluctuations, must be presumed rare or possibly absent in roots.
4. Response to Shoe/sing When large, 80-ms positive-going or negative-going pulses of voltage were applied to the blades of leaves and cotyledons through either a recording electrode or a separate electrode placed nearby, responses of the type illustrated in Fig. 9 were sometimes seen. Such responses could be of positive or negative sign and were highly variable in shape, size and duration. They might exhibit small shoulders on the return slope, and the return might overshoot the baseline slightly for a few seconds. The m a x i m u m duration (taken as attainment of and return to 10% amplitude) was about 30 s, and the m a x i m u m amplitude was about 50 inV. The threshold was usually above a volt but could be as low as 100 inV. I t was in general impossible to predict whether a response would occur at all, but if it did occur, rapidly succeeding shocks elicited progressively smaller responses. Periods of rest, however, frequently resulted in regained responsiveness. I t was usually true t h a t if the voltage of successive, widely spaced shocks was stepped up (beginning at a level near threshold), the magnitude of the responses increased. Recordings were also made with paired electrodes situated several cm apart on a petiole or on a stem of Ipomoea or Xanthium and with a shocking electrode placed some centimeters proximal or distal; the reference electrode was positioned between the shocking and the recording electrodes. Responses detected through the two recording electrodes might v a r y together to some extent, but could also v a r y independently. I~o consistent temporal separation of the two responses was detected. 8
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B.G. Pickard: Fig. 9. Response to a ~- 10-V shock administered through an electrode recording from a petiole. Time ticks, 1 s
Responses of the type illustrated in Fig. 9 could never be observed in roots.
5. Meandering of the Background Voltage The background voltage in aerial organs was rarely stable ~or long periods of time. The DC level frequently drifted slowly, and sometimes shifted or oscillated slowly and erratically. A good example is provided incidentally in Fig. 1. If paired pipette wicks were close together, the paired records tended to show meanderings identical in the finest detail. T h a t such synchrony was not due to misbehavior of the reference electrode was routinely checked b y shorting one of the paired pipettes to the agar of the ground tube. As pipettes were moved a little farther apart, the paired records showed less detailed correspondence, and when their separation exceeded 10 m m synchrony of meanderings was rare. Such meanderings as did occur on both channels when electrodes were separated widely could be of the same or different amplitude and sign. The voltage in intact roots tended to undergo meanderings of relatively low amplitude. However, voltage recordings from isolated roots usually showed only a little more drift than recordings made with the electrodes shorted to ground. Discussion
1. Repetitive Spiking The relatively fast rise and slow return of typical repetitive spikes, as well as their brief duration, gives them the appearance of action potentials. Though the function of the spontaneous trains of fluctuations is completely unknown, their great regularity contributes to the impression t h a t the spikes m a y indeed be genuine action potentials, serving in some signaling or regulatory role. This regularity suggests further t h a t each train might result from the activity of a single ceil (or possibly a synchronized group of cells). The increase in amplitude so frequently observed in the early moments of a train might well be an artifact in the sense t h a t it might arise due to an increased conductance of the extracellular fluid surrounding an active cell as it releases ions during the postulated action potential
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(cf. Williams and Pickard, in press, b) The decrease in amplitude characteristic of the end of a train might simply result from fatigue. The deceleration in firing rate which typically occurs at the end of a train might also be a s y m p t o m of fatigue. However, since acceleration and deceleration m a y occur in any part of the train, other factors might also regulate changes in rate of firing. The confusing fact t h a t the sign of a fluctuation m a y be either positive or negative could be explained in principle by assuming t h a t the fluctuations are relatively uniform action potentials of invariant polarity, but t h a t because the signals originate in a tissue which is electrically complicated and are scattered arbitrarily with respect to the recording salt wick, the area under the electrode m a y sometimes serve as a source of current for the signal and sometimes as a sink. The same explanations has been offered previously for other signals of unusual polarity but better known character (Wolbarsht, 1960; Williams and Piekard, in press, a, b 1971). The occasional simultaneous recording of spikes by two pipettes withtips separated by a centimeter or more tends to support this explanation, since sometimes such concurrent signals had the same sign but sometimes opposing signs. Fluctuations of unusual shape might well arise as summations of signals from a single excitable cell or unit which reached the area under the recording electrode b y multiple paths of drastically different electrical properties. There were indeed some cases in which it seemed likely t h a t such summations had occurred. However, it i s difficult to interpret solely b y this means shoulders such as illustrated in Fig. 4; putative action potentials must originate as signals exhibiting a great deal of variety. The complex behaviour of Fig. 4 is particularly hard to explain as simple distortion; it is possible that this and a few other recordings represent interaction of two or more kinds of electrical activity or perhaps of two or more excitable units.
2. Solitary Fluctuations I t is also possible t h a t the fluctuations which occurred singly or in small, irregular groups are action potentials. Often they had shapes and durations similar to those of the putative action potentials which occurred]n trains. Frequently, however, they exhibited slower time courses, and the rise and return were more often relatively more symmetric. They were also more frequently of relatively large size. The occasional inversion or mild distortion in one of two simultaneous fluctuations recorded from pipettes with salt wicks separated by 1 or 2 cm suggests t h a t some of the variations of shape, and perhaps all of the variation of sign, are consistent with the hypothesis that the fluctuations originate as reasonably uniform action potentials at sites separated from the S'*
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recording wicks by paths of varying degrees of electrical impedance. However, it seems unlikely that the wide range of shapes and time courses could result from distortion alone; individual fluctuations must in fact be quite variable at their site of origin. I t is equally possible that some or all of the solitary fluctuations may be incidental manifestations of cellular activities (for example, pinocytosis or extrusion of vesicles) rather than action potentials.
3. General Activity General activity could be postulated to result from the superposition of fluctuations from one or more populations of cells which are active during a given interval. Certainly, as illustrated in Fig. 7, individual spikes could be closely grouped. Whether under some conditions there might exist excitatory electrical feedback between cells or whether there are other ways in which the activities of cells might be phased together remains open to speculation. 4. Periods o/Quiet Lest the occurrence of fluctuations be overemphasized, it should be stressed that the recordings were extremely quiet much of the time. Part of the time they showed general activity consisting of erratic fluctuations tending to have rounded peaks and amplitudes of perhaps 50-200 ~V (e. g. Figs. 6, 7). Although such low-level activity is probably biological in origin because the small fluctuations disappeared when the electrodes were shorted, and were site-specific rather than electrodespecific, the tissue immediately surrounding the salt wick is doubtless inactive when apparent low activity is recorded. The occasional simultaneous appearance of individual and repetitive fluctuations in two recordings from pipettes about 1 cm apart suggests that electrical disturbances can be detected over considerable distance in the shoot, so perhaps very low-level general fluctuations may also be explained as due to activity of distant cells. This latter point is of course of minor importance; even in the unlikely event that the low level disturbances should prove merely to be cellularly or externally generated eletrical noise, the major point that the cells at any particular site on a blade or petiole are generally inactive most of the time would remain valid. Meandering of the background voltage may likewise be considered to be electrically quiet from the standpoint of analyzing the rapid fluctuations which are the subject of this paper. Since synchrony of voltage meandering in paired recordings is greater the closer the paired pipette wicks are placed, and since the activity disappears in either channel when the corresponding wick is shorted to ground, it is probable that the
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slow shifts and oscillations reflect some sort of biological activity. However, wick electrodes are not ideally suited for studying such fluctuations because of the changes which can occur as water evaporates from their surface and from the film surrounding the area of contact, and meandering was therefore not studied in its own right.
5. Response to Shocking The responses to electrical shocking resemble responses of plant tissue known to be excitable, such as the tentacle of a Drosera leaf (Williams and Pickard, in press, a, b) or the lamina of a leaf of Dionaea (BurdonSanderson and Page, 1876). Moreover, the occurrence of the elicited responses in the shoot, demonstrated to have three types of spontaneous putative action potentials, and their absence in the root, which when isolated could not be detected to show spontaneous voltage fluctuations, is consistent with the interpretation that the responses are compound action potentials. On the other hand, this interpretation requires careful evaluation since 1) not all excitable tissue responds to extracellular shocking, 2) Berry and H o y t (1943a), upon passing direct current through tips of onion root, observed responses comparable to those of the shoots ha the present study, and 3) it is possible for a passive nonlinear circuit to produce shock-stimulated voltage deflections which closely mimic those of excitable tissue (Newman and Pickard, unpubl.). Berry and Hoyt (1943a) believed the responses they observed in onion root tips to be action potentials: "The oppositely oriented potential changes following current flow would be expected.., only as a result of alternations of membranes or phase boundaries which serve as electrodes within the cell. Such an explanation is the well-known and generally accepted interpretation of muscle and nerve action potentials in biology." However, Berry and Hoyt (1943b) discussed the possibility that rectification by the roots might equally well explain observed responses to applied alternating currents. Should the responses reported by Berry and Hoyt indeed prove to be action potentials, it is not necessary that they result from the activity of cells which are spontaneously active. The excitable cells might instead serve a sensory function, perhaps similar to that reported by Piekard (1971) for the etiolated pea epicotyl.
6. Propagation The failure to observe propagation does not prove that it does not occur within some tissues. If conductive tissue were located within a sheath of other cells, or if the pathway of propagation was confined to a few ceils or to a narrow column of cells (especially if such conductive columns did not happen to lie directly under both of the paired electrodes), simple extracellular recordings might be inadequate to demonstrate propagation. I n the case of shock-elicited action potentials, recording difficulties might well be enhanced by responses from types of excitable
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cell which can produce action potentials but which do not participate in the normal propagation of action potentials. If, as might well be imagined, the excitable population was heterogeneous in threshold, in shape and size of elicited response, in refractoriness, and in fatigability, still further difficulties could be expected. Any spatial and temporal variation in the passive electrical properties of the tissue would also contribute confusion. The most characteristic property of the action potentials described in representative angiosperms by Sinyukhin and Gorehakov (1966a, b, 1968) was their propagation, which was thought to occur in the phloem of both roots and shoots at rates ranging from 1 to 20 mm s -1. The action potentials were triggered by applying heat or saline to almost any part of the plant. However, Sinyukhin and Gorchakov (1966 a, b, 1968) were themselves unable to measure electrical responses to heat or saline in the laboratory as opposed to the greenhouse, and I working in the laboratory have fared no better. Since action potentials have long been known to propagate in the phloem of mimosas which utilize the signals to coordinate leaf movements, the reports of Sinyukhin and Gorchakov deserve careful consideration. Similarly, the reports by Sinyukhin and Britikov (1967a, b) that action potentials propagate down the style following pollination, though not completely convincing, are extremely suggestive and call for further investigation of the possibility theat propagation is of widespread occurrence.
7. Speculations On the one hand, the fluctuations may ultimately prove to be merely incidental accompaniments to some physiological process or processes; if so, they should serve as valuable indices to facilitate quantitative measurements. On the other hand, the demonstration of rapid spontaneous electrical fluctuations by tissues of Ipomoea, Pisum, and Xanthium increases the plausibility of speculations that non-propagating and perhaps propagating electrical signals may play widespread roles in fundamental cellular processes. Obvious suggestions for the general nature of such roles include the control of release of chemical agents, and the transmission of messages independent of hormone movement. A particularly interesting possibility of long-distance transmission as well as of local electrical control is raised by continuing failures (Evans, 1969) to identify a chemical substance as the evocator of flowering. If the rapidly rising voltage fluctuations now demonstra'~ed to occur spontaneously in higher plants under ordinary circumstances may indeed be identified as action potentials, the behaviors of plant and animal
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cells e x h i b i t a m o r e g e n e r a l a n d f u n d a m e n t a l s i m i l a r i t y t h a n h a s b e e n widely appreciated. The painstaking assistance of Mu-lien H. Wu is gratefully acknowledged. For the use of the shielded room and advice on instrumentation, thanks are due William F. Piekard, Department of Electrical Engineering, Washington University. The work was supported by National Science Foundation Grant No. GB-8262 and by National Institutes of Health Grant No. 5 P10 ES 00139 to the Center re1 the Biology of Natural Systems. References Berry, L. J., Hoyt, R. C.: Polarization and stimulation of the onion root by direct current. Plant Physiol. 18, 372-396 (1943a). - - - - Stimulation of the onion root by alternating current. Plant Physiol. 18, 570-587 (1943b). Burden-Sanderson, J., Page, F. J . M . : On the mechanical effects and on the electrical disturbance consequent on excitation of the leaf of Dionaea muscipula. Prec. roy. See. 25, 4 1 1 4 3 4 (1876). Evans, L. T. (ed.): The induction of flowering: Some case histories. Ithaca, N. Y. : Cornell Univ. Press 1969. Pickard, B. G.: Action potentials resulting from mechanical stimulation of pea epicotyls. Planta (Berl.) 97, 106-115 (1971). Sibaoka, T.: Action potentials in plant organs. Symp. Soc. exp. Biol. ~0, 49-73 (1966). - - Physiology of rapid movements in higher plants. Ann. Rev. Plant Physiol. 20, 165-184 (1969). Sinyukhin, A. M., Britikov, E . A . : Action potentials in the reproductive system of plants. Nature (Lend.) 215, 1278-1280 (1967a). - - - - Generation of potentials in the pistils of Incarvillea and lily in conjunction with movement of the stigma and fertilization. Fiziol. Rast., Engl. trsln. 14, 393-403 (1967b). - - Gorchakov, V.V.: Action potentials of higher plants not possessing motor activity. Biofizika, Engl. trsln. 11, 966-975 (1966a). - - - - Characteristics of the action potentials of the conducting system of pumpkin stems evoked by various stimuli. Fiziol. Rast., Engl. trsln. 13, 727-733 (1966b). - - Role of the vascular bundles of the stem in long-distance transmission of stimulation by means of bioclectric impulses. Fiziol. Rast., Engl. trsln. 15, 400-407 (1968). Williams, S . E . , Pickard, B. G.: Receptor potentials and action potentials in Drosera tentacles. Planta (Berl.) (in press, a). Properties of action potentials in Drosera tentacles. Planta (Berl.) (in press, b). Wolbarscht, M . L . : Electrical characteristics of insect mechanoreceptors. J. gen. Physiol. 44, 105-122 (1960). -
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Corrigendum I t was earlier mentioned (Vol. 97, p. 114, Pickard, 1971) that A. E1-Gadi and I had found series of action potentials correlated with the slow coiling of mechanically stimulated pea tendrils and with the pollination of peas. I have now attempted