Plant Response as a Means of Physiological Investigation

In the case of Biophytum, moreover, we found that with high E.M.F., we arrived at a phase of response, the A stage, in which both the anode and the kathode caused excitation at make ; and in Desmodium in a state of standstill I obtained an exactly similar result, for, on now using a higher E.M.F. of forty-eight volts, with the same specimens as in the last experiment, I obtained excitation at make, at both anode and kathode. Multiple response caused by strong stimulation in Desmodium. — I next tried to find out whether Desmodium in a state of standstill would give multiple responses to a strong chemical stimulation, as I had found Biophytum to do. Remembering how successive twitches are produced in a frog's muscle, in a nerve muscle preparation, when the nerve is touched with salt, I applied a strong solution of the same reagent to the petiolule of the arrested Desmodium leaflet. This gave rise to a series of four vigorous mechanical pulsations.

In order again to show that multiple response could be initiated by strong thermal stimulus, in Desmodium as in Biophytum, I selected a plant whose leaflets were in a state of natural standstill. A fairly strong stimulus* was applied by "means of the thermal stimulator, at a point on the petiole half a centimetre above the insertion of the motile leaflet, in precisely the same manner as was ordinarily done with Biophytum. The Desmodium leaflet now gave a multiple series of responses, exactly similar to those obtained from Biophytum. The first occurred three and a half minutes after the application of stimulus. The successive rise and fall were then uninterrupted. The average period of each response in the series was approximately 4*5 minutes. The multiple responses gradually declined in amplitude, and came to a stop after the thirteenth oscillation. It is thus seen that a strong stimulus will give rise to a multiple series of responses in the case of Desmodium, precisely as in that of Biophytum.

It is now clear that there is no rigid line of demarcation between multiple and automatic responses. An ordinarily responding plant like Biophytum, which gives a single response to single moderate stimulus, and multiple response to strong stimulus, will, under very favourable circumstances, that is to say, when it has absorbed an excess of energy from without, become automatically responding ; and, conversely, the pronouncedly automatic Desmodium will, under unfavourable circumstances, that is to say, when the sum total of its latent

energy has fallen below par, be reduced to the condition of an ordinarily responding plant, giving single response to single moderate stimulus, and multiple response to strong stimulus. On application of a strong stimulus, of whatever nature, to a vegetable tissue, a multiple series of electromotive responses is produced. These multiple responses may also be observed by the electrotactile method. These multiple excitations, in consequence of a single strong stimulus, may be observed in Biophytum as multiple mechanical responses.

These multiple responses may be uniform in character, or may exhibit cyclic variations, similar to those observed in the rhythmic pulsations of animal tissues. As, in the case of a plant-tissue, a strong stimulus causes multiple excitations, so, in the retina, strong stimulus of light causes multiple visual excitations, seen in recurrent afterimages, which have the same characteristics as the multiple after-effects of stimulation in Biophytum. There is no strict line of demarcation between the phenomena of multiple and of automatic response. Under very favourable circumstances — that is to say, when it has absorbed an excess of energy from without — an ordinarily responding plant like Biophytum will become converted into an apparently automatically responding plant, like Desmodium.

Conversely, under unfavourable circumstances — that is to say, when the sum total of its energy is below par — an automatically responding plant like Desmodium, will become converted into an ordinarily responding plant like Biophytum. Its leaflets then come to a state of standstill. Desmodium leaflets in a state of standstill respond to stimulus in exactly the same way as do those of Biophytum. To moderate stimulus, both give single response ; the polar effects of currents in both are the same ; and strong stimulation causes a multiple series of responses in both.

Production of pulsatory movements as after-effect of energy absorbed — Physical analogue — Localisation of seat of automatic excitation in Desmodium — Demonstration of multiple response to a constant stimulus: (i) Chemical — (2) Electrical — (3) Stimulus of light — Multiple response to constant stimulus of light, in : (a) retina— (b) Biophytum — (c) Desmodium — (4) Thermal — Induction of automatism in Biophytum at favourable temperature — (5) Of internal hydrostatic pressure — Absorption of external energy and its absorption by the plant in latent form — True meaning of ' tonic ' condition — Cause of rhythmicity — After-effect, and its relative persistence.

In living tissues, both animal and vegetable, we find numerous cases of spontaneous periodic movements, to which no direct exciting cause is apparently assignable. We confess our inability to trace out the exciting cause by classing such phenomena as automatic. Among wellknown examples of automatic movements in animal tissues may be cited the pulsatory action of the heart. In the vegetable kingdom, also, such movements are very numerous, and are of various degrees of rapidity, from quick pulsations of some few seconds in duration, to others which occupy periods of several hours. It is to be remembered that these spontaneous movements take place in plants under favourable circumstances, i.e. under that totality of the optimum degrees of light, temperature, turgidity, and so on, which is vaguely referred to in vegetable physiology as the tonic condition. I intend to set forth presently experimental considerations which will, I hope, serve to make clear the precise significance of this term.

only one way of evading the difficulty of finding out their actual cause. When we see these responsive indications given by a moving leaf or leaflet, we can but feel it necessary to trace them to the impulses, internal or external, by which they must have been occasioned. How are such periodic impulses caused, and where is their seat? The investigations which follow are intended to throw light on this, one of the most obscure problems in Physiology. In previous chapters I have demonstrated the fact that the antecedent cause of a periodic effect need not itself be periodic. A stimulus may remain long latent in a tissue, and this latent stimulus may subsequently give rise to periodic excitations.

Such periodic expression of the absorbed energy is not without analogy in the world of physics. For example, we take a glass tube of moderate diameter and push into it, to a certain distance from the free end, a piece of wire gauze, which is then heated over a Bunsen burner. On removal of the heating flame, we observe the after-effect of this absorbed thermal energy, in pulsating movements of the air-column, which give rise to a musical note, whose pitch is determined by their periodicity. We have here a physical analogue to a case previously described, in which the thermal energy absorbed by a tissue of Biophytum was afterwards manifested in long-continued periodic movements of the leaflets. In the experiment with the glass tube, the periodicity of aerial pulsation is determined by the size, shape, and temperature of the pulsating column. Similarly, the periodicity of multiple response in the Biophytum leaflet is determined by the various constants of the cell-complex which is the seat of the movement.

We saw in Biophytum that the region to which strong external stimulus was applied, became the place in which it was accumulated, and the source of subsequent excitation. We further saw that there is no rigid line of demarcation between plants which exhibit multiple responses and those which show autonomous movements, the same plant passing from one category to the other, according to circumstances. Thus Biophytum, which normally speaking gives ordinary single or multiple response, may under favourable circumstances exhibit automatic response. On the other hand, Desmodium, which normally speaking exhibits automatic response, will under unfavourable circumstances become converted into an ordinarily responding plant.

As already stated, in the case of Biophytum, the point of application of stimulus becomes also the source of subsequent multiple excitations. An observer, unacquainted with the position of this point, might succeed in determining it, by watching the order of pulsation of the leaflets. For if we suppose the stimulus to have been applied at some point in the middle of the leaf, the observer will in that case notice periodic waves of excitation proceeding in opposite directions, and giving rise to the closure of successive leaflets from a common point outwards, thus clearly indicating the position of the point from which successive excitations are initiated. Had the stem, on the other hand, been stimulated, and thus become the source of excitation, these successive impulses would have begun by arriving at the first pair of leaflets, and thence would have passed through all the leaflets to the tip of the leaf, in a centrifugal order. Again, had the stimulus remained latent at the tip of a particular leaf, the successive excitatory waves would then have proceeded through the leaf in a centripetal order, and on reaching the stem would have radiated outwards, or in a centrifugal sequence once more, throughout the other leaves. There are other ways also, presently to be described, by whose means we can obtain an idea of the direction in which the excitatory wave is travelling.

Localisation of seat of origin of autonomous excitation in Desmodium. — Our next inquiry is into the very obscure question of the point of origin of the so-called autonomous excitation of Desmodium. I have already shown that, if the plant absorbs a certain amount of energy in excess of that required for immediate response, the surplus is stored up, to be given out subsequently in the form of pulsating waves of excitatory disturbance. But the difficulty is to determine the point at which the latent energy is stored up, and from which the excitatory disturbances subsequently proceed. As we are accustomed to think that stimulus must be due to some sudden variation, one is tempted to suppose, as the simplest explanation, that in the case of autonomous movements the stimulation is caused by variations in the rate of absorption of food material, which is probably being carried on, in an intermittent manner only, by the roots and leaves. In this case, if the stimulus proceed from the root, we may expect the excitation to travel to the motile leaflets through the stem, outwards, or in a centrifugal direction. If the assimilating leaves, however, be the source of excitation, we shall look to see the wave of excitation proceeding from without inwards, or centripetally.

In order, therefore, to localise the source of stimulation in Desmodium gyrans, for example, I first tried to find out in which direction the excitatory impulse proceeded. Unfortunately in this case, the leaf being provided with only a single pair of motile leaflets, it is impossible to obtain the indication of direction which is given by Biophytum, by the successive closure of leaflets in a definite order. I was therefore obliged to have recourse to other expedients.

I have already shown how the transmission of stimulus can be arrested by local application of ether, or by the anodic block. If the pulsating stimulus, therefore, should be central, that is to say, proceeding from the stem, we should then expect the application of ether, or the anodic block, at the junction of the leaf with the stem, to arrest the movement of the leaflets. If, on the other hand, the source of the stimulation should be peripheral, a block produced in the manner described, at a point between the terminal expanded leaflet and the small motile leaflets, would prevent the response of the latter. In carrying out these experiments, however, I found that no such arrest took place in either case.

The fact that the source of stimulation is not central, is also made evident by the following consideration. Had it been so, the responding movements would have been synchronous with that of the hypothetical source, and all the leaflets of the plant would in that case have had the same period of vibration. As a matter of fact, however, different leaflets have different periodicities. That the source of stimulation, again, is neither central nor peripheral, may be shown by applying two tight ligatures, one behind and the other in front of the motile leaflets. The passage of the excitatory impulse, should this be transmitted from either direction, will by this means be completely arrested. On carrying out this experiment, however, the periodic pulsation was not affected.

Again, the leaf may be completely detached from the plant, and the terminal leaflet amputated. But the periodic pulsation still proceeds as before, just like the persistent beat of the isolated heart of a frog. Thus we find that the isolated motile leaflet continues to manifest an evolution of energy in the form of pulsating movements, which cannot be derived from either of the hypothetical sources, whether central or peripheral. It maintains this activity for a long time, when kept under favourable conditions. It thus becomes clear that in the case of Desmodium the power of maintaining rhythmic pulsation is local, and resides in the tissue of the motile leaflets.

This does not exclude the possibility of other periodically moving organs obtaining the pulsating excitatory impulses from a distant point. Such a case may well happen when, the conductivity of the intervening tissue being very great, and the excitability of the motile organ high, stimulation, though enfeebled by transmission through a long tract, yet remains above that critical intensity which would cause effective response. In the previous chapter, I showed that if Desmodium be kept in the dark for a sufficiently long period — but not so long as to produce permanent rigor — or if a specimen be taken in the unfavourable season of the year, its autonomic movements would be found to have come to a stop for the

time. 'Phis was because the energy stored up in the tissue had become exhausted. If, then, a single stimulus were given, say, by condenser discharge, a single response would be found to ensue. This showed that in such a case the arrest of the automatic movements was not due to the abolition of motility in the leaflet, but only to exhaustion of the energy stored up, which would have given rise to oscillation. It was also shown that if Desmodium in a state of standstill were subjected to a single strong thermal stimulus, it exhibited a multiple series of rhythmic responses.

Multiple response to constant stimulus. — Having observed the production of multiple responses as an aftereffect of a single strong stimulus, I shall now proceed to demonstrate the production of similar multiple responses by the action of a constant excitatory condition. i. Chemical. — I have shown that in Biophytum, and in Desmodium at standstill, rhythmic pulsations are produced by the constant action of a chemical stimulant. Analogous phenomena are also known in animal tissues ; in the isolated heart in a state of standstill, rhythmic pulsation can be renewed by chemical stimulation.

2. Electrical. — I have often observed that when a strong current is sent through Averrhoa carambola, a number of rhythmic pulsations are found to take place. In animal tissues, again, similar rhythmic excitations have been observed, not only in cardiac muscle, whose rhythmicity is so marked a characteristic, but also in skeletal and other muscles. 3. Stimulus of light : (a) On retina. — And now before I describe the experimental demonstration of periodic excitation in plants, as caused by constant stimulus of light, I shall refer in detail to certain remarkable periodic effects which I have observed in the retina, under the action of the same stimulus. I showed in the last chapter that strong stimulus of light gives rise in the retina to multiple responses, in the form of recurrent after-images. I shall now prove that during the continuance of constant light, pulsatory visual effects are

produced. These pulsations are not usually noticed in visual sensation, owing firstly to the absence of a standard of comparison, and secondly to the fact that though the impressions on the individual retinae undergo variation, the sum total of the two remains constant. I have been able to provide the necessary comparison-standards by having two distinguishable images produced in the two eyes, the fluctuation in the visual excitation in one eye being thus capable of detection by comparison with that in the other. It would have been impossible to detect this fluctuation had the excitatory variation taken place in the two eyes simultaneously, i.e. if the maximum excitation in the one had occurred at the same moment as the maximum excitation in the other. But I have found that, as regards excitation, there is a relative difference of phase, of half a period, between the two eyes, so that the maximum effect at the given moment in one eye corresponds to the minimum in the other. It is owing to this fact that the periodic excitations in each retina are brought out in an unmistakable manner by the following experiment, which consists in looking at two slits through the modified stereoscope described in the previous chapter. One of the two slits inclines to the right and the other to the left, and on looking through these at a bright sky, the right eye perceives a bar of light turned, say to the right, and the left eye a bar turned to the left, the resultant impression being that of an inclined cross.

When the stereoscope is turned to a bright sky, and the cross looked at steadily for some time, it will be found, owing to pulsatory excitation in each eye, that when one arm of the cross begins to be dim, the other becomes bright, and vice versa. These periodic fluctuations areperceived continuously under the constant action of light.1 I shall now proceed to the demonstration of the very interesting rhythmic movements caused in plants under constant light-stimulus. As we wish to prove that the cause of automatic movements lies in the action of some

1 The experiment will be found described in detail in my book, Response in the Living and Non-Living, p. 175. continuous stimulation, it is necessary to satisfy ourselves that this continuous source is the sole cause of the rhythmic movements. This will appear conclusive, if it is made clear that the plant does not possess any intrinsic energy of selfmovement, but only the power to regulate rhythmically the overflow of energy supplied. The demonstration will then be made rigorous, if we take a plant which isnot manifesting automatic movement, and cause it to exhibit such action by the application of constant external stimulus. For the purpose of this experiment, then, we may take the leaflet of Desmodium in a state of standstill, or that of BiopJiytum under normal conditions, which, as we have seen, may be regarded as practically equivalent to Desmodium in a standstill condition. If therefore we can succeed in making Biophytum exhibit rhythmic movements under the continuous action of a given external energy, we shall not need to look for the explanation of the so-called automatic movements of Desmodium to some periodically varying stimulus, any constant stimulus being then proved fully competent to produce the rhythmic pulsations.

[b) On Biophytum. — I first subjected the motile leaflet of Biophytum to light of comparatively short duration, i.e. two minutes, by throwing sunlight upon it from a reflecting mirror. There was no immediate response, but after a latent period of one minute the leaflet gave a single response. I next subjected the plant to the continuous action of light during ten minutes. I obtained rhythmic pulsations, there being two responses in the given time. I then cut off the light, and the rhythmic action was stopped. After an interval of several minutes I again applied light for ten minutes, and this gave rise to similar rhythmic responses. The procedure was repeated once more with the same results. With another specimen, I applied light for a still longer period, i.e. twelve minutes, and I obtained three pulsations. It will thus be seen that we have here a regulated outflow of energy ; the plant absorbs energy continuously, but gives it out in a pulsating manner (fig. 123). In this way, a number

of autonomic pulsations may be obtained in Biophytum under the continuous stimulation of light. The recovery not being complete after each pulse, the result is a progressive folding downwards of the leaflets, and this makes it impossible after a time to observe further responses. I shall presently describe, however, another method of supplying the plant with energy, by thermal means, in which case the autonomous pulsation in Bio- Iphytum is prolonged indefinitely. (c) On Desmodium leaflet at standstill. — Turning now to the leaflet of Desmodium in a state of standstill, I find that application of sunlight initiates rhythmic movements. In a particular experiment, light was continuously applied for half an hour, and there were produced in that time seven vibrations. The responses were found to undergo 'staircase' increase by increasing absorption of energy, and the resultant increase of molecular mobility produced in the tissue. On the stoppage of light the rhythmic pulsations persisted for some time, the amplitude, however, undergoing diminution owing to the run-down of absorbed energy.

4. Thermal: (a) Induction of autonomous response in Biophytum. — I shall now describe an experiment of very great theoretical importance, by which I have able to convert a specimen of Biophytum from an ordinarily into an automatically responding plant. Guided by the theoretical inference which I have already stated, that it is an excess of energy that brings about the condition of automatism, I subjected a specimen of Biophytum to the constant stimulus of a moderately high temperature. I first placed the specimen in a chamber whose temperature was 370 C, and it was astonishing to see the younger (and therefore more excitable)

Fig. 123. Multiple Response of Biophytum under the Continuous Action of Light leaflets, at first quiescent, break into a sustained series of uninterrupted pulsatory movements, which they kept up throughout the maintenance of the condition of high temperature. It was interesting also to observe the quickening of vibration by absorption of energy. The pulses were at first slow, each having a period of four minutes, but they became steadily more rapid, till they had reached a frequency of two vibrations in a minute. A continuous record of these periods was made during one hour, and the following tabular statement exhibits the results :

Table showing Periods of Successive Pulsations in Biophytum when Temperature is Raised to 27° C. Having thus found that a high temperature was favourable to the initiation of automatic movements in Biophytum, I was next desirous of determining the minimum temperature at which such responses could be induced. For this purpose I took a fresh specimen of Biophytum, and cautiously raised the temperature of the chamber from 230 C. upwards. When 290 C. had been reached, I obtained the first pulse of autonomous response, the period being rather slow — that is to say, 8*5 minutes — and by the time the temperature had gone up to 350 C. this period had become shortened to two minutes. We find here a phenomenon identical with that of Desmodium, where the frequency of vibration is found to be increased with rising temperature.

In Desmodium, the autonomous movement is initiated at a certain more or less definite temperature, which is about 1 70 C. This we may call the critical thermo-tonic condition. Below this critical degree, Desmodium ceases to be autonomic, and becomes an ordinarily responding plant. In Biophytum, similarly, the critical thermo-tonic point is about 29°- C. Above this, the young leaflets are autonomic, and below it, ordinarily responding. The difference between Desmodium and Biophytum in this respect lies, therefore, in the fact that their critical thermo-tonic points are about twelve degrees apart.