Plant Response as a Means of Physiological Investigation

We have seen that when stimulus is applied to a sensitive organ like the pulvinus of Mimosa there is a fall of the leaf, which fall is due to the excitatory contraction of the more excitable lower half of the pulvinus. Ordinary plants are said to give no motile indications, hence they are usually regarded as insensitive.1 It is difficult, however, to conceive that while the protoplasm of certain plants reacts to stimulus, that of others should not do so. On the other hand, it may be that the absence of mechanical response in these ordinary plants is not due to any want of excitability, but rather to the fact that conditions favourable to the conspicuous exhibition of motile effects do not in

1 Vines has already drawn attention to the possibility of error here : We must be careful not to assume that irritability is restricted to growing and to motile organs. For all we know to the contrary, it is possessed by the protoplasm of all plant organs, and if in any case the action of a stimulus is not followed by a responsive movement, we must, before we assume the absence of irritability, assure ourselves that the structure of the organ is such that a movement is a mechanical possibility.' — Vines, Physiology of Plant 's, 1886, p. 372.

such cases exist. What these conditions are will be detailed in the next chapter, where it will also be shown that excitation of an organ may take place, even where there is little mechanical indication of the fact, owing to antagonistic and balanced contractions. Electrical response. — It is my intention, in the course of the present work, to offer a complete demonstration of all the phenomena of excitation in plants, by means of mechanical response alone. But the conclusions to which we shall be led by the study of this response will receive irrefragable support, if they can also be established independently by some mode of investigation altogether different. Such a mode of inquiry, namely the electrical, and the conclusions to which it leads, will be fully described in the companion volume to this work, on the Electro-Physiology of Plants. Meanwhile it is convenient in this place to enter upon a short elucidation of the principle of that method, in order that we may be able, while considering the results of mechanical response, to make casual references to confirmatory results of independent observations obtained by means of the electrical method.

It has been said that under the action of stimulus excited cells undergo contraction, and that owing to the consequent expulsion of water, the turgidity of the tissue is diminished. Thus one expression of the molecular change induced by stimulus is a negative variation of turgidity. But this molecular change may also be detected by means of other concomitant physical changes. For instance, the electrical level or potential of a given point may, owing to the excitatory molecular change, undergo variation, relatively to another point which is unexcited. A hydraulic model will serve to make this point clear (fig. 22).

Let us imagine a flexible pipe of india-rubber, with bent ends of glass-tube, filled with water, and held in the middle by a clamp c. It is also supported in the stable horizontal position by spiral springs. If a single blow, say upwards, be now given to the end A, the level of the pipe at that end will be raised, and there will be a resultant flow of water from A to B, or away from the struck end. The intensity of the current is determined by the height to which the struck end A has been raised, and this again depends on the intensity of the blow. Hence the intensity of the current is a measure of the intensity of the stimulus or disturbance. The flow subsides with the return of the pipe to its equilibrium position. If the pipe had been disturbed throughout, the level would have been raised equally at both ends, and there would have been no flow. The object of the clamp is, therefore, to confine the disturbance to one side. If the blow had been

Fig. 22. Hydraulic Model for Explanation of Electric Response When an upstroke is given to A, a responsive current flows from a to b, and vice versa. given on the B side, the direction of the responsive flow would have been reversed. The principle of electromotive response in plants is exactly similar to this. The plant tissue is clamped at C (fig. 23), and a stimulus is given at one end, say A. The electrical level of that side is now found to be raised, it becomes electro-positive, or like the copper in a voltaic combination. The responsive current thus flows in the tissue from A to B, or away from the excited point. In the external circuit containing the galvanometer, it flows, of

course, in the opposite direction, that is from u to A.1 The excited point A is thus electro-positive, but in physiological text-books it has been ambiguously termed negative. In order therefore to keep touch with the older terminology and yet avoid the implied error, I shall refer to the excited point as ( galvanometrically negative.' If the B end of the specimen be now excited, the direction of the responsive current will be reversed. With greater intensity of stimulus the electrical response will be found correspondingly increased. The record of such responses is

obtained on a revolving drum, by following the deflection of a spot of light reflected from the galvanometer mirror in a manner precisely similar to that employed with the Optic Lever (fig. 24). 23. Electrical Response in Plant Case just described, the tissue tion of a produces upward response, however, another method of transmitted stimulation, by which a stimulus, — say by application of cut, or hot wire, or electrical shock,— is given at a point X some distance away, say to the right. The excitation now travels with a velocity characteristic of the specimen, and when it reaches the proximal electrode produces galvanometric negativity of that point. The interval of time which elapses between the application of stimulus and response will therefore depend on the velocity of transmission and the distance of the point

1 The failure to understand this point, clearly has been the source of many grave errors in some physiological text books. From the fact that the current in the external circuit is seen to flow in the direction of A, it has been erroneously supposed that that point is negative, or zinc-like. See Bose, Response in the Living and Non- Living. of application. It will also be remembered that the state of excitation is attended by an expulsion of water, or negative turgidity-variation. After causing galvanometric negativity of the proximal contact, the excitation may reach the distal, and bring about reversal of response, thus constituting a

diphasic variation. If, however, the distal point be very far, the excitation may by transmission through the long tract become so enfeebled as to produce practically no effect at Stimulus applied to the right at x . Excitation reaches right contact first, causing galvanometric negativity of the point. that point, in which case we obtain only the monophasic response of the proximal point. Simultaneous mechanical and electrical events, ensuing on excitation. — We may prove that these electrical responses are undoubtedly signs of excitation, by choosing

for the electrical experiment a plant in which the state of excitation is independently manifested by mechanical response. If now these electrical and mechanical responses be indeed only two different expressions of the same thing — that is to say, of a molecular disturbance and recovery which is concomitant to excitation and recovery from excitation — then we should expect that, on taking a simultaneous record, the two responses would be shown to be initiated at the same moment, and to bear some general resemblance to each other. In the following record it will be seen that this is found to be the case (fig. 26).

To recapitulate : let us take the concrete example of the Mimosa leaf. When the pulvinus is excited, owing to the Fig. 26. Simultaneous Mechanical (m) and Electrical (e) Responses in Biophytum These responses are seen to take place at the same moment. molecular change induced by stimulus, there is an expulsion of water, or negative turgidity-variation, and in the absence of restraint this is attended by the normal negative mechanical response, or fall of the leaf If, now, electrical connections have been made, one with the pulvinus, and the other with a distant point on the stem, it will be found that the excitatory change is attended by a strictly concomitant electrical change, the current of response flowing away from the excited point, which in other words becomes galvanometrically negative.

All these events will perhaps be more easily realised if we remember that excitation, in the typical case of Mimosa gives rise simultaneously to (a) contraction of the cells, with concomitant negative turgidity-variation ; (b) negative mechanical response, or fall of the leaf; and (c) galvanometric negative variation. Had the leaf been physically restrained by any means, the mechanical response would have been prevented, although the negative turgidity-variation concomitant to excitation would have taken place just the same. But this internal change would have been imperceptible, and in that case we could still have detected the effect of excitation by means of the electromotive response. As a matter of fact it is found that the electrical response always takes place in answer to effective stimulation, even in cases where the mechanical response is rendered impossible. We thus see that galvanometric negativity is a certain indication of the excitatory contraction of a cell,, whether or not the effect of such contraction be outwardly manifested by mechanical movement. The detection of the state of excitation by the electric test is thus unfailing, and of universal application. By the employment of this electric mode of investigation, I have shown that not sensitive plants alone, but every plant, and also every organ of every plant, is excitable.

True excitatory negative versus hydrostatic positive variation. — It has been supposed that the galvanometric negativity consequent on stimulation may be due to mechanical movement of water in the tissue. But I have shown that this cannot be the case. For while it is true that the production of water-movement by sudden forcing of water into a tissue does cause electrical variation, yet it must be noted that the sign of this electrical change is always one of galvanometric positivity, which is opposite to that of the true excitatory response. The intensity of the true negative electrical response, moreover, varies with the physiological activity of the tissue, and is abolished with its death. The electrical variation due to mere water-movement, however, may take place even in a dead tissue, and is, as has been said, of positive sign.

tion — that is to say, be locally disturbed, say by torsional vibration — two effects will be produced : first, the negative turgidity-variation, which is the true excitatory effect, with its attendant negative electrical variation ; and, second, the electrical effect due to hydrostatic disturbance or watermovement, which is positive. Of these two opposed electrical effects, the first, or true excitatory variation, is generally speaking much the stronger. It therefore completely masks the second, or effect of water-movement, and the resultant response is the normal negative variation. The water-movement effect may, however, be unmasked by killing the tissue, and then applying the same torsional vibration as before. The result is now a positive electrical response.

Or the positive effect may be made to exhibit itself separately, under favourable conditions in a living tissue, by the method of indirect stimulation, that is to say, by the application of stimulus at a distance. When such a distant point is stimulated, there is a sudden expulsion of water from that point, due to stimulation. This gives rise to a wave of increased hydrostatic pressure (with its attendant positive turgidity-variation), which travels with a relatively great velocity. The true excitatory variation, travelling at its slower rate, reaches any given distant point much later. The two effects ought thus to be divided from each other by some interval of time.

We should therefore expect on stimulation of a sensitive plant to find the hydrostatic disturbance, with its attendant positive turgidity-variation— reaching the distant motile organ the earlier of the two. And since the negative turgidity-variation due to excitation causes a fall of the leaf, the positive turgidity-variation due to hydrostatic disturbance should be expected to produce an abnormal positive or erectile movement, and the same positive turgidity-variation should also find a simultaneous electrical expression in the abnormal positive response. The true excitatory response — with its attendant negative turgidity-variation — should cause, la er the normal negative mechanical response,

and also the normal negative electrical response. We have already seen, in fig. 17, an instance of this abnormal positive followed by the normal negative mechanical response, in experiments with Biophytum. This abnormal positive response, being a matter of the intensity of the blow delivered by the water-movement, can only exhibit itself under favourable conditions. It is thus possible in mechanical response to have either the normal response preceded by the abnormal, or the normal response alone. But whenever we have an abnormal mechanical response, due to positive turgidityvariation, we have also simultaneously an abnormal positive 'electrical response. In fig. 27 is shown a simultaneous record of the two, in which there is a preliminary abnormal positive mechanical response, and a synchronous positive electrical response, followed in both cases by the normal responses.1 It should be stated here that this positive turgidityvariation, which is referred to as abnormal, is

of very great importance, and will be seen in Chapter XXX. to be directly responsible for growth. It will be found Fig. 27. The Abnormal Positive preceding the Normal Negative in Mechanical and Electrical Responses in Biophytum : represents the moment of application of stimulus. The upper is the mechanical and the lower the electrical record. The records downward indicate erection of the leaf or galvanometric positivity. 1 It must be understood that this positive electrical response, being dependent on the excitatory expulsion of water at a distant point, is, in a certain sense, a physiological response. For it is the excitability of that distant point which determines the positive turgidity and concomitant positive electrical variation of the point under examination. The contraction of the excited point gives rise to a hydrostatic disturbance, by which a movement of water is brought about. Such a disturbance, then, will be indifferently designated as the hydrostatic, or hydraulic, wave.

helpful in the future if we uniformly distinguish (i) the true excitatory or normal as the direct, and (2) this positive or abnormal as the indirect effects of stimulation. Discrimination of differences of excitability by electric test.— Not only does the electrical response enable us to detect the state of excitation, but I have been able further to devise an electrical test by which differences in the natural excitability of two points might be distinguished. The demonstration of the existence of two electrical responses, one of which alone, namely the negative, constitutes the true excitatory effect, is of much theoretical interest. For the hydraulic, or positive electrical effect, has been mistaken for the true excitatory response in plants. As the excitatory effect in animal tissues, moreover, was known to be negative, this fact was supposed to indicate a difference between the protoplasmic reactions of animal and vegetable. But the experiments which I have just described conclusively prove that such a difference does not exist, the sign of response in animal and vegetable being the same. They offer us an explanation, further, of the source of error. A more detailed account of this subject will be found in my forthcoming work on the Electro-Physiology of Plants.

Many of the motile phenomena of mechanical response which we shall have to study in the course of the present work are modified by differences of excitability at different parts of a tissue. In the case of the primary pulvinus of Mimosa for example, we can see how the responsive fall is brought about by the evidently greater excitability of the lower half of the pulvinus. But in organs which apparently exhibit little motility, it is impossible from inspection to know whether all parts of the tissue are equally excitable, and, if not, which parts exhibit the greater excitability. Such variation of excitability is often due to invisible molecular differentiation, and eludes visual scrutiny. Fortunately, as already said, I have been successful in devising a mode of electrical investigation by which this differentiation is detected with the greatest certainty. This method will be

found fully described elsewhere, but the results may be summarised as follows : On simultaneous excitation of two points, the current of response flows in the tissue from the more excitable point a to the less excitable point B ; conversely, if the direction of the responsive current is from A to B, the point A may be taken as the more excitable. By means of the unfailing discrimination of the differences of excitability in a tissue which this method renders possible, it will be shown in the course of the present work that many ot the anomalies of growth-curvature receive a most complete and satisfactory explanation.

Fig. 28. Response of Selenium to the Stimulus of Light (Conductivity variation method.) The electrical response given by plant-tissues in general, as described in this chapter, is obtained by means of the difference of electrical potential or electromotive variation, induced as between the excited and unexcited portions of the tissue. There is another method of detection by means of those changes of electrical conductivity which are concomitant to excitation in the substance under experiment.1 It should be borne in mind that the various responses, obtained by the mechanical, by the electromotive, or by the conductivity-variation method, are merely different expressions of

1 Bose, Molecular Changes produced in Matter by Electric Waves, Roy. Soc. Proc. 1 00. that fundamental molecular change which underlies excitation, and which disappears on the restoration of molecular equilibrium. Universality of responsiveness in matter. — If we take a plant-tissue and subject it to a sufficient degree of cold, its responsive power will be found to disappear. It reappears, however, on the return of the tissue to the normal temperature. The power of response is thus seen to depend on the molecular condition of the substance.

Now this irritability, or power of responding to stimulus, may be vaguely regarded as a characteristic property of living substances ; and we may evade the difficulty of any attempt at a real explanation by describing it as a ' vital ' phenomenon. But if we regard all such phenomena as due ultimately to physico-chemical actions, we cannot rest satisfied with what is, after all, a mere descriptive phrase. Progress can only be made in scientific inquiry by attempting gradually to discard all such assumptions of the working of mystical forces in favour of simpler and more rational explanations. By following the electrical method of inquiry which has just been described, I have been able to prove that the power of responding to stimulus, and, under certain conditions, the arrest of this power, is the characteristic not of organic matter only, but of all matter, both organic and inorganic ; ' and that in general the various agencies which bring on the modifica-

Fig. 29. Response of Metal abolished by the action of ' Poison ' (Oxalic Acid) The record to the left is the normal response, and the line to the right shows abolition of response on application. tion of response in one case — such as fatigue, temperature changes, stimulating or depressing chemical reagents — act in the same way in the other. The capability of responding, so long regarded as the peculiar characteristic of the organic, is also found in the inorganic, and seems to depend in all cases, both qualitatively and quantitatively, on the condition of molecular mobility. In the course of the present work, then, the term ' physiological ' is to be understood as a convenient expression for the phenomena of plant or animal tissues under investigation, and not as in any sense opposed to the word 1 physical.'

Stimulus causes molecular derangement in matter. The conditions of molecular upset and return to the state of equilibrium correspond to the state of excitation and recovery from that state. The molecular disturbance is attended by various physicochemical changes in the properties of the substance, among the most important of which in a living tissue may be mentioned (1) contraction of the excited cell, and expulsion of water ; (2) electromotive variation ; (3) conductivityvariation.

The true excitatory change causes negative variation of turgidity, with depression or negative mechanical response of the leaf, and galvanometric negativity. The intensity of these effects varies with the physiological condition, being totally abolished by those molecular changes which are concomitant with the death of the tissue. By means of electrical response it is found that, not sensitive plants alone, but every plant and every organ of the plant is excitable.

Positive turgidity-variation, by whatever means produced, causes positive mechanical response — or, in the case of Mimosa, erection of the leaf — and galvanometric positivity. A pulse of positive turgidity-variation is often propagated in consequence of the sudden expulsion of water from a stimulated point at a distance. The laws of true electric response to excitation may be summarised as follows : A. The indirect effect of stimulation is a positive electrical variation, indicating a positive turgidity-variation.

P>. The direct effect of stimulation causes negative turgidityvariation, concomitant with a negative electrical variation. (i) The direction of this responsive current in planttissues, as in animal, is from the more to the less excited portions of the tissue ; or, the excited point is galvanometrically negative. of response in the tissue is from the more to the less excitable. Differences of degree of motile sensibility in sensitive plants so called — Response of anisotropic organ brought about by differential contraction — Production ot response by artificial variation of turgidity — Variation and counter-variation of turgescence, causing two opposite responsive movements — Differences between hydrostatic and true excitatory effects— Distinction of plants as ordinary and sensitive, arbitrary - Sensitive plants may be excited, yet give no mechanical response — Certain conditions necessary to exhibition of differential response — Balanced action as result of diffuse stimulus on radial organ — Slight differential contraction of pulvinus magnified by long petiolar index.