Plant Response as a Means of Physiological Investigation

Investigation on supposed abolition of excitability by amputation of root-tip. — The question as to whether amputation of the tip abolishes the general excitability of the growing region of the root, can only be decided finally by the direct observation of the variation, if any, in the mechanical response of the root to stimulus after amputation. Should there be no such variation, then —the researches of Ciesielski and Darwin having proved that a root deprived of its tip does not respond to gravity — it will follow that the tip of the root alone is geotropically sensitive. That this is so — that is to say, that amputation does not permanently modify the general sensibility of the root — I shall next proceed to demonstrate.

The experiments that have been carried out by different observers, for the purpose of determining whether amputation of the root-tip produces any modification of the rate of growth, have led to very various results. Some have found that it has the effect of inducing an accelerated rate of growth ; others, again, state that it induces no change whatever in the rate ; and still others find that it causes retardation of growth. All these discrepancies are reconciled, however, when we remember, as I have demonstrated, that indirect stimulus causes increase, and direct stimulation retardation, of growth. Moderate stimulation, therefore, by section of the extreme tip, giving rise to the transmitted effect of indirect

stimulus, would accelerate growth ; while a section made in a better-conducting tissue, or nearer the growing region, with its stronger stimulation, would transmit the direct effect, with concomitant retardation of growth. But a stimulation of intermediate intensity would initiate no change. I shall, however, describe an investigation by which the question of the variation of responsive excitability in the growing region by amputation is put to the test of direct experiment. For this purpose I mounted a straight waterroot of Bindweed in my Crescograph, and took a record of its normal rate of growth. It should be stated that the root was attached to the Optic Lever at a distance of 3 mm. below the tip, which was thus left free for amputation. The normal rate of growth was considerable, being '0105 mm. per minute. I next took a record of three successive contractile responses to thermal shocks (fig. 227). After this, without disturbing the arrangement, the root-tip was amputated, to a distance of 2 mm., and a record of the growth was again taken after an interval of fifteen minutes. It was now found to be practically the same as before amputation, the rate being -oioo mm. per minute. The contractile responses to thermal shocks of the same intensity as before were also, to all intents and purposes, the same. With another specimen the rate of growth before section was '0066 mm. per minute ; after amputation of 3 mm. of tip, the rate was •005 5 mm. The mechanical responses before and after section were almost the same. These experiments, then, show that amputation of the root-tip does not abolish the general excitability of the responding region. The fact that the gravitational response of the root, however, is abolished by the removal

Fig. 227. Curves showing Effect of Amputation on Rate of Growth and Response in Root of Bindweed (a) continuous line indicates normal rate of growth. Dotted curves represent three responses to three successive uniform stimuli ; (b) exhibits the rate of growth and responses after amputation. It will be seen that there is practically very little variation. of the tip, proves that, as pointed out by Darwin, the tip is, with regard to gravity, the perceptive organ of the root.

The tip of the root the organ of gravi-perception. — The inference that it is the root-tip which is the perceptive organ has also been arrived at by Pfeffer and Czapek, pursuing an independent method of investigation which discarded amputation. There is again the very suggestive fact that the starch grains whose weight, according to the statolithic theory, brings about stimulation, occur generally at the root-tips alone. Thus three different lines of research lead to the conclusion that it is In that region that the gravi-perceptive power resides, the power of response by curvature being behind, in the region of growth.

The perceptive versus the responding organ. — In view of "much existing vagueness on this subject, it is desirable here to obtain clear and well-defined conceptions as to how far the differences commonly insisted on between perceptive and responding organs are justified. To take the familiar instance of Mimosa, we know that when stimulus is applied, for example, on the stem, there is no perceptible motile effect in the stem itself, but excitation is markedly manifested at the pulvinus. From this it has been erroneously supposed that the stem merely perceived, and did not respond to, stimulus. It has, however, been shown in previous chapters that every part of the plant-tissue responds to stimulus by contraction, the visible and striking manifestation seen at the pulvinus being simply the result of certain accessory anatomical facilities which happen to exist at that point.

Thus, every part of the plant-tissue is both perceptive and responsive to external stimulus, to a greater or less extent. When we come to the growing organ, responsive curvature is induced by the concavity of the excited side. And as the zone of growth is, generally speaking, the region of the greatest excitability, the greatest curvature usually takes place there, that region functioning in this respect as a diffuse pulvinoid. It should be remembered, however, that the excitability of a tissue does not disappear on the cessation of growth, and responsive curvatures are thus sometimes seen to extend even beyond that zone.

Contraction takes place, as we have seen, in all tissues, but it is relatively much less in the very young and the very old. The responsive contraction at the tip is therefore slight and comparatively inconspicuous. Indeed, it can only be made visible under very strong stimulation. That contraction is induced, however, is shown in the fact that a wave of positive turgidityvariation reaches the zone of growth by excitation of the tip. This can only be brought about by the expulsion of water, which is the result of excitatory contraction at that point. Under direct stimulation, then, every tissue, whether at the tip or in the growing region, undergoes a similar contraction. The sensitiveness, or sign of response, of all tissues is therefore the same ; but about these signs we are liable to be deceived if we pay attention to the conspicuous effects alone, for the local application of a unilateral stimulus at the tip gives rise to little perceptible concavity, while its transmitted indirect effect, lower down in the growing region, induces a relatively great convexity. Moreover, the whole length of the organ above the point of greatest convexity acts as a kind of leverindex, causing high magnification of the responsive movement. The tip, on the other hand, possesses no such magnifying index. These considerations will clearly show that there is no difference in kind between the sensitiveness of the tip and that of the growing region, both alike having the power of perceiving and responding, in different degrees, to external stimulation in general. Just as the tip itself merges physically into the growing region, so these respective responses pass imperceptibly into one another. Though moderate unilateral stimulus of the root-tip causes the characteristic movement away from stimulus, yet strong stimulation induces movement towards it.

Stimulus applied on other parts of the plant also will induce movements similar to that caused by moderate stimulation of the tip, provided the effect transmitted to the growing region be indirect. True perceptive region. — There is a sense, however, in which it is true that the root-tip alone is the perceptive region, but this statement applies exclusively to the stimulation caused by gravity ; for, in order that this may take place, the presence of statolithic or other elements through which it can act is necessary. But if such elements be concentrated at the root-tip, it is clear that that region alone can become the seat of stimulation. This is not the case with other forms of stimulus, which act directly, the presence or absence of statoliths being a matter of indifference. This distinction is important, since the outward resemblance of effects at the root-tip, in the two cases of stimulation by light and stimulation by gravity, has sometimes led to the assumption that statolithic bodies formed the medium of excitation in both. That such, however, is not the case is now evident.

In the geotropic root, then, it is the indirect effect of moderate stimulus, unilaterally transmitted from the distant tip, that causes the responsive curvature. In the case of the apogeotropic stem, on the other hand, it is the direct stimulation of the statolithic elements distributed throughout that region that induces the responsive curvature ; and since I have shown that the curvature caused by direct is opposite to that which is the result of indirect stimulation, it will be seen that the opposite curvatures observed in root and shoot are not due to different sensibilities possessed by the two organs.

It is to be noted, moreover, that although the statolithic or radial pressure theory affords a very rational explanation of the mode in which stimulation is brought about by gravity, yet the explanation which I have offered, of the occurrence of opposite geotropic effects in root and shoot, does not in any way depend upon the ultimate validity of this or any other particular theory. The aim of my demonstration has been to show that, through whatever means the stimulus of gravity may act, it is inevitable— from the fact

that the stimulation of the shoot is direct, and that of the root indirect — that a single identical stimulus should in the two cases induce opposite responsive effects. Thus, though for the sake of convenience it is necessary to distinguish the upward curvature of the stem from the downward curvature of the root, by such terms as apogeotropic and geotropic, yet these words must not be allowed to connote two different sensibilities to the action of gravity ; for the sensibility of irritable tissues to stimulus is always of one kind, and of one kind only.

The responses induced by stimulation of the tips of shoot and root are similar under similar circumstances. The amputation of the tip of the root does not, normally speaking, abolish the excitability of the growing region of the root. The abolition of geotropic action in the root, after amputation of the tip, shows therefore, as pointed out by Ciesielski and Darwin, that with regard to gravity it is the tip that is the perceptive organ. This conclusion is also supported by the experiments of Pfeffer and Czapek, in which amputation was not included. The statolithic particles, again, through whose weight stimulation by gravity is probably brought about, are, generally speaking, found concentrated at the root-tip.

Direct stimulation has been shown to induce a responsive movement in one direction, and indirect stimulation in the opposite. The opposite geotropic curvatures in apogeotropic and geotropic organs are therefore due, not to the possession of different sensibilities, but to the fact that in the former stimulation is direct, and in the latter indirect. General difficulties of the investigation — How to overcome these difficulties- Three distinct methods of testing results: (i) by variation of longitudinal growth ; (2) by responsive movement of pulvinus ; and (3) by growth-curvature— Method of application of chemical reagent — Effect of alkali — Effect of acid — Effect of copper sulphate — Action of sugar solution— Chemo-tactic movements — Explanation of anomalous osmotic or plasmolytic action — Excitatory versus plasmolytic reaction in pulvinus of Mimosa : ( 1 ) Favourable tonic condition — (2) Ordinary tonic condition— Polar effects of currents inducing growth-curvatures — Localised polar effects on pulvinus — Anodic and kathodic effects on longitudinal growth — Generalised law of polar excitation in plants — Galvano-tropic response— The indirect effect of polar excitation — The effect on growth of ' electrification ' of soil.

It will be understood that growth-curvatures take place so slowly, that the effects of various external agents in inducing them can only be observed visually after the lapse of considerable intervals of time, which may be a matter of hours, or even of days. This fact is sufficient of itself to introduce elements of complication into the problem ; for (i) the plant may in that time undergo unknown spontaneous variations ; (2) the fundamental effect of any given stimulus is subject, when too long continued, to reversal, as we have seen in the case of the leaf of Mimosa, which from normal contraction passes into fatigue-relaxation, under the longcontinued action of stimulus ; (3) the subjection of a plant during too long a period to an abnormal condition may sometimes cause derangement of its general functions ; and (4) there is the further element of variation which depends upon the point of application of stimulus itself, since we have seen that stimulation, acting directly on the responding organ, may produce one effect, and indirectly exactly the opposite. It

has doubtless been due to the action of these sources of variation that the observations made by investigators have so often been contradictory. All these uncertainties disappear, however, when we employ the high magnification, and the method of continuous record, which have been described ; for by these means we are enabled to follow the different phases of effect which occur immediately on the application of stimulus, and during its continuation. As these curvatures, moreover, are the results of the unilateral application of stimulus, expressed in the one-sided modification of longitudinal growth, the previous demonstration of the effect of the diffuse application of the same agent on growth itself enables us to infer the result to be looked for. The experiment is thus resolved into a verification of the inference.

Again, I have shown that the growing region acts like a diffuse pulvinoid. Experiments then, by unilateral application of the particular stimulus to a true pulvinus, offer us another and independent means of testing the results arrived at. Thus we have no fewer than three distinct methods of testing the action of a given agent in the induction of growthcurvature, which, if they corroborate each other, may be regarded as affording a rigorous demonstration of the results. These are : first, the variation of longitudinal growth under the diffuse action ; second, the responsive movement of a pulvinus under the unilateral action ; and, third, the curvature which represents the modification of growth induced by the unilateral action of a given stimulus or agent.

Method of application of chemical reagent. — In dealing with problems involving the unilateral application of chemical reagents particularly, the experimental arrangements which I am about to describe will be found suitable. In order to obviate the complications which might result from the indirect effect of stimulus applied at a distance, the chemical reagent should be applied directly on the growing region. For this purpose the petals of various flowers, while in an active state of growth, are very appropriate, and I have used particularly

those of the Indian Champaca [Michelia Champaca). The claw of a detached petal is held in a clip, and the petal is immersed in the inverted position in a small glass trough of water, of cubical shape. Inside the trough, and almost touching one face of the petal, say with its own right surface, is a partition of mica, in which is a long narrow slit. Thus the petal is in the left-hand compartment of the cube. A microscope of low magnification is focussed on the marginal point of its tip. The required chemical solution is now dropped by means of a pipette into the right-hand compartment. Thus it is diffused through the narrow slit, and acts directly and virtually unilaterally on the proximal side of the petal. If the particular agent should be one which accelerates growth, that is to say induces expansion, a growth-curvature will be induced, the proximal side becoming convex ; l but if its action is to induce depression or retardation of growth, the permanent effect will be a concavity of the proximal. The movements induced are observed by means of the microscope.

Effect of alkali. — In studying the effect of various drugs on growth in Chapter XXXV. we saw that the characteristic effect of alkali was to produce a contraction and retardation. In the present experiment, on the unilateral application of sodium hydrate in the manner described, the proximal side of the petal was found to become concave, the tip being carried towards the agent. Effect of acid. — The general effect of acid was found, as will be remembered, to be opposite to that of alkalis, namely relaxation. On now applying solution of HC1 unilaterally to a petal of Champaka. the result was the convexity of the proximal side.

Effect of copper sulphate. — This I have found to arrest growth, and its unilateral application in the present case was observed to induce concavity of the proximal side of the petal. 1 The sudden introduction of a reagent may occasionally give rise to a transient excitatory contraction, but the effects described here are the permanent growtheffects. Action of sugar solution. — A dilute solution of this agent has already been shown to accelerate growth. Its unilateral application was found, in the present case, to bring about convexity of the proximal side of the specimen ; but very strong solutions of sugar were found, as we have seen, to retard growth, and the unilateral application of such a solution was now found to induce concavity of the proximal side.

Chemo-tactic movements. — We have now seen that the excitatory or depressing action of various agents is indicated, in the case of the growing organ, by a curvature one way or the other. Similar movements are induced also in pulvinated organs ; but in organs which are capable of multiple response we shall expect to find that the corresponding effects induced by such agents will consist in similar movements often repeated— that is to say, in the initiation of such multiple response, or in its appropriate modification. It will be shown in Chapter XLIX. that the swimming movements executed by ciliated organisms form an instance of such multiple response. It will further be shown in that chapter that the organs respond to stimulus, whether chemical, photic, thermal, or electrical, by swimming either towards or away from it.

Explanation of anomalous osmotic or plasmolytic action. — An attempt has sometimes been made to explain the responsive movements of plant-organs as the result of supposed osmotic variations within the tissue. Thus it was held by De Vries that the growth-curvatures of multicellular organs were brought about by an increase of osmotic substances on the convex side, giving rise to augmented hydrostatic pressure. It was afterwards ascertained, however, that the convex side of the curved organ did not contain any greater quantity of osmotically active substances than the concave. The method of plasmolysis of De Vries is often used for the determination of the differential osmotic activity and turgescence on the two sides of a curved organ. After plasmolysis, the curve induced in the organ is flatter than before, and from this it has been inferred that the cell-

sap of the convex side of the organ is more powerfully osmotic than that on the concave. But various effects have been observed, of which the occurrence of plasmolysis alone affords no explanation. Thus, the diminution of curvature, which De Vries showed to be a consequence of plasmolysis, was demonstrated by Noll, in the case of recently curved organs, to be only the second phase of the effect ; for at the beginning of the operation in these cases, as he pointed out, the curvature of the organ was actually increased. This opposition of effects, the increase of curvature, followed by the flattening of the curve, as the result of plasmolysis, has not hitherto met with any satisfactory explanation.

These obscurities in plasmolytic reactions have arisen from the fact that the excitatory action of some of the plasmolysing reagents has not hitherto been recognised. That such an influence may be exercised, and sometimes in opposition to osmosis, was, however, demonstrated in my experiments on suctional response, where it was seen that a strong solution of sodium chloride applied at the roots, instead of arresting suction by the withdrawal of cell-sap, actually enhanced the rate of suction for some time (p. 383). It was also shown in a later chapter, dealing with the effects of chemical reagents on growth, that when favourable tonic conditions had been induced, fairly strong salt solutions, instead of the usual retardation of growth, brought about a temporary enhancement, followed by depression (p. 486). We thus see that the responsive movement is modified (1) by the condition of the tissue, and (2) by the duration of the application of the solution. Now, in an already curved organ, we have an induced anisotropy, or difference of condition, on the two opposite sides. The effects of chemical reagents, then, on such an organ will be complex, for they will differ on the two sides, in intensity, and also in phase of reaction. The observed responsive movement in increasing or diminishing the existing curvature will thus represent the algebraical summation of the effects on the two sides.

Excitatory versus plasmolytic reaction in pulvinus of Mimosa: (1) Favourable tonic condition. — I have already stated that a moderately strong solution of sodium chloride is a stimulating reagent. It is therefore to be expected that its diffuse application on the pulvinus will bring about a fall of the leaf ; and this, as we shall presently see, is found to be the case. We saw, however, in studying the effect of sodium chloride on growth-response, that under the favourable tonic condition induced by the optimum temperature of 340 to 35° C. it gave rise to a preliminary relaxation, followed later by contraction. From this it occurred to me, that in the parallel response of the pulvinus of Mimosa we might expect, under similar favourable conditions, to observe two opposite responsive movements : first, a preliminary expansion, exhibited by erection of the leaf, and afterwards a contraction, exhibited by the depression of the leaf.

In carrying out the experiment, I cautiously raised the temperature of the pulvinus to 34° C. and then applied to it a 3 per cent, solution of sodium chloride at the same temperature. The responses were automatically recorded by the leaf on smoked paper wrapped about the revolving drum. It will be seen (fig. 228) that the preliminary effect was a movement of relaxation or erection, which was completed in the course of one minute, and was followed by the opposite movement, or fall of the leaf. In these two opposite responsive movements, then, occurring in succession to each other, we have an analogous case to that of the two opposite and succeeding modifications of curvature, observed by Noll in a curved organ, immersed in salt solution.

Fig. 228. Response of Leaf of Mimosa in Favourable Tonic Condition to Chemical Stimulus of 3 per Cent. Salt Solution (2) Ordinary tonic condition. — It will thus be seen that inferences drawn from experiments on plasmolysis may lead us to very wrong conclusions unless we take full account of the possible excitatory action of the particular solution. This is made strikingly evident by the following experiment. If we apply a strong solution — say 10 per cent. — of sodium chloride to the pulvinus of Mimosa, then, arguing entirely from the theory of plasmolysis, we must expect that the withdrawal of water will cause flaccidity of the tissue, and so bring about the fall of the leaf. This flaccidity, further increasing with the duration of application, would tend also to increase the fall of the leaf progressively. But a similar fall

Fig. 229. Response of Leaf of Mimosa in Ordinary Tonic Condition to the Chemical Stimulus of 10 per Cent. Solution of Salt Under continuous stimulation the normal responsive fall is followed by fatigue-relaxation. may, on the other hand, be due to an excitatory reaction caused by strong salt, and there is nothing at first sight which would enable us to distinguish the one from the other. We know, however, as regards Mimosa, that under a continuous application of stimulus-- as, for instance, rapidly succeeding mechanical or electrical shocks — the first fall of the leaf is succeeded by a return to the erect position. If the predominant effect of salt, therefore, in the given instance, be to induce a responsive fall by excitation, then its continuous operation should give rise to a subsequent erection. And from the automatic curve recorded on the smoked drum this is found to be the case (fig. 229) ;

for it demonstrates that the fal) of the leaf of Mimosa under the action of a strong solution of salt was due, not to plasmolytic, but to excitatory action. Polar effects of currents inducing growth-curvatures.— The galvano-tropic effects noticed by various observers in the case of growing plant-organs have been contradictory, because of the many complicating factors which might be expected to arise, not only from the differences of anodic and kathodic effects, but also from the varying points of application, and intensity and duration of current. All the obscurities and anomalies in connection with this subject will, however, be found to disappear, when they are related to the fundamental polar effects, which have already been established as applying to the excitation of plant-organs (Chapter XVI.). I have shown that the kathode excites at make, and the anode at break. I shall now be able to show further that the galvanotropic effects observed are all deducible from these. Incidentally, too, owing to certain special advantages afforded by growth-response, we shall be able to discover additional effects which could not have been detected by the mechanical response of the pulvinus alone. We have hitherto investigated the polar effects of an electrical current acting on the pulvinus as a whole ; but in order to bring these fundamental effects into the clearest prominence, and to establish their universality, I shall now study (1) the reaction induced in a limited area of the pulvinus ; (2) the variations of longitudinal growth which result from the electrical actions of anode and kathode ; and (3) the growth-curvature induced by the unilateral action of anode or kathode on a growing region.