Plant Response as a Means of Physiological Investigation

In animal tissues, such rhythmic movements may be observed in their perfection in the case of cardiac muscle. The isolated heart, when brought to a state of temporary standstill, will give, in answer to a. single stimulus, a single response, or to a sufficiently strong stimulus a multiple series of rhythmic responses ; and under favourable circumstances it will give automatic responses for a considerable length of time. Similarly, the plant Biophytum, under exceptionally favourable circumstances, exhibits what are apparently automatic responses ; and again, under ordinary conditions it

gives a single response to a single stimulus, or if the stimulus be sufficiently strong, a multiple series of rhythmic responses. In the plant Desmodium, under favourable tonic conditions we observe automatic movements ; but when under less favourable circumstances, as for instance owing to the unfavourable season, it is brought to a state of standstill, it gives a single response to a single stimulus. When the stimulus, however, is strong, we have seen that it gives rise to a multiple series of responses, in a manner precisely like that of Biophytum under similar circumstances. It will thus be seen that Biophytum in its ordinary condition may be regarded as equivalent to Desmodium in a state of standstill. Similarities, in their fundamental characteristics, of rhythmic tissues, animal and vegetable : (i) In responses. — In the matter of response, we have found that the rhythmic automatic movements of cardiac muscle are repeated in Desmodium under ordinary tonic conditions, and in Biophytum under exceptionally favourable circumstances. In a state of standstill, all three give a single response to a single moderate stimulus, and a multiple series of rhythmic responses to a sufficiently strong stimulus. The following tabular statement exhibits this parallelism in a concise form :

Tabular Statement showing Similarities in the Responses of Rhythmic Animal and Vegetable Tissues (2) In possession of long refractory period. — In order to studyin detail the characteristics of response in Desmodium, I took a plant in which the leaflets had come to a state of natural standstill. To such a specimen I applied the stimulus of a condenser discharge ; it was found, as stated already, that a rather high electromotive charge (twenty-four volts) was required to produce response. The first few responses were somewhat feeble, owing to the sluggish condition of the tissue ; they then increased in a ' staircase ' manner till they became uniform, the period of a complete response being now about six minutes. From this point on, the responses were the maximal possible, and a higher E.M.F. produced no noticeable increase. The most characteristic feature of these responses was the possession of a long refractory period, which we have also found to be characteristic of the response of Biophytum. With this specimen of Desmodium I found that when a second stimulus was given after three minutes, there was no further response. But a stimulus given after three and a half minutes was effective. It may be mentioned here that the length of the refractory period varies somewhat with the condition of the tissue, being relatively longer when that is sluggish.

(3) In incapability of tetanus. — The rhythmic tissue of Desmodium thus resembles cardiac tissue, in the possession Fig. 145. Record showing that Rhythmic Tissue of Desmodium is Incapable of being Tetanised After the first two pulsations, strong tetanising electric shocks were applied continuously. No tetanic effect was produced, but the pulsation became somewhat irregular. of a marked refractory period. There is again another interesting similarity. A rhythmically beating cardiac tissue cannot be thrown into tetanus by quickly recurring electric

shocks. An automatically moving Desmodium leaflet is also incapable of being thrown into a state of tetanus (fig. 145). Rapidly succeeding shocks do not produce tetanic contraction, though some irregularity may occur in the pulsation ; excessively strong shocks kill the plant, and the pulsation is then permanently arrested. Theories regardingthe causation of heart-beat. — Having thus seen how similar are the phenomena of rhythmicity in cardiac muscle and in plants, we may proceed to inquire into the theories which have been proposed to account for the automatic pulsation of the heart. It has been suggested :

(1) That discrete impulses are sent out from certain motor nerve-centres in the heart to the muscular tissue, thus causing the periodic heart-beat. Assuming the correctness of this theory, however, the difficulty is merely transferred, for we have still to account for the rhythmic excitation of the nerve. But that the rhythmic heart-beat is not fundamentally due to rhythmic impulses from nerve-centres, has been proved from facts discovered by various observers : (a) that the isolated ganglion-free apex of the frog's heart may be thrown into rhythmic activity by stimulus ; it has also been shown by Gaskell (b) that the apex of the tortoise-heart, which is free from nerve-cells, is capable of rhythmic movements ; and (c) it is found that even in the embryo, before any connection with the central nervous system has been established, there is a rhythmic heart-pulsation.

(2) That cardiac muscle may have the inherent property of rhythmicity. This explanation, however, by itself, is incomplete, for it takes no account of the stimulus which must exist, in order to give rise to rhythmic expression. (3) That the pulsation of the heart is maintained by some ■ inner stimuli,' its rhythmicity being brought about by the long refractory period peculiar to cardiac muscle. Independent light, however, may be expected to be thrown on the question of the causation of spontaneous

rhythmic action in cardiac muscle, by the consideration of similar phenomena in plants, especially since it can be shown that their similarity is manifested under numerous varying conditions, and extends to fundamental characteristics. We have already seen that there is a similarity of fundamental characteristics between the response of cardiac muscle and that of rhythmic vegetable tissue. Similarities of rhythmic tissues, animal and vegetable.— We shall now, therefore, observe in detail those other and more special similarities which are exhibited in a common modification of response under varying external conditions, by Desmodium and cardiac muscle alike, and I shall first describe the remarkable effect produced on both by internal pressure.

( I ) The effects of in ternal hydrostatic p ressu re. — 1 1 i s fou n d that a heart which has come to a condition of standstill may be set into rhythmic activity by filling the cavity of the heart with liquid. Endo-cardiac pressure is thus found to act as a stimulus. In Desmodium, when the season is favourable, the tissue is in a turgid condition, and there is a considerable internal hydrostatic pressure, which we have seen to be advantageous to the maintenance of rhythm. This turgid condition depends on the ascent of sap, which, as I shall show in Chapter XXVIII., depends again on the rhythmic activity of certain tissues. Thus, in the summer season, we have the conditions most favourable for the maintenance of rhythmic activity in Desmodium. But with the approach of winter the vigour of the plant and its turgid condition undergo a marked decline, and the autonomous movement of the leaflet then comes to a stop.

It appeared to me that this cessation of movement might to a great extent be due to the diminution of internal hydrostatic pressure, and I undertook experiments to see whether the pulsatory movement could be renewed by an increase of this pressure, just as increased endo-cardiac pressure was found to renew the beating of the heart. The Fig. 146. Curve showing Relation between Temperature and Period of Pulsation in Desmodiitm Abscissa represents temperature, and ordinate time, in tenths of a minute.

experiment was carried out by mounting a detached petiole containing the motile leaflets at one end of a limb of a U-tube. Any desired pressure could be exerted by varying the height of the second limb, which was connected with the first by india-rubber tubing. By exerting internal pressure in this manner, I was able to produce vigorous rhythmic movements of leaflets which were, before this, in an absolutely quiescent condition. The beneficial effect of this constant internal pressure was further seen demonstrated by the extreme regularity and persistency of the rhythmic beats. Even in the best season of the year, the pulsations are irregular, and come to an occasional stop ; but under the action of internal pressure, I have found the detached leaflet to maintain its rhythmic activity unimpaired for nearly one hundred hours. In connection with this question of the increase of internal pressure, it should be mentioned here that, after the normal condition of

turgidity has been established, a further increase of internal pressure is found to increase the frequency of pulsation. Fig. 147. Curve showing Relation between Temperature and Period of Pulsation in the Heart of a Frog Excessive pressure, however, brings on irregularity, or even stoppage, of autonomous movement. (2) The effects of variation of temperature. — I have shown in the last chapter how perfectly similar are the effects of temperature in causing variations of the amplitude and frequency of pulsation in rhythmic tissues, both vegetable and animal. As regards the effect of temperature on the period, the similarity is strikingly exhibited in the two curves given above, showing the relation between temperature and period in Desmodium and in cardiac muscle. It will be seen that in both (figs. 146 and 147) the fall of period with increase of temperature is at first rapid and then slow. (3) Pei-iodic groupings of response. — Another very remarkable similarity between the pulsations of Desmodium and of cardiac muscle lies in their exhibition of periodic groupings, very simple types of which, given by Desmodium, are seen in the photographic records (figs. 148, 149). In fig. 146 we have an alternate waxing and waning of pulsation, and a remarkably similar record, given by frog's

Fig. 150. Simple Alternation of Pulsation in Frog's Heart (Pembrey and Phillips) heart, is seen in fig. ] 50. These groupings are of various degrees of complexity, one type of such heart-beats being that known as Luciani's groups, where the successive groups are separated by a long pause. Now, similar groups with intermediate pause are also seen in the pulsation of Deswodinm. I have again shown that periodic rhythms of various degrees of complexity are seen, not only in the pulsations of Desmodium, but also in the multiple responses of Biophytum, and even in the electrical responses in plants.

(4) Effects of barium salt. — There are agencies, such as internal pressure and certain chemical reagents, which induce regularity of pulsation in irregularly pulsating rhythmic tissues. Conversely, there are others, which produce the opposite effect, that is to say, a regular pulsation, after such an application becomes irregular. When the heart pulsations are regular, it is found that addition of Veratrin disturbs the uniformity. Remembering the general similarity of the action of Veratrin and barium salts, I applied a 5 per cent, solution of this substance to the pulvinus of Desmodium leaflet, which was executing very regular vibrations. The application of this reagent disturbed the regularity of the pulsation, and somewhat irregular

The regular pulsation, seen to the left, becomes irregular after application of this reagent. Fig. 152. Arrest of Beat of Ventricle of Frog at Diastole by Application of Acid at Arrow groupings were at once established (fig. 151). In another experiment with the same reagent, as the application of heat is known to neutralise the action induced by Veratrin and barium salts, I raised the temperature of the plant chamber, with the result that the beats became regular once more.

(5) Antagonistic actions of acid and alkali. — But the most remarkable of all the similarities seen in the pulsations of rhythmic tissues of animal and vegetable, is that of the antagonistic actions of acid and alkali. Acid induces in the case of the heart a relaxed or diastolic standstill, whereas i Fig. 153. Systolic Arrest of Heart-beat by Dilute NaHO Solution (Gaskell) alkali induces an effect exactly the opposite, the standstill being brought about at systolic contraction (figs. 152 and 153). It is also known that the standstill caused by one of these reagents can be counteracted by the antagonistic action of the other.

It is astonishing to find that exactly the same effects are produced by these reagents on the tissue of Desm odium. I first tried the effect of dilute hydrochloric acid, which, as will be seen, produced an arrest of pulsation in the ' diastolic ' or relaxed position (fig. 154). I next tried the effect of alkali — dilute solution of sodium hydrate — and it will be seen that this produced an arrest of pulsation in the ' systolic ' or contracted position (fig. 155). In records of the effect of this reagent on other specimens, in which its action had not proceeded so far, there was a continuous diminution of pulsation with a shifting towards the systole ; and when an acid was now applied, the antagonistic character of its action to alkali was clearly shown, by a gradual revival of response, with a shifting towards the diastole. In the present record, the systolic standstill caused by alkali was allowed to proceed far, and

Fig. 154. Arrest of Desmodium Pulsation at ' Diastole ' by Application \ of Acid yet on application of the acid there is seen to be a slight revival of pulsation and a final arrest towards diastole. Identical nature of rhythmic phenomena in animal and vegetable tissues. —We have thus found the responsive phenomena of cardiac muscle to be in every respect similar to those observed in the rhythmic tissues of Desmodium and FlG. 155. Arrest of Pulsation of Desmodium at ' Systole ' by Application of Dilute Alkali at T

Acid was next applied at 1, and the record shows its antagonistic action. Biophytum, the response of the latter being regarded as practically that of Desmodium in a state of standstill. We have seen that in these rhythmic animal and vegetable tissues the fundamental characteristics are identical : (1) In all of them stimulus gives either maximum response or none. (2) They all exhibit a long refractory period, during which additional stimulus produces apparently no effect.

And further, as regards the influence of various external agencies on the two classes of rhythmic tissues, animal and vegetable, the effects are also remarkably similar. Internal hydrostatic pressure renews in them rhythmic activity. All of them exhibit under certain circumstances similar cyclic groupings. The effects of chemical reagents are similar on both classes. Rise of temperature quickens the rhythm and reduces the amplitude of pulsation in a manner exactly similar in both ; and, finally, the effects of chemical reagents, even in the matter of antagonistic actions, are alike in the two classes. From a consideration of all these, it would appear that in studying response in rhythmic animal and vegetable tissues, we are dealing, not with two distinct but with a single class of phenomena. We are thus justified in ascribing the rhythmic action of the heart to those same causes which we have found to originate and maintain the rhythm of Desmodium or Biophytum. We have seen that these plants absorb energy continuously from the various forms of stimulus — mechanical, thermal, chemical, and other — to which they are subjected. This absorbed energy remains latent in the tissue, and determines its tonic condition, which is simply the sum total of these latent stimulating factors. When the sum of these factors exceeds a certain value, it will find expression outwardly in the form of excitatory discharges. This discharge, however, is not single and continuous, but intermittent. After each partial excitatory discharge, there is a diminution of conductivity and excitability which are only restored gradually. The long refractory period is merely an expression of this peculiar property. Owing to this periodic oscillation of conductivity and excitability, the constant latent stimulus finds expression in a rhythmic manner. Under favourable circumstances, there is a large surplus of accumulated energy, and longcontinued responses, apparently automatic, are thus produced.

Under less favourable conditions, when the stored-up energy is not great, a single stimulus gives rise to a single response, or, when the stimulus is stronger, to a multiple series of rhythmic responses ; and this statement is true of tissues exhibiting ' spontaneous movements,' not only in the case of the plant, but also in that of cardiac muscle. It is impossible to conceive that there could be movement without an exciting cause. Automatism is said to be one of the properties of protoplasm. It will be seen, however, from the evidence which I have adduced in cases where experimental investigation is possible, that, strictly speaking, there is no such thing as automatism. Only under the action of a stimulus can a living tissue give responsive indications. The impact of an external stimulus may give rise to an immediate expression, or it may partly or wholly be reserved in latent form for subsequent manifestation. ' Inner stimuli ' are simply external stimuli absorbed previously and held latent. An animal or a plant is thus an accumulator which is constantly storing up energy from external sources, and numerous manifestations of life — often periodic in their character — are but responsive expressions of energy which has been derived from external sources and held latent in the tissue.

The rhythmic tissue of Biophytum may be regarded as equivalent to that of Desmodium in a state of standstill. Both alike, when at standstill, give a single response to a single moderate stimulus, and multiple response to a strong stimulus. Both, when the sum total of latent energy is above par, give apparently ' automatic ' responses. The rhythmic tissues of both plants exhibit a long refractory period. The automatically responding leaflet of Desmodium is incapable of being tetanised.

An artificial increase of internal hydrostatic pressure renews pulsation in a Desmodium which was previously in a state of standstill. The effect of rise of temperature on Desmodium is to produce a shortening of period and decrease of amplitude of oscillation. The automatic responses of Desmodium often exhibit periodic groupings, of various degrees of complexity. Certain reagents tend to make irregular pulsations in Desmodium regular. Conversely, other reagents, like barium salts, induce irregularity in regular pulsations. The irregularity induced by the latter reagent may, however, be counteracted by a rise of temperature.

The effects of acids and alkalis on the pulsatory movements of Desmodium are antagonistic, acids inducing arrest of pulsation in a relaxed position, while alkalis induce its arrest in a contracted position. The standstill induced by one reagent may therefore be counteracted by the use of the other. By all these, the rhythmic phenomena of the plant are seen to be identical with those of the animal. The pulsation of the animal heart is thus to be ascribed to the same causes as bring about and maintain the rhythmic pulsations of Desmodium.

Inadequacy of existing theories of ascent of sap— General considerations regarding cellular activity and resultant propulsion of water — The Shoshungraph — Balanced Shoshungraph for determining variations of suction — Hydrostatic and Hydraulic Methods of Balance. THERE are few phenomena in plantlife which have attracted keener interest and inquiry than that process of transport by which water is carried, from below the surface of the earth to the tops of the tallest trees. The obscurity of the subject is so great, and the secondary co-operating agencies so numerous, that the inquirer is apt to be led into the error of confining his attention to some one of them alone, imagining it to be the principal element in the problem. In studying this subject, then, our first effort must be to distinguish between the essential factor and others which are merely subsidiary.

For a statement of the inadequacy of these subsidiary factors to the solution of the problem, it is only necessary to refer to the summary of Strasburger and PfefTer regarding existing theories of the ascent of sap : l The theory of atmospheric pressure is discredited, inasmuch as water is known to be lifted, in certain cases, to many times the height of the water-barometer. The theory of capillarity is inadequate, inasmuch as continuous capillaries are absent, and the height to which

(liquids could be raised by such means would not, moreover, approach that of an ordinary tree. The theory of osmotic action cannot be considered satisfactory, since such action is too slow ; besides which, there is no fixed distribution of osmotic substances, such as would account for the necessary transportation-current. The theory of root-pressure \ again, is open to the objection that it cannot possibly account for the maintenance of a sufficient force during the process of active transpiration, when root-pressure is found to be negative. Moreover, this rootpressure itself requires an explanation.

There is, however, another theory, due to Dixon, Joly, and Askenasy, which has apparently more to support it than any of those yet mentioned. According to this, the ascent is brought about by transpiration from the leaves. The fluid in the mesophyl cells of the leaves becomes concentrated by evaporation ; thus osmotic attraction is set up by the leaves, and the suction thereby exerted is supposed to be transmitted backwards, as far as the roots, through cohering columns of water. The difficulties in the way of this theory lie (1) in explaining how a slow osmotic action could produce so rapid a water-current ; (2) in the absence of any conclusive proof that, under actual conditions within the plant, the watercolumn could have sufficient tensile strength ; and, lastly, (3) in the fact which I shall demonstrate, that, when evaporation is not taking place in the leaves, the transport of water is still very considerable, and that, besides, other related phenomena, like exudation pressure, continue to take place even in the complete absence of evaporative activity in the leaves. I shall, moreover, be able to show that the movement of water often takes place in the plant in a direction opposite to that which would be the case if osmotic action were alone involved.

1 Thus,' to quote Pfeffer, ' a satisfactory explanation of the means by which the transpiration-current is maintained has not yet been brought forward. If no vital actions take part in it, then it is obvious that we have only an incomplete knowledge of the causes at work, and of the relationship of the different factors concerned.' J There then remains the question as to whether living cells by some unknown physiological activity might not be instrumental in effecting this transport of water. But the experiments of Hartig, Bohm, and Strasburger have been held to contradict such a possibility. Thus, Strasburger set the cut ends of trees in tubs of poison, such as copper sulphate solution. The poison ascended to the leaves, a distance, in the tallest trees, of twenty-one metres. Now, if such violent protoplasmic poisons ascend the trunk, it is clear that they must kill all the cells lying in their path. That the living cells of the stems could not be necessary to the rise of sap was taken to be a necessary inference from this experiment. Strasburger also killed portions of the stems of living trees by heat, and yet the upper living and leafy portion was found to remain turgid for a few days. Another well-known experiment which was held to negative the theory of protoplasmic activity, was that in which boiling water was poured on the roots, when the plant continued to transpire, in spite of the roots having been killed.

From such considerations, Strasburger was led to conclude that ' the supposition that the living elements in any way co-operate in the ascent of the transpiration-current is absolutely precluded.' l I shall nevertheless show that the ascent 6f sap is fundamentally due to the physiological activity of living cells, and that the experiments described above in no way negative this, being, on the contrary, capable of a different, and very satisfactory, explanation. Many difficulties connected with the problem of the ascent of sap will be found to disappear, when the physiological activity of living tissues is once clearly established as the essential factor. But a vague assumption of protoplasmic activity will not be sufficient for the elucidation of the phenomenon. It will be necessary to show further how this excitatory activity is initiated, and by what means a definite-directioned flow is imparted to the sap.