Plant Response as a Means of Physiological Investigation

Fig. 127. Photographic Record of Uniform Pulsations in Desmodium speaking, the down movement is more rapid, yet at times the up and down movements approximate to each other in this respect. I have occasionally found similar instances in the pulsatory movement of Desmodium, when there would be only a slight difference between the rates of the up and down movements. The normal rates again may even become reversed under the influence of external agencies.

Responsive significance of up and down movements. — A question of some difficulty and importance arises here as to the significance, whether of contraction or relaxation, of these up and down movements. We saw in the cases of Mimosa and Biophytum that the down movement was due to the relatively greater contraction of the lower half of the pulvinus, and the up movement to recovery or relaxation. It must be borne in mind that the upper half of the pulvinus of Mimosa, like the lower, is also excitable, though in a minor degree, as will be shown in a subsequent chapter. We have further seen that the more excitable half responds earlier to stimulus. The depression of the leaf, then, is due to the earlier and greater contraction of the lower half of the organ ; and its subsequent erection, to a natural expansive recovery, possibly aided by the later and feebler contraction of the upper half of the pulvinus. Arguing from analogy, we may regard the movement of the Desmodium leaflet as essentially similar to this. For here too we find, generally speaking, that the down movement is the more energetic, and the up movement relatively the slower, of the two. Hence we may infer that in Desmodium the down movement is due to contraction and the up movement to relaxation.

Test by increased internal hydrostatic pressure.— It will be shown on page 349 that when Desmodium in a subtonic condition undergoes arrest of pulsation, an increase of internal hydrostatic pressure is found to renew the rhythmic activity. With fairly high internal pressure, the frequency of the pulsation is increased, though the amplitude is decreased. In order now to test the responsive significance of the up and down movements respectively, I tried the effect of an increase

of internal hydrostatic pressure in shifting the vibration-limits of the Desiuodium leaflet. It will be remembered that in the case of Mimosa the effect of this increased internal hydrostatic pressure was to cause the erection of the leaf above its normal position. If, then, the same result should follow in the case of Desmodium, we should be justified in inferring that the mechanics of the motile organ were similar in the two cases. The increase of internal hydrostatic pressure was in this case effected by the same method as was employed in that of Mimosa — that is to say, the cut end of the petiole of Desmodium was placed in one limb of a U-tube, filled with water, and the pressure was increased by adding water, so as to raise the level at the free end of the tube. As the leaflet of Desmodium, however, is in constant oscillation, we must regard the mean of its vibration-limits as the normal mean, and the extent of the leaflet's erection under increase of hydrostatic pressure must be measured by the shifting upwards of this mean.

In carrying out this experiment, I found, on applying the increase of internal pressure exerted by a water-column of 10 cm., that, as will be seen in fig. 128, the lower limit of oscillation was displaced upwards in the record by 11*5 mm. and the upper limit by 3*5 mm. The mean position was thus raised by 7*5 mm. As the magnification of the record was in this particular case five times, it will be seen that the pressure exerted by a height of 10 cm. of

Fig. 128. Displacement of Mean Position of Vibration of Desmodium Leaflet by Increased Internal Hydrostatic Pressure U, upper limit ; L, lower limit ; M, mean position under normal conditions ; u', l', m', corresponding positions under increased internal hydrostatic pressure ; mm' is the extent of displacement upwards. In- creased internal pressure, generally speaking, produces a movement towards expansion, and tends to diminish the amplitude of the pulse, while increasing the frequency.

water had thus produced an absolute displacement of the mean position of the leaflet, of 1-5 mm. Considering these facts, it becomes reasonable to regard the motile indications of Desmodium as similar to those of Mimosa ; hence the down position of the Desmodiurn leaflet may be regarded as one of contraction, the up position being one of relaxation. Thus in Desmodium the down position of the leaflet corresponds to the systolic contraction, and the up position to the diastolic expansion of the heart.

Mode of application of chemical reagents. — Having now to some extent determined the character of the movements of the Desmodium leaflet, we shall proceed to observe in detail the modification of their movements by the action of various chemical reagents. Three different methods of application may be employed. In the first place, the chemical reagent may be dissolved in the water in which the specimen is placed. The solution will thus reach the motile

organ by the same process as that which brings about the ascent of sap. The characteristic action of the chemical reagent will be demonstrated in the subsequent modification of the responses. This method of applying a reagent at one point — in this instance the cut end of the petiole — and observing the subsequent physiological effect on the distant motile organ, is of special interest and importance in the case of poisons. For it serves to elucidate the obscure question of the ascent of sap through tissues that have been killed by poison (p. 385).

Fig. 129. Method of Application of Chemical Agent to Cut End of Petiole given plant, before and after the application of the solution, it is necessary that the continuity of the record should be in no way disturbed. For this purpose I insert the specimen in the arrangement shown in the diagram (fig. 129). One end of the tube is connected with a funnel, F, by means of an india-rubber tubing ; the other end is provided with a stopcock. The tube is first filled with water, and the stop-cock, S, closed. The normal responses of the leaflet are now taken, with the petiole in water. Next, by proper manipulation of the stop-cock, the water is allowed to run out, and its place is taken by the chemical solution which is passed in at the funnel. The record which is now taken exhibits the effect of the drug.

The second of our three methods of experiment is that of direct application — that is to say, touching the motile organ itself with a drop of the solution. In this case, the modification of response takes place rapidly. And, lastly, gaseous substances may be introduced or withdrawn from the plant chamber, by means of suitable inlet and outlet pipes. Action of chemical reagents modified by : tonic condition of tissue ; strength of solution ; duration of application.— There are certain general considerations of a very significant kind which it will be well to specify at this point. Though the fundamental effect of any given reagent is definite, yet certain minor variations of this effect, due to the constitution of the individual plant, are liable to occur, and these are often extremely suggestive. Two successive experiments, for example, were performed to determine the action of the poison mercuric chloride on different specimens ; one of these was extremely vigorous, and the other the reverse. The effect of the poison on the robust specimen consisted in depressing the pulsation, and reached its maximum some fifteen minutes after application. But subsequently the plant appeared to shake off the influence of the poison, and the pulses slowly recovered, till in the course of an hour they had once more become normal. The effect on the weakly specimen, however, was somewhat different. Instead of depression, the immediate result of application was a transi-

tory exaltation of the amplitude of oscillation, which was doubled, though at the same time the rhythm became slowed. After ten minutes, however, these pulsatory movements grew irregular and depressed, and the plant succumbed to the action of the toxic agent, its pulsation undergoing complete arrest forty-five minutes after application. In the case of a weakly specimen, again, on filling the chamber with an atmosphere of carbonic acid gas, the pulsations soon come to a stop, and unless fresh air be quickly introduced, this arrest becomes permanent. Butwith a vigorous specimen, the depression produced by this gas is very slow, and the permanent arrest does not take place till after a considerable lapse of time.

The effect of an agent, again, depends on the strength of the solution, and the duration of application. A solution which, in larger quantities, would produce depression, will often, if given in very diluted form, cause the exaltation of response. The sudden introduction of an agent which may ultimately produce arrest, may act as a transitory stimulus, bringing about a preliminary augmentation of response, to be followed later by depression and arrest. The application of a deleterious substance, again, for a short time, will cause a temporary depression, from which there is revival ; but too long-continued action of the same reagent will cause permanent arrest. Besides all these, there is the interesting phenomenon of accommodation, by which the plant becomes gradually accustomed to the action of any adverse circumstance, and is thus rendered immune.

Effect of anaesthetics. — Taking a specimen of Desmodium, I passed ether-vapour into the plant chamber. The pulsatory movement which had hitherto been fairly uniform now showed a transitory exaltation, and then fell with remarkable regularity of decrement. The response next showed an equally regular tendency towards the gradual recovery of its previous amplitude, but with longer period, this being protracted, from the normal two to four minutes. Finally, the pulsation of this specimen was abolished, the leaflet remaining in a position of relative relaxation, thirty

minutes after the first introduction of ether into the plant chamber. On blowing off the ether-vapour, there was in this case no revival of response. In those cases, however, in which the ethervapour is more diluted with air, or applied for a shorter period, the depression is temporary only, and is followed by revival. But if a larger quantity of ethervapour be at once introduced, a permanent arrest, in the relaxed position, quickly ensues. Before this happens, there may be one or two

Fig. 130. Photographic Record of Effect of Ether Vapour, Large Dose Arrow marks moment of application. Pulsation arrested in up, or relaxed, position. It has to be remembered that the up movement of the record corresponds to a down movement of the leaf. spasmodic flutterings, but these quickly subside, as will be seen in the photographic record given (fig. 130). Effect of alcohol. — The effect of this reagent is much modified by the tonic condition of the specimen. For example, in the case of a weaklier plant, the application, even of dilute solutions, induces rapid diminution and arrest of pulsation. More vigorous specimens can, however, withstand the deleterious effect of this drug, and bear stronger doses. In the photographic record here given (fig. 131) a 5 per cent, solution is seen to induce a greater regularity and amplitude of pulsation. The subsequent application of a 10 per cent, solution causes a moderate, and 15 per cent, a still greater, depression. The application of a 5 per cent, solution to a weakly specimen, however, is found, as said before, to induce a depression so great as to cause a speedy arrest.

Effect of carbonic acid. — The first effect of the introduction of this gas is sometimes one of exaltation. This is however, brief, and is followed by depression and sudden permanent arrest of pulsation in weaklier specimens. In other cases, again, an earlier re-introduction of fresh air into the chamber is sufficient to restore the specimen to its Pulsations to left show normal response, f Marks application of 5 per cent, solution ; f ' application of 10 per cent. ; f " application of 15 per cent.

natural pulsatory activity. I give here (fig. 132) a photographic record of the effect of this gas on a vigorous specimen, in which its action is seen to be somewhat gradual. One curious phenomenon which I have no* ticed in connection with the effect of this gas, is that when it remains stagnant in the chamber its depressing effect is much more rapid than when a current is allowed to stream through. In the manner which I have just described, I have carried out further a number of experiments on the action of various gases and vapours, such as ammonia, carbon disulphide, and others, all of which

are found to cause an arrest of the rhythmic movements of Desmodium. It is not necessary to go into these in detail, the experiments already given affording sufficient information for their successful repetition. I will only mention here the very interesting and important fact, that I find acids and alkalies, generally speaking, to produce effects which are in a certain physiological sense antagonistic. These effects, together with the influence of temperature on rhythm, and the action of tetanising electric shocks on the autonomous movements of Desmodium, will be found fully described in Chapters XXVI. and XXVII., where the remarkable parallelism of their influence on rhythmic animal and vegetable tissues will be demonstrated. I shall conclude the present chapter by describing the action of a strongly poisonous reagent on the pulsatory movements of Desmodium. Effect of copper sulphate solution. — I carried out two experiments on similar specimens to test the effect of

this reagent. In the first, the solution was applied directly on the pulvinus, and produced a very quick arrest of its rhythmic activity (fig. 133). In the second case, the application was made at the cut end of the petiole, as already described, which was at a distance of 2 cm. from the pulvinus. In this case the arrest took place much later, that is to say, thirty minutes after the application. This delay was due to the fact that the poisonous solution had to ascend the intervening distance before it could affect the rhythmic activity of the tissue at the pulvinus. This experiment will

Fig. 133. Photographic Record of Effect of Copper Sulphate Solution Applied on the Pulvinus Arrow marks moment of application. be found important, as touching a later investigation on the ascent of sap. It is to be noticed that the petiole allows the poisonous solution which kills it to pass upwards through it. Spark-record of pulsation of Desmodium.— Before ending this chapter, I shall give a spark-record of a single pulsation in a leaflet of Desmodium. The successive sparks were produced at intervals of 5 seconds, and a glance at the record affords a visual demonstration of the peculiar characteristics of the movement of the leaflet. The up line as usual indicates down movement. It is thus seen that, after a pause in the highest position, a sudden excitatory impulse is developed, which is gradually exhausted, as the lowest position is reached. The up movement takes place more gradually, and at a much slower rate. The results are shown

Fig. 134. Spark-record of Single Pulsation in Leaflet of Desmodium Tahle showing Rates of Movement at Different Stages of Pulsation in Desmodium. Total period . . 45 seconds Average rate . -6i mm. per sec. Maximum rate . 7 ,, ,, ,, Duration of pause . 40 seconds Total period . . 70 seconds Average rate . -4 mm. per sec. Maximum rate . '5 >> >> m Duration of pause . 35 seconds The effect of a chemical reagent on a plant is modified to some extent by the tonic condition of the tissue. A vigorous plant will, generally speaking, withstand for a considerable time the action of deleterious agents ; a weakly specimen succumbs more quickly.

The effect of a reagent depends also on the strength of the solution. A reagent which, in strong solution, induces depression, may, if given in small quantities, cause exaltation. The effect of a reagent depends also on the duration of application. The temporary depression produced by a short application is overcome by the self-accommodation of the plant. But it will succumb to too long or too strong an application of the same reagent. The effects of the various reagents on autonomous response are, generally speaking, similar to their effects on simple response.

The depressed position of the leaflet of Desmodium represents a ' systolic ' contraction, and the up position a 1 diastolic ' relaxation, of the motile organ. Increased internal hydrostatic pressure increases the extent of the relaxed or ' diastolic ' limit. The effect of too strong an application of ether is to abolish response, the arrest of pulsation usually taking place in a relaxed position. The immediate effect of application is generally a transient exaltation of response.

The effect of vapour of alcohol is usually a transient exaltation. If the application be prolonged, the result is a permanent arrest of pulsation. Carbonic acid sometimes produces a transient exaltation of response, and always a subsequent depression, which, under the long-continued action of this gas, may pass into permanent arrest. Copper sulphate solution, when applied directly on the pulvinus, quickly causes arrest of pulsation ; but if the cut petiole be allowed to absorb the solution, the final arrest does not take place till after the lapse of a certain period, required for the solution to ascend to the motile organ.

Increase of frequency and diminution of amplitude of pulsation with rising temperature — Converse effect of fall of temperature — Similar effect in cardiac pulsation — Effect of the reduction of temperature to the thermo-tonic minimum — Explanation of diminution of amplitude of pulsation with rise of temperature — Anomalous use of the word ' relaxation ' — Simple versus additive character of individual pulsation. It is my intention in the course of the next chapter to make a comprehensive review of the similarities in all their characteristics of rhythmically responding tissues, both vegetable and animal. In the present chapter, then, we shall confine our attention to a study in detail of the influence of temperature in modifying the amplitude and period of rhythmic autonomous responses, exemplified in the case of plant-tissues by Desmodium and in that of the animal bycardiac muscle.

As then we are about to study the effect of temperature on the period and amplitude of vibration of the Desmodium leaflet, it is clear that our first difficulty must be the securing of a specimen in which both are, to begin with, more or less uniform ; for the pulsation of Desmodium, like that of the isolated frog's heart used for experiments, is often irregular. It is thus only by careful selection that one can obtain suitable experimental subjects. A moderate increase of the internal hydrostatic pressure, however, will often have the effect of rendering the responses sufficiently uniform.

Regulation of temperature. — The second difficulty in this investigation lies in subjecting the plant to the required rise or fall of temperature. A rise of temperature may be secured by any one of three different methods, (i) A spirit flame may be applied underneath a bath of water in which the leaflets are placed. The temperature is thus gradually and continuously raised, and the successive pulsations, corresponding to different temperatures, are recorded in the usual manner by means of the Optic Lever. (2) Water at the required temperature may be syphoned into the bath, and the responses taken in the ordinary manner. (3) The air chamber in which the specimen is placed may be subjected to electric heating. The use of temperature may now be very accurately regulated by adjustment of the current, and records of pulsations may be taken at different and determinate temperatures.

This last is the most perfect method, the two former, dependent as they are on the immersion of the specimen in a bath of water, having as compared with it many disadvantages. For the natural freedom of movement of the leaflet is hampered by the water, and more troublesome still is the difficulty which at times arises from capillary action in the partially immersed cocoon-thread, by which the leaflet is attached to the Optic Lever outside the bath.

But we have not the same perfect facilities for lowering temperature in a gradual and continuous manner, as for raising it. This may be accomplished, however, sufficiently well for our purposes: (1) by placing fragments of ice in the air chamber ; or (2) the pulvinus of the leaflet may be touched with cold water which has been reduced to the required temperature by means of ice. I find, however, (3) that a much better method is that of placing in the air chamber a coil of thin-walled metallic tubing, preferably of highly conducting copper. When cooled brine is made to circulate through this coil, the temperature of the chamber is lowered, and by regulation of the flow, by means of stopcocks, it is possible to produce an adjustment of cooling.