Life Movements in Plants, Volume I

So far I have merely described the observed diurnal variation of ex­cit­abil­ity. We may next inquire whether there is any causal relation between the change of external conditions and the observed variation of ex­cit­abil­ity. It has been shown that the moto-ex­cit­abil­ity is greatly influenced by temperature. In order to find in what manner the diurnal variation of ex­cit­abil­ity was influenced by the daily variation of temperature, I took special care to secure by means of the thermograph a continuous record of temperature variations. The table which follows shows the relation between the hours of the day, temperature, and amplitude of response, in a typical case of diurnal variation of ex­cit­abil­ity.

TABLE II.—SHOWING THE RELATION BETWEEN HOUR OF THE DAY, TEMPERATURE, AND EXCITABILITY. (SPRING SPECIMEN.)

From the data given in the table, two curves have been obtained. One of these shows the relation between the hours of the day and temperature; the other exhibits the relation between the hours of the day and the ex­cit­abil­ity as gauged by the amplitude of response (Fig. 28). It will be seen that there is, broadly speaking, a marked resemblance between the two curves, which demonstrate the predominant influence of temperature on diurnal variation of ex­cit­abil­ity.

It has been shown (page 59) that owing to physio­logic­al inertia, the change of ex­cit­abil­ity, generally speaking, lags behind the inducing cause. This fact finds striking illustration in the lag exhibited by the curve of ex­cit­abil­ity in reference to the temperature curve. The minimum temperature was attained at about 4 A.M., but the ex­cit­abil­ity was not reduced to a minimum till four hours later and again there is a marked fall of temperature after 5 P.M., but the ex­cit­abil­ity did not become depressed till two hours later.

There is again the factor of variation of light, the effect of which is not so great as that of temperature. The periods of maximum of light and temperature are, however, not coincident.

We may now discuss in greater detail the diurnal variation of ex­cit­abil­ity in Mimosa, taking the typical case, the record of which is given in Fig. 23. The temperature here is seen to remain almost constant, and at an optimum, from 1 to 5 P.M., the condition of light is also favourable. Hence the ex­cit­abil­ity is found to be constant, and at its maximum between these hours. The temperature begins to fall after 6 P.M., and there is, in addition, the depressing action of gathering darkness. Owing to the time-lag, the fall of ex­cit­abil­ity does not commence immediately at 6 P.M., but an hour afterwards, and continues till the next morning. During this period we have the cumulative effect of twelve hours’ darkness and the increasing depression due to cold, the temperature minimum occurring at 4 A.M. On account of the combined effects of these various factors, and phenomenon of lag, the period of minimum ex­cit­abil­ity is in general reached about 8 A.M. In certain other cases this may occur earlier. After the attainment of this minimum, the ex­cit­abil­ity is gradually and continuously increased, under the action of light and of rising temperature, till the maximum is reached in the afternoon.

EFFECT OF SEASON.

It was said that temperature exerted a predominant influence in inducing variation of ex­cit­abil­ity. We may, therefore, expect that the diurnal period would be modified in a certain way according to the season. In winter the night temperature falls very low; hence the depression of ex­cit­abil­ity is correspondingly great, and results in the complete abolition of ex­cit­abil­ity. The after-effect of intense cold is seen in the condition of inexcitability persisting for a very long period in the morning. In summer the prevailing high temperature modifies the diurnal periodicity in a different manner. When the night is warm, the fall of ex­cit­abil­ity is slight. In the day, on the other hand, the temperature may rise above the optimum, bringing about a depression. In such a case the ex­cit­abil­ity in the earlier part of the evening may actually be greater than in the middle of the day. These modi­fi­ca­tions are shown in a very interesting way in the following record (Fig. 29) taken at the end of April. The temperature of Calcutta at this season often rises above 100°F. or 38°C. Table III also exhibits, in the case of the summer specimen, the relation between the hours of the day, temperature, and ex­cit­abil­ity.

An inspection of the record given in Fig. 29 shows that the amplitude of response was enhanced after 4 P.M. The temperature up to that time was unusually high (38°C.), and there was in consequence a depression of ex­cit­abil­ity. After that hour there was a mitigation of heat, the temperature returning towards the optimum. Hence we find that the maximum ex­cit­abil­ity was attained between the hours 4 and 6 P.M. The minimum temperature at night was higher in the present case than that of the experiment carried out in February; in the former the minimum was 25.5°C., while in the latter it was 19.5°C. On account of this difference the night record in summer shows a fall of ex­cit­abil­ity which is far more gradual than that obtained in spring. The ex­cit­abil­ity is here not totally abolished in the morning, but reaches a minimum after 8 A.M.; the sensitiveness is then gradually enhanced in a staircase manner.

TABLE III—SHOWING THE RELATION BETWEEN HOURS OF THE DAY, TEMPERATURE, AND EXCITABILITY. (SUMMER SPECIMEN.)

SUMMARY.

The moto-ex­cit­abil­ity of Mimosa was gauged every hour of the day and night, by the amplitude of the response to a testing stimulus. This is effected by means of automatic devices which excite the plant periodically by an absolutely constant stimulus, and record the corresponding mechanical response.

From the record thus obtained, it was found that the ex­cit­abil­ity of the plant is not the same throughout the day, but undergoes a variation char­ac­ter­is­tic­al­ly different at different times of the day. In a typical case in spring the ex­cit­abil­ity attained its maximum value after 1 P.M. and remained constant for several hours. There was then a continuous fall of ex­cit­abil­ity, the minimum being reached at about eight in the morning. The plant at this time was practically insensitive. The moto-ex­cit­abil­ity was then gradually enhanced in a staircase manner till it again reached a maximum next afternoon.

The effect of sudden darkness was found to induce a transient depression, followed by revival of ex­cit­abil­ity. The effect of persistent darkness was to induce a depression.

Exposure to light from darkness caused a transient depression, followed by an enhancement of ex­cit­abil­ity.

Excessive turgor induced a diminished response.

Lowering of temperature induced a depression of ex­cit­abil­ity, culminating in an abolition of response. The after-effect of excessive cold was a prolonged depression of ex­cit­abil­ity.

Excitability was enhanced by rising temperature up to an optimum; above this point a depression was induced.

Owing to physio­logic­al inertia the change of ex­cit­abil­ity induced by variation of external condition lags behind the inducing cause.

The diurnal variation of ex­cit­abil­ity is primarily due to diurnal variation of temperature. The effect is modified in a minor degree by variation of light.

V.—RESPONSE OF PETIOLE-PULVINUS PREPARATION OF MIMOSA PUDICA

By

Sir J. C. Bose,

Assisted by

Surendra Chandra Das, M.A.

The most suitable plant for researches on irritability of plants is Mimosa pudica, which can be obtained in all parts of the world. An impression unfortunately prevails that the excitatory reaction of the plant can be obtained only in summer and under favourable circumstances; this has militated against its extensive use in physio­logic­al experiments, but the misgiving is without any foundation; for I found no difficulty in demonstrating even the most delicate experiments on Mimosa before the meeting of the American Association for the Advancement of Science held during Christmas of 1914. The prevailing outside temperature at the time was considerably below the freezing point. With foresight and care it should not be at all difficult to maintain in a hot-house a large number of these plants in a sensitive condition all the year round.

In order to remove the drawback connected with the supply of sufficient material, I commenced an in­ves­ti­ga­tion to find whether a detached leaf preparation could be made as effective for the study of irritability as the whole plant. Here we have at the central end of the leaf the pulvinus, which acts as the contractile organ; the conduct­ing strand in the interior of the petiole, on the other hand, is the vehicle for trans­mission of excitation. The problem to be solved is the rendering of an isolated petiole-and-pulvinus of Mimosa as efficient for researches on irritability as the nerve-and-muscle preparation of a frog. On the success of this attempt depended the practical opening out of an extended field of physio­logic­al in­ves­ti­ga­tion which would be unhampered by any scarcity of experimental material.

In connection with this it is well to note the surprising difference in vegetative growth as exhibited by plants grown in soil and in pots. A pot-specimen of Mimosa produces relatively few leaves, but one grown in the open ground is extremely luxuriant. As an instance in point, I may state that for the last five months I have taken from a plant grown in a field about 20 leaves a day for experiment, without making any impression on it. A large box containing soil would be practically as good as the open ground, and the slower rate of growth in a colder climate could be easily made up by planting half a dozen specimens. The protection of the plants from inclemencies of weather can be ensured by means of a glass cover with simple heat-regulation by electric lamps, in place of an expensive green-house.

Returning to the question of the employment of an isolated leaf, which I shall designate as a petiole-pulvinus preparation, instead of the entire plant, the first attempts which I made proved unsuccessful. The cut leaf kept in water would sometimes exhibit very feeble response, at other times all signs of ex­cit­abil­ity appeared to be totally abolished. It was impossible to attempt an in­ves­ti­ga­tion on the effect of changing environment on ex­cit­abil­ity when the normal sensitiveness itself underwent so capricious a change

These difficulties were ultimately overcome from knowledge derived through systematic in­ves­ti­ga­tion on the relative importance of the different parts of the motor apparatus, on the immediate and after-effect of section on the ex­cit­abil­ity of the leaf, and on the rate of decay of this ex­cit­abil­ity on isolation from the plant. The experience thus gained enabled me to secure long-continued and uniform sensibility under normal conditions. It was thus possible to study the physio­logic­al effects of changing external conditions by observing the responsive variation in the isolated petiole-pulvinus preparation. I propose to deal with the different aspects of the in­ves­ti­ga­tion in the following order:—

1. The effect of wound or section in modi­fi­ca­tion of normal ex­cit­abil­ity.

2. The change of ex­cit­abil­ity after immersion in water.

3. Quantitative determination of the rate of decay of ex­cit­abil­ity in an isolated preparation.

4. Effect of amputation of the upper half of pulvinus.

5. Effect of removal of the lower half.

6. Influence of the weight of leaf on rapidity of responsive fall.

7. The action of chemical agents.

8. Effect of “fatigue” on response.

9. The influence of constant electric current on recovery.

10. The action of light and darkness on ex­cit­abil­ity.

The isolated petiole-pulvinus preparation is made by cutting out a portion of the stem bearing a single lateral leaf. The four diverging sub-petioles may also be cut off. In order to prevent rapid drying the specimen has to be kept in water. Preparations made in this way often appeared to have lost their sensibility. I was, however, able to trace this loss to two different factors: first, to the physio­logic­al depression due to injury caused by section, and, second, to the sudden increase of turgor brought on by excessive absorption of water. I shall now proceed to show that the loss of sensibility is not permanent, but is capable of restoration.

EFFECT OF WOUND OR SECTION IN MODIFICATION OF NORMAL EXCITABILITY.

In connection with the question of effect of injury, it is to be borne in mind that after each excitation the plant becomes temporarily irresponsive and that the ex­cit­abil­ity is fully restored after the completion of protoplasmic recovery. A cut or a section acts as a very intense stimulus, from the effect of which the recovery is very slow. If the stem be cut very near the leaf, the excitation of the pulvinus is very intense, and the consequent loss of ex­cit­abil­ity becomes more or less persistent. But if the stem be cut at a greater distance, the transmitted excitation is less intense, and the cut specimen recovers its ex­cit­abil­ity within a moderate time. I have also succeeded in reducing the excitatory depression by previously benumbing the tissue by physio­logic­al means. The isolated specimen can be made still more compact by cutting off the sub-petioles bearing the leaflets; the preparation now consists of a short length of stem of about 2 cm. and an equally short length of primary petiole, the motile pulvinus being at the junction of the two.

For the restoration of sensitiveness, and to meet working conditions, the lower end of the cut stem is mounted on a T-tube, with funnel-attachment and exit-tube, as shown in Fig. 30. The other two cut ends—of the stem and of the petiole—may be covered with moist cloth or may be closed with collodion flexile to prevent rapid evaporation and drying up of the specimen. A slight hydrostatic pressure maintains the specimen in a moderately turgid condition. A preparation thus made is insensitive at the beginning, but if left undisturbed it slowly recovers its ex­cit­abil­ity. The history of the depression of ex­cit­abil­ity after shock of preparation and its gradual restoration is graphically illustrated by a series of records made by the plant (Fig. 31).

The petiole-pulvinus preparation thus made offers all facilities for experiment. Owing to its small size it can be easily manipulated; it can be enclosed in a small chamber and subjected to varying conditions of temperature and to the action of different vapours and gases. Drugs are easily absorbed at the cut end, and poison and its antidote can be successively applied through the funnel without any disturbance of the continuity of record. In fact, many experiments which would be impossible with the entire plant are quite practicable with the isolated leaf.

The arrangement for taking records of response is seen in Fig. 30, which is reproduced from a photograph of the actual apparatus. For recording the response and recovery of the leaf under stimulation, I use my Resonant Recorder fully described in the ‘Philosophical Transactions’ (1913). The petiole is attached to one arm of the horizontal lever. The writer, made of fine steel wire with a bent tip, is at right angles to the lever, and is maintained by electro­magnetic means in a state of to-and-fro vibration, say, ten times in a second. The record, consisting of a series of dots, is free from errors arising from friction of continuous contact of the writer with the recording surface. The successive dots in the record at definite intervals of a tenth of a second also give the time-relations of the response curve.

On account of its small size, the petiole-pulvinus preparation offers great facilities for mounting in different ways suitable for special in­ves­ti­ga­tions. Ordinarily, the cut stem with its lower end enclosed in moist cloth is supported below. A very suitable form of stimulus is that of induction shock from a secondary coil, the intensity of which is capable of variation in the usual manner by adjusting the distance between the primary and the secondary coils. The motile pulvinus, P, may be excited directly. For in­ves­ti­ga­tions on velocity of trans­mission of excitation, stimulus is applied on the petiole at some distance from the pulvinus, by means of suitable electrodes. Excitation is now transmitted along the intervening length of petiole, the conduct­ing power of which will be found appropriately modified under the action of chemical and other agents. In this normal method of mounting, the more excitable lower half of the pulvinus is below; excitatory reaction produces the fall of the petiole, gravity helping the movement. The preparation may, however, be mounted in the inverted position, with the more excitable lower half of the pulvinus facing upwards. The excitatory movement will now be the erection of the petiole, against gravity.

Under natural conditions the stem is fixed, and it is the petiole which moves under excitation. But a very interesting case presents itself when the petiole is fixed and the stem free. Here is presented the unusual spectacle of the plant or the stem “wagging” in response to excitation.

THE CHANGE OF EXCITABILITY AFTER IMMERSION IN WATER.

The isolated specimen can be kept alive for several days immersed in water. The ex­cit­abil­ity of the pulvinus, however, undergoes great depression, or even abolition, by the sudden change of turgor brought on by excessive absorption of water. The plant gradually accommodates itself to the changed condition, and the ex­cit­abil­ity is restored in a staircase manner from zero to a maximum.

In studying the action of a chemical solution on ex­cit­abil­ity, the solution may be applied through the cut end or directly on the pulvinus. The sudden variation of turgor, due to the liquid, always induces a depression, irrespective of the stimulating or the depressing action of the drug. The difficulty may be eliminated by previous long-continued application of water on the pulvinus and waiting till the attainment of uniform ex­cit­abil­ity which generally takes place in the course of about three hours. Subsequent application of a chemical solution gives rise to char­ac­ter­is­tic variation in the response.

QUANTITATIVE DETERMINATION OF THE RATE OF DECAY OF EXCITABILITY IN AN ISOLATED PREPARATION.

Variation of ex­cit­abil­ity after section: Experiment 23.—In order to test the history of the change of ex­cit­abil­ity resulting from the immediate and after-effect of section, I took an intact plant and fixed the upper half of the stem in a clamp. The response of a given leaf was now taken to the stimulus of an induction shock of 0.1 unit intensity, the unit chosen being that which causes a bare perception of shock in a human being. The specimen was vigorous and the response obtained was found to be a maximum. The stem bearing the leaf was cut at the moment marked in the record with a cross, and water was applied at the cut end. The effect of section was to cause the maximum fall of the leaf, with subsequent recovery. After this, successive responses to uniform stimuli at intervals of 15 minutes show, in (1) of Fig. 31, that a depression of ex­cit­abil­ity has been induced owing to the shock caused by section. In course of an hour, however, the ex­cit­abil­ity had been restored almost to its original value before the section. This was the case with a vigorous specimen, but with less vigorous ones a longer period of about three hours is required for restoration. In certain other cases the response after section exhibits alternate fatigue; that is to say, one response is large and the next feeble, and this alternation goes on for a length of time. The isolated specimen, generally speaking, attains a uniform sensibility after a few hours, which is maintained, with very slight decline under constant external conditions, for about 24 hours. On the third day the fall of ex­cit­abil­ity is very rapid, and the sensibility declines to zero in about 50 hours after isolation [Fig. 31 (2)]. We may describe the whole cycle of change as follows: by the shock of operation the isolated preparation is rendered insensitive for nearly an hour, the ex­cit­abil­ity is then gradually restored almost to its normal value before operation. Under constant external conditions, this ex­cit­abil­ity remains fairly constant for about 24 hours after which depression sets in. The rate of fall of ex­cit­abil­ity becomes very rapid 40 hours after the operation, being finally abolished after the fiftieth hour. It is probable that in a colder climate the fall of ex­cit­abil­ity would be much slower. The most important outcome of this inquiry is the demonstration of the possibility of obtaining persistent and uniform sensibility in isolated preparations. On account of this, not only is the difficulty of supply of material entirely removed but a very high degree of accuracy secured for the in­ves­ti­ga­tion itself.

EFFECT OF AMPUTATION OF UPPER HALF OF PULVINUS.

Experiment 24.—The determination of the rôle played by different parts of the pulvinus in response and recovery is of much theoretical importance. Our knowledge on this subject is unfortunately very scanty. The generally accepted view is that on excitation “the actual downward curvature of the pulvinus is partly due to a contraction of the walls of the motor cells consequent upon the decrease of turgor, but is accentuated by expansion of the insensitive adaxial half of the pulvinus—which was strongly compressed in the unstimulated condition of the organ—and also by the weight of the leaf.”[H] According to Pfeffer, after excitation of the organ, “the original condition of turgor is gradually reproduced in the lower half of the pulvinus, which expands, raising the leaf and producing compression of the upper half of the pulvinus, which aids in the rapid curvature of the stimulated pulvinus.”[I]

It was held, then, that the rapidity of the fall of leaf under stimulus is materially aided (1) by the expansion of the upper half of the pulvinus, which is normally in a state of compression, and (2) by the weight of the leaf. So much for theory. The experimental evidence available regarding the relative importance of the upper and lower halves of the pulvinus is not very conclusive. Lindsay attempted to decide the question by his amputation experiments. He showed that when the upper half was removed the leaf carried out the response, but rigor set in when the lower half was amputated. Pfeffer’s experiments on the subject, however, contradicted the above results. He found that “after the upper half of the pulvinus was carefully removed, no movement was produced by stimulation, whereas when the lower half is absent a weakened power of movement is retained.” Pfeffer, however, adds, “since the operation undoubtedly affects the irritability, it is impossible to determine from such experiments the exact part played by the active contraction of the lower half of the pulvinus.”[I]

The cause of uncertainty in this in­ves­ti­ga­tion is twofold. First, it arises from the unknown change in irritability consequent on amputation; and, secondly, from absence of any quantitative standard by which the effect of selective amputation of the pulvinus may be measured. As regards the first, I have been able to reduce the depressing action caused by injury to a minimum by benumbing the tissue before operation, through local application of cold, and also allowing the shock-effect to disappear after a rest of several hours. As regards the physio­logic­al gauge of efficiency of the motor mechanism, such a measure is afforded by the relation between a definite testing stimulus and the resulting response with its time-relations, which is secured by my Resonant Recorder with the standardised electrical stimulator.

Experiment 25.—The shock-effect caused by the amputation of the lower half was found to be very great, and it required a long period of rest before the upper half regained its ex­cit­abil­ity. The excitatory reaction of the upper half is by contraction, and the response is, therefore, the lifting of the petiole. Thus, in an intact specimen, excitation causes antagonistic reactions of the two halves. But the sensibility of the upper half is very feeble and the rate of its contractile movement, relatively speaking, very slow. The record of the response of the upper half of the pulvinus, seen in Fig. 33, was taken with an Oscillating Recorder, where the successive dots are at intervals of 1 sec.: the mag­ni­fi­ca­tion employed was about five times greater than in recording the response of the lower half (Fig. 32). The intensity of stimulus to evoke response had also to be considerably increased. Taking into account the factors of mag­ni­fi­ca­tion and the intensity of stimulus for effective response, the lower half I find to be about 80 times more sensitive than the upper. Thus, under feeble stimulus the upper half exerts practically no antagonistic reaction. The excitatory response of the upper half is also seen to be very sluggish.

Experiment 26.—It is obvious that the mechanical moment exerted by the weight of the leaf must help its responsive fall under excitation. But the relative importance of the factors of active contraction of the lower half of the pulvinus and of the weight, in the rate of the responsive down-movement, still remains to be determined. A satisfactory way of solving the problem would lie in the study of the characteristics of response-records taken under three different conditions: (1) When the leaf is helped in its fall by its weight; (2) when the action of the weight is eliminated; and (3) when the fall has to be executed against an equivalent weight. An approximation to these conditions was made in the following manner. We may regard the mechanical moment to be principally due to the weight of the four sub-petioles applied at the end of the main petiole. In a given case these sub-petioles were cut off, and their weight found to be 0.5 grm. The main petiole was now attached to the right arm of the lever, and three successive records were taken: (1) With no weight attached to the petiole; (2) with 0.5 grm. attached to its end; and (3) with 0.5 grm. attached to left arm of the lever at an equal distance from the fulcrum. In the first case, the fall due to the excitatory contraction will practically have little weight to help it; in the second case, it will be helped by a weight equivalent to those of the sub-petioles with their attached leaflets; and in the third case, the fall will be opposed by an equivalent weight. We find that in these three cases there is very little difference in the time taken by the leaf to complete the fall (Fig. 34).

The chemical solution may be applied directly to the pulvinus, or it may be absorbed through the cut end, the absorption being hastened by hydrostatic pressure. The normal record is taken after observing precautions which have already been mentioned. The reaction of a given chemical agent is demonstrated by the changed character of the record. The effect of the drug is found to depend not merely on its chemical nature, but also on the dose. There is another very important factor—that of the tonic condition of the tissue—which is found to modify the result. The influence of this will be realised from the account of an experiment to be given presently, where an identical agent is shown to produce diametrically opposite effects on two specimens, one of which was in a normal, and the other in a sub-tonic, condition. The experiments described below relate to reactions of specimens in a normal condition.

Hydrogen Peroxide: Experiment 27.—This reagent in dilute solution exerts a stimulating action. Normal records, were taken after long-continued application of water on the pulvinus. The peroxide, as supplied by Messrs. Parke Davis & Co., was diluted to 1 per cent., and applied to the pulvinus; this gave rise to an enhancement of response. Re-application of water reduced the amplitude to the old normal value (Fig. 35).