III.—ACTION OF STIMULUS ON VEGETABLE TISSUES
By
Sir J. C. Bose,
Assisted by
Narendra Nath Sen Gupta.
The leaf of Mimosa pudica undergoes a rapid fall when subjected to any kind of shock. This plant has, therefore, been regarded as “sensitive,” in contradistinction to ordinary plants which remain apparently immobile under external stimulus. I shall, however, show in course of this Paper that there is no justification in regarding ordinary plants as insensitive.
Let us first take any radial organ of a plant and subject it to an electric shock. It will be found that the organ undergoes a contraction in length in response to the stimulus. On the cessation of excitation the specimen gradually recovers its original length. Different organs of plant may be employed for the experiment, for example, the tendril of Cucurbita, the pistil of Datura, or the flower bud of Crinum. The shortening may be observed by means of a low power microscope. Greater importance is, however, attached to the detailed study of response and its time relations. The pull exerted by a delicate organ during its excitatory contraction is slight; hence arises the necessity of devising a very sensitive apparatus, which would give records magnified from ten to a hundred times.
RESPONSE RECORDERS.
The magnification of movement is produced by a light lever, the short arm of which is attached to the plant organ, the long arm tracing the record on a moving smoked plate of glass. The axis of the lever is supported by jewel bearings. The principal difficulty in obtaining accurate record of response of plant lies in the friction of contact of the recording point against the glass surface. This difficulty I have been able to overcome by providing a device of intermittent instead of continuous contact. For this, either the writer is made to vibrate to and fro, or the recording plate is made to oscillate backwards and forwards.
1. The Resonant Recorder.—In this the writing lever is made of a fine steel wire. One end of this wire is supported at the centre of a circular electromagnet; this latter is periodically magnetised by a coercing vibrator, which completes an electric circuit ten hundred, or two hundred times in a second. The writing lever is exactly tuned to the vibrating interrupter and is thus thrown into sympathetic vibration. Successive dots in the record thus measure time from 0.1 to 0.05 second. The employment of the Resonant Recorder enables us to measure extremely short periods of time for the determination of the latent period or the velocity of transmission of excitation.[D]
2. The Magnetic Tapper.—Measurement of very short intervals is not necessary in ordinary records of response. In this type of recorders, the circular magnet is therefore excited at longer intervals, from several seconds to several minutes; this is done by completion of the electric circuit at the required intervals, by means of a key operated by a clock.
3. The Mechanical Tapper.—In this, magnetic tapping is discarded in favour of mechanical tapping. The hinged writing lever is periodically pressed against the recording plate by a long arm, actuated by clock-work.
4. The Oscillating Recorder.—Here the plate itself is made to oscillate to-and-fro by eccentric worked by a clock. The frame carrying the plate moves on ball-bearings. The advantage of the Oscillating Recorder lies in the fact that a long lever, made of fine glass fibre, or of aluminium wire, may be employed for giving high magnification. A magnification of a hundred times may be easily obtained by making the short arm 2.5 mm. and the long arm 25 cm. in length.[E]
RESPONSE OF A RADIAL ORGAN.
Fig. 10. Response of a straight tendril of Passiflora to electric shock. Successive dots at intervals of 5 seconds. The vertical lines below are at intervals of a minute. In this and in all following records (unless stated to the contrary) up-curve represents contraction, and down-curve expansion or recovery.
Experiment 10.—As a typical example I shall describe the response of a straight tendril of Passiflora. A cut specimen was mounted with its lower end in water. Suitable electric connections were made for sending a feeble induction shock of short duration through the specimen. In this and all other records, unless contrary be stated, up-curve represents contractile movement. On application of stimulus of electric shock, an excitatory movement of contraction occurred which shortly reached its maximum; the apex-time was one minute and forty seconds, and recovery was completed after a further period of five minutes (Fig. 10). Stronger shocks induce greater contraction with prolongation of the period of recovery. The specimen was afterwards killed by application of poisonous solution of potassium cyanide; this brought about a permanent abolition of response. The experiment just described may be taken as typical of response of radial organs.
In a radial organ contraction takes place equally in all directions; it therefore shortens in length, there being no movement in a lateral plane. But if any agency renders one side less excitable than its opposite, diffuse stimulation will then induce greater contraction on the more excitable side which will therefore become concave.
RESPONSE OF AN ANISOTROPIC ORGAN.
Excessive stimulation is found to reduce the excitability of an organ. Under unilateral mechanical stimulation a tendril of Passiflora becomes hooked or coiled, the concave being the excited side. From what has been said, the unexcited convex side will relatively be the more excitable.
Fig. 11. Response of a hooked tendril of Passiflora to electric shock. Successive dots at intervals of 5 seconds.
Experiment 11.—I took a specimen of hooked tendril, and excited it by an electric shock. The response was by the greater contraction of the more excitable convex side, on account of which the curved specimen tended to open out. The record of this response is seen in Fig. 11; the apex-time was nearly two minutes, and the recovery was completed in the further course of 15 minutes.
From the responses of organs rendered anisotropic by the differential action of the environment we pass to others which show certain amount of anatomical and physiological differentiation between their upper and lower sides. I find that many petioles of leaves show movement in response to stimulus. Many pulvini, generally regarded as insensitive, are also found to exhibit responsive movements.
RESPONSE OF THE PULVINUS OF MIMOSA PUDICA.
Fig. 12. Response of the main pulvinus of Mimosa pudica.
The most striking and familiar example of response is afforded by the main pulvinus of Mimosa pudica of which a record is given in Fig. 12. It is generally assumed that sensibility is confined to the lower half of the organ. It will be shown in a subsequent Paper that this is not the case. The upper half of the pulvinus is also sensitive though in a feeble degree, its excitability being about 80 times less than that of the lower half. On diffuse stimulation the predominant contraction of the lower half causes the fall of the leaf, the antagonistic reaction of the upper half being, in practice, negligible. In order to avoid unnecessary repetition, I shall ignore the feeble antagonistic reaction of the less excitable half of the organ, and shall use the word ‘contraction’ for ‘relatively greater contraction.’
It is interesting in this connection to refer to the response of the leaf of Water Mimosa (Neptunia oleracea). Here the reaction is very sluggish in comparison with that of Mimosa pudica. A tabular statement of contractile response of various radial, anisotropic and pulvinated organs will show a continuity in the contractile reaction; the difference exhibited is a question of degree and not of kind.
TABLE 1—PERIODS OF MAXIMUM CONTRACTION AND OF RECOVERY OF DIFFERENT PLANTS.
| Specimen | Period of maximum contraction | Period of recovery. |
| Radial organ: Tendril of Passiflora | 100 seconds | 4 minutes. |
| Anisotropic organ: Hooked tendril of Passiflora | 120 " | 13 " |
| Pulvinated organ: Pulvinus of Neptunia Oleracea | 180 " | 57 " |
| Pulvinus of Mimosa pudica | 3 " | 16 " |
As regards the excitatory fall of the leaf of Mimosa pudica, Pfeffer and Haberlandt are of opinion that this is due to the sudden diminution of turgor in the excited lower half of the pulvinus. The weight of the leaf, no longer supported by the distended lower cells, causes it to fall. This is accentuated by the expansion of the upper half of the pulvinus which is normally in a state of compression. According to this view the excitatory fall of the leaf is a passive, rather than an active, movement. I have, however, found that in determining the rapidity of the fall of Mimosa leaf the factors of expansive force of the upper half of the pulvinus and the weight of the leaf are negligible compared to the active force of contraction exerted by the lower half of the pulvinus ([p. 87]).
With regard to the fall of turgor, it is not definitely known whether excitation causes a sudden diminution in the osmotic strength of the cell-sap or an increase in the permeability of the ectoplast to the osmotic constituents of the cell. Pfeffer favours the former view, while others support the theory of variation of permeability.[F]
RESPONSE OF PULVINUS OF MIMOSA TO VARIATION OF TURGOR.
Whatever difference of opinion there may be in regard to the theories of osmotic and permeability variations, we have the indubitable fact of diminution of turgor and contractile fall of the pulvinus of Mimosa under excitation. The restoration of the original turgor brings about recovery and erection of the leaf. In connection with this the following experiments on responsive movements of the leaf under artificial variation of turgor will be found of interest:—
Effect of Increased Turgor: Experiment 12.—A young Mimosa plant was carefully transplanted and the root embedded in soil placed in a linen bag. This was held securely by a clamp, and one of the leaves of the plant attached to the recorder. Withholding of water for a day caused a general loss of turgor of the plant. A vessel full of water was now raised from below so that the linen bag containing the roots was now in water. The effect of increased turgor by suction of water by the roots became apparent by the upward movement of the leaf. The distance between the immersed portion of the plant and the leaf was 2 cm. and the up-movement of the leaf was indicated within 10 seconds of application of water (Fig. 13). The velocity with which the effect of increased turgor travelled was thus 2 mm. per second. The leaf exhibited increasing erection with absorption of water.
Fig. 13. Response of Mimosa pulvinus to variation of turgor. Increased turgor by application of water at point marked with vertical arrow induced erectile movement. Diminution of turgor by application of KNO3 solution at the point marked with the horizontal arrow, brought about the fall of the leaf within 80 seconds. Successive dots at intervals of 5 seconds (The down curve represents up-movement and vice versâ.)
Effect of Diminution of Turgor: Experiment 13.—While the leaf in the above experiment was in process of erection, a quick change was made by substituting KNO3 solution for the water of the vessel in which the roots were immersed. The plasmolytic withdrawal of water at the roots gave rise to a wave of diminished turgor, the effect of which became perceptible within 40 seconds by the movement of fall of the leaf. (Fig. 13.)
DIFFERENT MODES OF STIMULATION.
In Mimosa excitation is manifested by the contraction of the pulvinus and the consequent movement of the leaf. But in most plants, excitatory movement cannot be realized on account of the rigidity of the plant structure, the thickness of the cell-wall and the want of facility for escape of water from the excited cells. I shall show later how excitation may be detected in the absence of mechanical movement.
As regards stimulation of vegetable tissues, there are various agencies besides electric shock, which induce excitatory contraction; these agencies I shall designate as stimuli. Excitation is detected in Mimosa by the downward movement of the leaf. It will be found that such excitatory movement is caused by a mechanical blow, by a prick or a cut, by the application of certain chemical agents, by the action of electric current and by the action of strong light. The study of the action of these stimuli will be given in greater detail in subsequent Papers.
I shall give below a general classification of different stimuli which cause excitation in vegetable tissues.
Electric Stimulus.—Induction shock, condenser discharge, the make of kathode and the break of anode.
Mechanical Stimulus.—Mechanical blow, friction, prick or cut.
Chemical Stimulus.—Effect of certain acids and of other chemical substances.
Thermal Stimulus.—Sudden variation of temperature; application of heated wire.
Radiation Stimulus.—Luminous radiation of the more refrangible portion of the spectrum; ultra-violet rays; thermal radiation in the infra-red region.
All these different forms of stimulus induce an excitatory contraction, a diminution of turgor, and a negative mechanical response or fall of a motile leaf.
SUMMARY.
A radial organ responds to stimulus by contraction in length; as all its flanks are equally excitable there is no lateral movement under diffuse stimulus.
Physiological anisotropy is induced in an organ, originally radial and isotropic, by the unequal action of the environment on its different sides. Diffuse stimulus induces a greater contraction of the more excitable side.
In a curved tendril the concave side is less excitable than the convex. Diffuse stimulus tends to straighten the curved tendril.
In the pulvinus of Mimosa pudica, the lower half is eighty times more excitable than the upper, and the fall of the leaf is due to the predominant contraction of the more excitable lower half.
A diminution of turgor takes place in the excited cells. Restoration of turgor brings about recovery of the leaf to its normal erect position. Independent experiments show that the fall of the leaf may be brought about by an artificial diminution of turgor, and the erection of the leaf by an increase of turgor.
IV.—DIURNAL VARIATION OF MOTO-EXCITABILITY IN MIMOSA
BY
Sir J. C. Bose.
Several phenomena of daily periodicity are known, but the relations between the recurrent external changes and the resulting periodic variations are more or less obscure. As an example of this may be cited the periodic variation of growth. Here the daily periodicity exhibited by a plant is not only different in varying seasons, but it also differs in diverse species of plants. The complexity of the problem is very great, for not only are the direct effects of the changing environment to be taken into consideration but also their unknown after-effects. Even in the case of direct effect, different factors, such as light, temperature, turgor, and so on, are undergoing independent variations; it may thus happen that their reactions may sometimes be concordant and at other times discordant. The nyctitropic movement of plants affords another example of daily periodicity. The fanciful name of ‘sleep’ is often given to the closure of the leaflets of certain plants at night. The question whether plants sleep or not may be put in the form of the definite inquiry: Is the plant equally excitable throughout day and night? If not, is there any definite period at which it practically loses its excitability? Is there, again, another period at which the plant wakes up, as it were, to a condition of maximum excitability?
In the course of my investigations on the irritability of Mimosa pudica, I became aware of the existence of such a daily periodicity; that is to say, the moto-excitability of the pulvinus was found to be markedly diminished or even completely abolished at a certain definite period of the day; at another equally definite period, the excitability was observed to have attained its climax. The observations on the periodic variation of excitability appeared at first to be extremely puzzling. It might be thought, for example, that light would prove to be favourable for moto-excitability; in actual experiment the results apparently contradicted such a supposition: for the excitability of the plant was found much higher in the evening than in the morning. Favourable temperature, again, might be regarded as an important factor for the enhancement of the moto-excitability; it was, nevertheless, found that though the excitatory response was only moderate at that period of night when the temperature was at its minimum, yet the excitability was altogether abolished at another period when the temperature was several degrees higher. The obscurities which surrounded the subject were only removed as a result of protracted investigation and comparison of continuous automatic records made by the plant itself during several months, beginning with winter and ending in summer.
The question whether a plant like Mimosa exhibits diurnal variation of excitability can be experimentally investigated by subjecting the plant at every hour of the day and night to a test-stimulus of uniform intensity, and obtaining the corresponding mechanical responses. Under these circumstances the amplitude of response at any time will serve as a measure of the excitability of the plant at the particular time. Any periodic fluctuation of response will then demonstrate the periodic character of variation of excitability.
The investigation thus resolves itself into:—
The successful construction of a Response Recorder which will automatically record the response of the plant to uniform periodic stimulation at all hours of day or night;
the study of the effects of various external conditions on excitability;
the diurnal variation of excitability and its relation to the changes of external conditions.
I will first give a diagrammatic view of the different parts of the apparatus which I devised for this investigation.[G] The leaf of Mimosa is attached to one arm of a light aluminium lever, L, by means of thread. At right angles to the lever is the writing index W, which traces on a smoked glass plate allowed to fall at a definite rate by clockwork the responsive movement of the leaf. Under a definite stimulus of electric shock the leaf falls down, pulling the lever L, and moving the writer towards the left. (Fig. 14.) The amplitude of the response-curve measures the intensity of excitation. The leaf re-erects itself after a time, the corresponding record exhibiting recovery. A second stimulus is applied after a definite interval, say an hour, and the corresponding response shows whether the excitability of the plant has remained constant or undergone any variation.
Fig. 14. Diagrammatic representation of the complete apparatus for determination of diurnal variation of excitability. Petiole of Mimosa, attached by thread to one arm of lever L; writing index W traces on smoked glass plate G, the responsive fall and recovery of leaf. A, primary, and S, secondary, of induction coil. Exciting shock passes through the plant by electrodes E, E′. A, accumulator. C, clockwork for regulating duration of tetanizing shock. Primary circuit of coil completed by plunging rod, V, dipping into cup of mercury M.
UNIFORM PERIODIC STIMULATION.
Electric mode of excitation.—I find that one of the best methods of stimulating the plant is by means of tetanizing induction shock. The sensitiveness of Mimosa to electric stimulation is very great; the plant often responds to a shock which is quite imperceptible to a human subject. By the employment of a sliding induction coil, the intensity of the shock can be regulated with great accuracy; the secondary if gradually brought nearer the primary till a stimulus is found which is minimally effective. The intensity of stimulus actually employed is slightly higher than this, but within the sub-maximal range. When the testing stimulus is maintained constant and of sub-maximal intensity, then any variation of excitability is attended by a corresponding variation in the amplitude of response.
The exciting value of a tetanizing electric shock depends (1) on the intensity, (2) on the duration of shock. The intensity may be rendered uniform by placing the secondary at a fixed distance from the primary, and keeping the current in the primary circuit constant. The constancy of the current in primary circuit is secured by the employment of an accumulator or storage cell of definite electromotive force. It is far more difficult to secure the constant duration of the tetanizing shock in successive stimulations at intervals of, say, one hour during twenty-four hours. The duration of the induction shock given by the secondary coil depends on the length of time during which the primary circuit is completed in successive excitations. I have succeeded in overcoming the difficulty of securing uniformity of duration of shock by the employment of a special clockwork device.
The clockwork plunger.—The alarum clock can be so arranged that a wheel is suddenly released and allowed to complete one rapid revolution at intervals of, say, one hour. There is a fan-governor by which the speed of the revolution can be regulated and maintained constant. This will specially be the case when the alarum spring is long and fully wound. The succession of short releases twenty-four times during the day produce relatively little unwinding of the spring. On account of this and the presence of the fan-governor, the period of a single revolution of the wheel remains constant. By means of an eccentric the circular movement is converted into an up and down movement. The plunging rod R thus dips into a cup of mercury M, for a definite short interval and is then lifted off. The duration of closure can be regulated by raising or lowering the cup of mercury. In practice the duration of tetanizing shock is about 0.2 second.
The same clock performs three functions. The axis which revolves once in twelve hours has attached to it a wheel, and round this is wound a thread which allows the recording glass plate to fall through six inches in the course of twenty-four hours. A spoke attached to the minute hand releases the alarum at regular and pre-determined intervals of time, say once in an hour. The plunging rod R, actuated by the eccentric, causes a tetanizing shock of uniform intensity and duration to be given to the plant at specified times.
Constancy of resistance in the secondary circuit.—In order that the testing electric stimulus shall remain uniform, another condition has to be fulfilled, namely, the maintenance of constancy of resistance in the secondary circuit, including the plant. Electric connections have to be made with the latter by means of cloth moistened with dilute salt solution; drying of the salt solution, however, gives rise to a variation of resistance in the electrolytic contact. This difficulty is overcome by making the electrolytic resistance negligible compared to the resistance offered by the plant. Thin and flexible spirals of silver tinsel attached to the electrodes E, E′ are tied round the petiole and the stem, respectively. In order to secure better electric contact, a small strip of cloth moistened with dilute salt and glycerine is wound round the tinsel. As the resistance of contact is relatively small, and as drying is to a great extent retarded by glycerine, the total resistance of the secondary circuit undergoes practically no variation in the course of twenty-four hours. This will be seen from the following data. An experiment was commenced one day at 1 P.M., when the resistance offered by 8 cm. length of stem and 2 cm. length of petiole was found to be 1.5 million ohms. After twenty-four hours’ record, the resistance was measured the next day and was found unchanged. The fact that the stimulus remains perfectly uniform will be quite apparent when the records given in the course of this paper are examined in detail.
THE RESPONSE RECORDER.
The amplitude of response affords, as we have seen, a measure of the excitability of the plant. In actual record friction of the writer against the glass surface becomes a source of error. This difficulty I have been able to overcome by the two independent devices, the Resonant Recorder and the Oscillating Recorder. In the former the writer is maintained by electric means in a state of continuous to and fro vibration, about ten times in a second. There is thus no continuous contact between the writer and the smoked glass surface, friction being thereby practically eliminated. The writer in this case taps a record, the successive dots occurring at intervals of 0.1 second. The responsive fall of the leaf is rapid, hence the successive dots in this part of the record are widely spaced; but the erection of the leaf during recovery takes place slowly, hence the recovery part of the curve appears continuous on account of the superposition of the successive dots. The advantage of the Resonant Recorder is that the curve exhibits both response and recovery. This apparatus is admirably suited for experiments which last for a few hours. There is, however, some drawback to its use in experiments which are continued for days together. This will be understood when we remember that for the maintenance of 10 vibrations of the writer in a second, 10 electric contacts have to be made; in other words, 36,000 intermittent electric currents have to be kept up per hour. This necessitates the employment of an electric accumulator having a very large capacity.
In the Oscillating Recorder the recording plate itself moves to and fro, making intermittent contact with the writer about once in a minute. The recording smoked glass plate is allowed to fall at a definite rate by the unwinding of a clock wheel. By an electromagnetic arrangement the holder of the smoked glass plate is made to oscillate to and fro, causing periodic contact with the writer.
Fig. 15. The Oscillator. Electromagnet M, M′, periodically magnetized by completion of electric current by clockwork C. Periodic attraction of soft iron armature A moves attached glass plate G to left, making thereby electric contact with writer.
The Oscillator is diagrammatically shown in Fig. 15. M, M′ are the two electromagnetic coils, the free ends of the horseshoe being pointed. Facing them are the conical holes of the soft iron armature A. This armature carries two rods which slide through hollow tubes. The distal ends of the rods support the holder H, carrying the smoked glass plate. Under normal conditions, the plate-holder is held by suitable springs, somewhat to the right of, and free from contact with, the writer. A clockwork C carries a rotating arm, which makes periodic contact with a pool of mercury contained in the vessel V, once in a minute. On the completion of the electromagnetic circuit, the armature A is attracted, the recording glass plate being thereby moved to the left making contact with the writer. The successive dots in the record thus take place at intervals of a minute. Only a moderate amount of electric current is thus consumed in maintaining the oscillation of the plate. A 4-volt storage cell of 20 amperes capacity is quite sufficient to work the apparatus for several days.
The responsive fall of the leaf of Mimosa is completed in the course of about two seconds. The leaf remains in the fallen or ‘contracted’ position for nearly fifteen seconds; it then begins to recover slowly. As the successive dots of the Oscillating Recorder are at intervals of a minute, the maximum fall of leaf is accomplished between two successive dots. The dotted response record here obtained exhibits the recovery from maximum fall under stimulation (cf. [Fig. 23]). The recovery of the leaf in one minute is less than one-tenth the total amplitude of the fall, and is proportionately the same in all the response records. Hence the successive amplitudes of response curves that are recorded at different hours of the day afford us measures of the relative variations of excitability of the plant at different times. This enables us to demonstrate the reality of diurnal variation of excitability. In my experimental investigations on the subject I have not been content to take my data from any particular method of obtaining response, but have employed both types of recorders, the Resonant and Oscillating. It will be shown that the results given by the different instruments are in complete agreement with each other.
EFFECTS OF EXTERNAL CONDITIONS ON EXCITABILITY.
Before giving the daily records of periodic variation of excitability, I will give my experimental results on the influence of various external conditions in modifying excitability. The conditions which are likely to affect excitability and induce periodicity are, first, the effects of light and darkness: under natural conditions the plant is subjected in the morning to the changing condition from darkness to light; then to the action of continued light during the day; and in the evening to the changing condition from light to darkness. A second periodic factor is the change in the condition of turgidity, which is at its maximum in the morning, as evidenced by the characteristic erect position of the petiole. Finally, the plant in the course of day and night is subjected to a great variation of temperature. I will now describe the effects of these various factors on excitability. It should be mentioned here that the experiments were carried out about the middle of the day, when the excitability, generally speaking, is found to remain constant.
EFFECTS OF LIGHT AND DARKNESS.
Fig. 16. Effect of cloud. Dotted up-curve indicates responsive fall, and continuous down-line exhibits slow recovery. First four responses normal; next three show depression due to diminution of light brought on by cloud, the duration of which is indicated by horizontal line below. Last three records show restoration of excitability brought on by clearing of sky. All records read from left to right.
I have frequently noticed that a depression of excitability occurred when the sky was darkened by passing clouds. This is clearly seen in the above records obtained with the Resonant Recorder. Uniform sub-maximal stimuli had been applied to a specimen of Mimosa at intervals of fifteen minutes. The dotted up-line represents the responsive fall, and the continuous down-line, the slow recovery. The first four are the normal uniform responses (Fig. 16). The next three show the depressing effect of relative darkness due to cloudy weather. The sky cleared after forty-five minutes, and we notice the consequent restoration of normal excitability.
Effect of sudden darkness and its continuation. Experiment 14.—In the next record (Fig. 17) is shown the immediate and continued action of darkness. The first two are the normal uniform responses in light. By means of screens, the plant was next subjected to sudden darkness; this brought about a marked depression of excitability. Subjection to sudden darkness thus acts as a stimulus inducing a marked but transient fall of excitability. Under the continuous action of darkness, however, the excitability is at first restored and then undergoes a persistent depression.
Fig. 17. Effect of sudden darkness. Plant subjected to sudden darkness beyond horizontal line seen below. First two responses normal. Note sudden depression of excitability, revival and final depression under continued darkness.
Effect of transition from darkness to light: Experiment 15.—Here we have to deal first with the immediate effect of sudden transition, and then with the persistent effect of continuous light. In the record given in Fig. 18 the plant had been kept in the dark and the responses taken in the usual manner. It was then subjected to light; the sudden change from darkness to light acted as a stimulus, inducing a transient depression of excitability. In this connection it is interesting to note that Godlewski found that in the phenomenon of growth, transition from darkness to light acted as a stimulus, causing a transient decrease in the normal rate. The effect of continued light on Mimosa is an enhancement of excitability.
Fig. 18. Effect of change from darkness to light. The first three records are normal under darkness. Horizontal line below indicates exposure to light. Note preliminary depression followed by enhancement of excitability.
EFFECT OF EXCESSIVE TURGOR.
I have often found that the moto-excitability is depressed under excessive turgor. Thus the “over-turgid” leaf of Biophytum sensitivum does not exhibit any mechanical response on rainy days.
Fig. 19. Effect of enhanced turgor, artificially induced. First two responses normal. Application of water, at arrow, induces depression of moto-excitability.
Experiment 16.—The effect of excessive turgor on moto-excitability may be demonstrated in the case of Mimosa by allowing its main pulvinus to absorb water. The result is seen in the above record (Fig. 19), where water was applied on the pulvinus after the second response. It is seen how a depression of moto-excitability results from excessive turgor brought on by absorption of water. In such cases, however, the plant is found to accommodate itself to the abnormal condition and gradually regain its normal excitability in the course of one or two hours.
INFLUENCE OF TEMPERATURE.
The moto-excitability of the pulvinus of Mimosa is greatly modified under the influence of temperature. For the purpose of this investigation I enclosed the plant in a glass chamber, raising the temperature to the desired degree by means of electric heating. Responses to identical stimuli were then taken at different temperatures. It was found that the effect of heightened temperature, up to an optimum, was to enhance the amplitude of response. Thus with a given specimen it was found that while at 22°C. the amplitude of response was 2.5 mm., it became 22 mm. at 27°C., and 52 mm. at 32°C. The excitability is enhanced under rising, and depressed under falling temperature. The moto-excitability of Mimosa is practically abolished at the minimum temperature of about 19°C.
Fig. 20. Effect of moderate cooling during a period shown by horizontal line below. Moderate depression followed by quick restoration.
Effect of lowering of temperature: Experiment 17.—A simple way of exhibiting the effect of lowering of temperature is by artificial cooling of the pulvinus. This cannot very well be done by application of a stream of cooled water, because, as we have seen, absorption of water by the pulvinus is attended by a loss of excitability: diluted glycerine has, however, no such drawback. This fluid at ordinary temperature was first applied on the pulvinus, and after an interval of half an hour records were taken in the usual manner. Cooled glycerine was then applied and the record taken once more; the results are seen in Figs. 20 and 21. In the former, the first response was normal at the temperature of the room, which was 32°C.; the next two exhibit depression of excitability under moderate cooling; the duration of application of moderately cooled glycerine is there indicated by the horizontal line below. On the cessation of application, the normal temperature was quickly restored, with the restoration of normal excitability.
In the next record (Fig. 21) is shown the effect of a more intense cold. It will be noticed that the first effect was a depression, and subsequently, a complete abolition of excitability. Thick dots in the record represent applications of stimulus which proved ineffective. It will also be noticed that even on the cessation of cooling, and the return of the tissue to normal temperature the induced abolition of excitability persisted as an after-effect for a considerable time. I have likewise found that the after-effect of cold in abolishing the conduction of excitation is also very persistent. These experiments show that owing to physiological inertia, the variations of excitability in the plant often lag considerably behind the external changes which induce them.
Fig. 21. Effect of application of more intense cold. Note sudden depression followed by abolition of excitability, also persistent after-effect.
Effect of high temperature: Experiment 18.—It has been shown that the moto-excitability is enhanced by rising temperature; there is, however, an optimum temperature above which the excitability undergoes a depression. This is seen in the following record (Fig. 22), where the normal response at 32°C. was depressed on raising the temperature to 42°C.; the excitability was, however, gradually restored when the plant was allowed to regain the former temperature.
Fig. 22. Effect of temperature above optimum. Note depression of excitability induced by high temperature, and gradual restoration on return to normal.
I may now briefly recapitulate some of the important results: darkness depresses and light exalts the moto-excitability. Excessive turgor depresses motility. Still more marked is the effect of temperature. Lowering of temperature depresses and finally abolishes the moto-excitability: rise of temperature enhances it up to an optimum temperature, but beyond this point the excitability undergoes depression. The change in excitability induced by the variation of external condition is not immediate; the induced effect, generally speaking, lags behind the inducing cause.
DIURNAL VARIATION OF EXCITABILITY.
I will now give automatic records of responses taken once every hour for twenty-four hours. They prove conclusively the diurnal variation of excitability in Mimosa. After studying in detail the variations characteristic of particular times of the day, I will endeavour to correlate them with the effects brought on by the periodic changes of the environment.
Experiment 19.—As a typical example I will first give a record obtained in the month of February, that is, say, in spring. From this it will not be difficult to follow the variations which take place earlier in winter or later in summer.
Fig. 23. Record for twenty-four hours, exhibiting diurnal variation of excitability (spring specimen). The displacement of base-line is due to nyctitropic movement.
The record given in Fig. 23 was commenced at 5 P.M. and continued to the same hour next day. The first thing noticeable is the periodic displacement of the base-line. This is due to the nyctitropic movements of the leaf. It should be remembered that the up-movement of the leaf is represented by down-curve, and vice versâ. After the maximum fall of the leaf, which in this case was attained at 9 P.M., there followed a reverse movement: the highest erection, indicative of maximum turgor, was reached at 6 A.M. The leaf then fell slowly and reached a middle position at noon. The extent of the nyctitropic movement varies in individual cases; in some it is slight, in others very large. The erectile movement began, as stated before, at about 9 P.M.; in some cases, however, it may occur as early as 6 P.M.
In following the characteristic variations of response occurring throughout the day, we find that while they are practically uniform between the hours of 5 and 6 P.M., a continuous decline is manifested after setting in of darkness (7 P.M.); the fall of excitability continues even after sunrise (6-30 A.M.), response being practically abolished at 8 A.M. The excitability is then gradually restored in a staircase manner, the maximum being reached after 12 noon. After attaining this, the excitability remains more or less constant till the evening. It will be noticed that the amplitude of response at 5 P.M. on the second day was the same as the corresponding response on the previous day.
The results of this and numerous other records taken in spring may be summarized as:—
1. The maximum excitability of Mimosa is attained between 1 and 3 P.M., and remains constant for several hours. In connection with the constancy of response at this period, it should be remembered that when the response is at its maximum a slight increase of excitability cannot further enhance the amplitude of response.
2. The excitability, generally speaking, undergoes a continuous decline from evening to morning, the response being practically abolished at or about 8 A.M.
3. From 8 A.M. to 12 noon, the excitability is gradually enhanced in a staircase manner, till the maximum excitability is reached after 1 P.M.
I have obtained numerous records in support of these conclusions, some of which are reproduced in the following figures. In these cases responses to uniform stimuli at intervals of half an hour were taken at different parts of the day, the recorder employed being of the Resonant type.
Mid-day record: Experiment 20.—The record of daily periodicity previously given shows that the excitability reaches its maximum after 12 noon, and that it remains constant at the maximum value for several hours. This fact is fully borne out in the following record obtained with a different specimen (Fig. 24). The responses were taken here from noon to 3 P.M., once every half-hour.
Fig. 24. Mid-day record from noon to 3 P.M. exhibiting uniform excitability. Responses taken once every half-hour.
Evening record: Experiment 21.—The record given in [Fig. 23] shows that the amplitude of response falls continuously after 6 P.M. It might be thought that the diminished amplitude in the first part may be due to the natural nyctitropic fall of the leaf. The range of the pulvinar movement being limited, it is clear that the extent of the responsive fall must become smaller on account of the natural fall of the leaf during the first part of the night. That this is not the whole explanation of the decline of response in the evening will be clear from certain facts which I will presently adduce. It was stated that the leaf of Mimosa exhibits nyctitropic fall from 6 to 9 P.M., after which there is a reverse movement of erection. In certain specimens, however, the erectile movement commenced as early as 6 P.M. It is obvious that in these latter cases diminution of amplitude of response cannot be due to the reduction of the range of movement of the leaf. In Fig. 25 is given a series of records from 6 to 10 P.M. obtained with a leaf in which erectile movement had commenced early in the evening. Though the full range of responsive movement was in this case available, yet the amplitude of successive responses is seen to undergo continuous diminution.
Fig. 25. Evening record from 6 to 10 P.M., showing gradual depression of excitability.
Record in the morning: Experiment 22.—The excitability is, as we have seen, nearly abolished about 8 A.M., after which there is a gradual restoration. This gradual enhancement of excitability to a maximum in the course of the forenoon is seen well illustrated in the record below (Fig. 26).
Fig. 26. Morning record from 8 A.M. to 12 noon, exhibiting gradual enhancement of excitability.
The record of daily periodicity given in [Fig. 23] may be regarded as a typical example. Modifications may, however, be observed which are traceable to individual peculiarities. As an example of this, I give a record ([Fig. 27]) obtained with a specimen in which nyctitropic movement was very pronounced. The periodic variation of excitability exhibited here is practically the same as shown by other specimens. The interesting variation is in the character of the recovery from stimulus; the leaf was falling from 6 to 9 P.M.; owing to the shifting of the base-line upwards the recovery appears to be incomplete. After 9 P.M. the leaf was erected, at first slowly, then at a very rapid rate. The consequent fall of the base-line late at night is very abrupt; hence there is an apparent over-shooting in the line of recovery.
Fig. 27. Record of diurnal variation of excitability; it exhibits marked nyctitropic movement.
EFFECT OF TEMPERATURE ON VARIATION OF EXCITABILITY.
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.)
| Hours of day. | Temperature. | Amplitude of Response. | Hours of day. | Temperature. | Amplitude of Response. | ||||||
| 5 | p.m. | 28°.0 | C. | 28.0 | mm. | 5 | a.m. | 20°.0 | C. | 5.0 | mm. |
| 6 | " | 25.5° | " | 28.0 | " | 6 | " | 20.5° | " | 4.2 | " |
| 7 | " | 24.5° | " | 27.0 | " | 7 | " | 21°.0 | " | 3.5 | " |
| 8 | " | 23°.0 | " | 23.5 | " | 8 | " | 22°.0 | " | 2.5 | " |
| 9 | " | 22°.0 | " | 21.5 | " | 9 | " | 24°.0 | " | 0.0 | " |
| 10 | " | 21°.0 | " | 18.0 | " | 10 | " | 26°.0 | " | 6.0 | " |
| 11 | " | 20.5° | " | 15.0 | " | 11 | " | 26.5° | " | 15.5 | " |
| 12 | " | 20°.0 | " | 13.0 | " | 12 | " | 28°.0 | " | 22.5 | " |
| 1 | a.m. | 20°.0 | " | 10.0 | " | 1 | p.m. | 28°.0 | " | 26.0 | " |
| 2 | " | 20°.0 | " | 8.0 | " | 2 | " | 28.5° | " | 28.0 | " |
| 3 | " | 20°.0 | " | 7.5 | " | 3 | " | 28.5° | " | 28.0 | " |
| 4 | " | 19.5° | " | 6.0 | " | 4 | " | 29°.0 | " | 28.0 | " |
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.
Fig. 28. The continuous curve shows the relation between the hour of the day and temperature. The dotted curve exhibits relation between the hour of the day and excitability.
EFFECT OF PHYSIOLOGICAL INERTIA.
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.
Fig. 29. Diurnal variation of excitability exhibited by summer specimen.
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.)
| Hours of day. | Temperature. | Amplitude of Response. | Hours of day. | Temperature. | Amplitude of Response. | ||||||
| 1 | p.m. | 38°.0 | C. | 22.0 | mm | 1 | a.m. | 26°.0 | C. | 21.5 | mm. |
| 2 | " | 38°.0 | " | 23.0 | " | 2 | " | 26°.0 | " | 20.0 | " |
| 3 | " | 38°.0 | " | 24.5 | " | 3 | " | 25.5° | " | 18.5 | " |
| 4 | " | 37°.0 | " | 28.0 | " | 4 | " | 25.5° | " | 17.0 | " |
| 5 | " | 35.5° | " | 29.0 | " | 5 | " | 25.5° | " | 16.0 | " |
| 6 | " | 33°.0 | " | 27.0 | " | 6 | " | 26°.0 | " | 15.0 | " |
| 7 | " | 31°.0 | " | 26.0 | " | 7 | " | 27°.0 | " | 14.0 | " |
| 8 | " | 30°.0 | " | 26.0 | " | 8 | " | 29°.0 | " | 13.0 | " |
| 9 | " | 29°.0 | " | 25.0 | " | 9 | " | 30.5° | " | 11.0 | " |
| 10 | " | 27°.0 | " | 24.5 | " | 10 | " | 33°.0 | " | 16.0 | " |
| 11 | " | 27°.0 | " | 24.0 | " | 11 | " | 35°.0 | " | 17.0 | " |
| 12 | " | 26.5° | " | 22.5 | " | 12 | " | 37°.0 | " | 21.0 | " |
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.
Fig. 30—The Resonant Recorder, with petiole-pulvinus preparation. (From a photograph.)
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.
Fig. 31—Variation of excitability after section. (1) Immediate effect; (2) variation of excitability in a second specimen during 50 hours: (a) response 4 hours after section; (b) response after 24 hours; (c) after 49 hours. Up-line of record represents responsive fall of the leaf, down-line indicates recovery from excitation.
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.
Fig. 32—Effect of amputation of upper half of pulvinus. Upper record gives normal response before amputation, and the lower, response after amputation. (Successive dots at intervals of 0.1 sec). Apex-time 11 sec, in both.
In carrying out this investigation I first took the record of normal response of an intact leaf on a fast moving plate. A second record, with the same stimulus, was taken after the removal of the upper half of the pulvinus, having taken the necessary precautions that have been described. Comparison of the two records (Fig. 32) shows that the only difference between them is in the exhibition of slight diminution of excitability due to operation. But, as regards the latent period and the quickness of attaining maximum fall, there is no difference between the two records before and after the amputation of the upper half. The upper part of the pulvinus is thus seen practically to have little influence in hastening the fall.
EFFECT OF REMOVAL OF THE LOWER HALF.
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.
Fig. 33.—Response after amputation of lower half of pulvinus. (Successive dots at intervals of a second; vertical lines mark minutes.) Apex-time, 40 secs.
INFLUENCE OF THE WEIGHT OF LEAF ON RAPIDITY OF RESPONSIVE FALL.
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).
Fig. 34.—Effect of weight on rapidity of fall. N, without action of weight; W, with weight helping; and A, with weight opposing.
It has been shown that the presence or absence of the upper half of the pulvinus makes practically no difference in the period of fall; it is now seen that the weight exerts comparatively little effect. We are thus led to conclude that in determining the rapidity of fall, the factors of expansive force of the upper half of the pulvinus and the weight of the leaf are negligible compared to the active force of contraction exerted by the lower half of the pulvinus.
ACTION OF CHEMICAL AGENTS.
In connection with this subject it need hardly be said that the various experiments which I had previously carried out with the intact plant can also be repeated with the isolated preparation. I will only give here accounts of experiments which are entirely new.
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.
Fig. 35.—Stimulating action of hydrogen peroxide.
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).
Fig. 36.—Incomplete recovery under the action BaCl2 and transient restoration under tetanisation at T.
Barium Chloride: Experiment 28.—The action of this agent is very characteristic, inducing great sluggishness in recovery. The preparation had been kept in 1-per cent. solution of this substance for two hours. After this the first response to a given test-stimulus was taken; the response was only moderate, and the recovery incomplete. The sluggishness was so great that the next stimulation, represented by a thick dot (Fig. 36), was ineffective. Tetanising electric shock at T, not only brought about response, but removed for the time being the induced sluggishness. This is seen in the next two records, which were taken under the old test-stimulus. There is now an enhanced response and a complete recovery. Beneficial effect of tetanisation disappeared, however, on the cessation of stimulus. This is seen in the next two records which were taken after two hours. The amplitude of response was not only diminished, but the recovery also was incomplete.
Fig. 37.—Antagonistic action of alkali and acid. Arrest of response in contraction under NaOH (↑), restoration and final arrest in expansion under lactic acid. (↑)
Antagonistic actions of Alkali and Acid: Experiment 29.—Alkali and acid are known to exert antagonistic actions on the spontaneous beat of the heart; dilute solution of NaOH arrests the beat of the heart in systolic contraction, while dilute lactic acid arrests the beat in diastolic expansion. I have found identical antagonistic reactions in the pulsating tissue of Desmodium gyrans, the telegraph plant. It is very interesting to find that these agents also exert their characteristic effects on the response of Mimosa in a manner which is precisely the same. This is seen illustrated in Fig. 37, where the application of NaOH arrested the response in a contracted state; after this, the antagonistic effect of dilute lactic acid is seen first, in its power of restoring the excitability; its continued application, however, causes a second arrest, but this time in a state of relaxed expansion.
CuSO4 Solution.—This agent acts as a poison, causing a gradual diminution of amplitude of response, culminating in actual arrest at death. Certain poisons, again, exhibit another striking symptom at the moment of death, an account of which will be given in a separate paper.
EFFECT OF “FATIGUE” ON RESPONSE.
Fig. 38.—“Fatigue” induced by shortening intervening period of rest.
With Mimosa, after each excitation the recovery becomes complete after a resting period of about 15 min. With this interval of rest the successive responses for a given stimulus are equal, and are at their maximum.
Experiment 30.—When the resting interval is diminished the recovery becomes incomplete, and there is a consequent diminution of amplitude of response. There is thus an increased fatigue with diminished period of rest. This is illustrated in Fig. 38, where the first two responses are at intervals of 15 min.; the resting interval was then reduced to 10 min., the response undergoing a marked diminution. Conversely, by increasing the resting interval, first to 12 and then to 15 min., the extent of fatigue was reduced and then abolished.
THE INFLUENCE OF CONSTANT ELECTRIC CURRENT ON RECOVERY.
Fig. 39.—Action of constant current in removal of fatigue by hastening recovery; N, curve of response in fatigued specimen; C, after passage of current.
Experiment 31.—From the above experiment it would appear that since the incompleteness of recovery induces fatigue, hastening of recovery would remove it. With this idea I tried various methods for quickening the recovery of the excited leaf. The application of a constant electric current was found to have the desired effect. Two electrodes for introduction of current were applied, one on the stem and the other on the petiole, at some distance from the pulvinus. In order to avoid the excitatory effect of sudden application, the applied current should be increased gradually; this was secured by means of a potentiometer slide. In my experiment a current having an intensity of 1.4 micro-ampère was found to be effective. Responses at intervals of 10 min., as we have seen, exhibit marked fatigue. Two responses were recorded on a fast-moving plate, N before, and C after, the application of the current. It will be seen (Fig. 39) how the application of current has, by hastening the recovery, enhanced the amplitude of response and brought about a diminution of fatigue. In connection with this, I may state that the tonic condition is, in general, improved as an after-effect of the passage of current. This is seen in some cases by a slight increase in excitability; in others, where the responses had been irregular, the previous passage of a current tends to make the responses more uniform.
ACTION OF LIGHT AND DARKNESS ON EXCITABILITY.
In taking continuous records of responses I was struck by the marked change of excitability exhibited by the intact plant under variation of light. Thus the appearance of a cloud was quickly followed by an induced depression, and its disappearance by an equally quick restoration of excitability. This may be explained on the theory that certain explosive chemical compounds are built up by the photosynthetic processes in green leaves, and that the intensity of response depends on the presence of these compounds. But the building up of a chemical compound must necessarily be a slow process, and it is difficult on the above hypothesis to connect the rapid variation of excitability with the production of a chemical compound, or its cessation, concomitant with changes in the incident light.
Fig. 40.—Stimulating action of light, and depressing action of darkness. Horizontal line below represents period of darkness.
Experiment 32.—In order to find out whether photo-synthesis had any effect on excitability, I placed an intact plant in a dark room and obtained from it a long series of responses under uniform test-stimulus. While this was being done the green leaflets were alternately subjected to strong light and to darkness, care being taken that the pulvinus was shaded all the time. The alternate action of light and darkness on leaflets induced no variation in the uniformity of response. This shows that the observed variation of excitability in Mimosa under the alternate action of light and darkness is not attributable to the photo-synthetic processes.
I next took a petiole-pulvinus preparation from which the sub-petioles bearing the leaflets had been cut off, and placed it in a room illuminated by diffused daylight. The normal responses were taken, the temperature of the room being 30°C. The room was darkened by pulling down the blinds, and records were continued in darkness. The temperature of the room remained unchanged at 30°C. It will be seen from records given in Fig. 40, that in darkness there is a great depression of excitability. Blinds were next pulled up and the records now obtained exhibit the normal excitability under light. The sky had by this time become brighter, and this accounts for the slight enhancement of excitability. This experiment proves conclusively that light has a direct stimulating action on the pulvinus, independent of photo-synthesis.[J]
SUMMARY.
On isolation of a petiole-pulvinus preparation, the shock of operation is found to paralyse its sensibility. After suitable mounting the excitability is restored, and remains practically uniform for nearly 24 hours. After this a depression sets in, the rate of fall of excitability becomes rapid 40 hours after the operation, sensibility being finally abolished after the fiftieth hour.
Experiments carried out on the effect of weight, and the influence of selective amputation of the upper and lower halves of the pulvinus, show that in determining the rapidity of fall of leaf, the assumed factors of the expansive force of the upper half of the pulvinus and the weight of the leaf are negligible compared to the force of active contraction exerted by the lower half of the pulvinus. The excitability of the lower half is eighty times greater than that of the upper.
Chemical agents induce characteristic changes in excitability. Hydrogen peroxide acts as a stimulant. Barium chloride renders the recovery incomplete: but tetanisation temporarily removes the induced sluggishness. Acids and alkalis induce antagonistic reactions, abolition of excitability with alkali taking place in a contracted, and with acid in an expanded condition of the pulvinus.
The responses exhibit fatigue when the period of rest is diminished. The passage of constant current is found to remove the fatigue.
Response is enhanced on exposure to light, and diminished in darkness. Light is shown to exert a direct stimulating action on the pulvinus, independent of photo-synthesis.