Plant Response as a Means of Physiological Investigation

We have next to study the relation between the intensity of the stimulus and the extent of response under varying conditions ; that is to say, we have to determine the ' threshold of response,' in other words, the minimum intensity of stimulus that will be just sufficient to initiate reaction. We have then to observe the repeated response of the plant to repeated stimulation, whether uniform or gradually increasing. We have to detect the signs of fatigue if there be any, and

discover after what period of rest this disappears. We have also to record the exact time-relations of these phenomena. And further we have to study the effects of various external agencies in modifying the response. In order to carry out these investigations, it will be necessary first to arrange for placing the plant under suitable conditions for experiment. The next point is the devising of facilities for applying a stimulus of known intensity, which can be repeated, or increased by definite amounts, at will. And, lastly, there must be some means of obtaining an exact record of the response, from which the absolute movement of the responding organ and its time-relations may be deduced.

Experimental plant chamber. — As regards the first of these, it is advisable to have a special plant chamber within which the specimen can be subjected to the necessary conditions. This chamber may consist of a base-board and a movable cover. The framework of the latter is of wood, with glass panes. In order to give easy access to the plant during experiment, one side of the cover has a hinged window. The recording Optical Lever is placed inside the chamber, and the glass cover protects the recorder from any accidental disturbance caused by air-currents.

In connection with this, it is also important to provide arrangements for producing changes of temperature, and maintaining the changed condition uniform, for the required length of time. This is most satisfactorily accomplished by means of a heating coil placed inside the chamber, the temperature being regulated by suitable adjustment of the electrical current, sent into the coil through proper electrodes. Other necessary accessories consist of appliances for the purpose of stimulation, and facilities by which a constant current can be made to flow through the tissue, in experiments on the effect of electric currents on the excitability of plants. Details regarding these will be given later. The plant may be maintained in favourable humid conditions by placing wet blotting-paper inside the chamber (fig. 12).

The most important question with regard to the application of suitable stimulus is, as has been said, that it should be capable of exact measurement, of uniform repetition, and of definite increase or decrease at will. Another point which must be borne in mind is that the application of stimulus should not, by causing injury, change the excitability of the organ. As, moreover, a magnified record of the responsive movement is to be made immediately after the application, any stimulus which causes the slightest jar must necessarily

Fig. 12. Plant Chamber and Recorder The glass cover is not shown. be avoided. And for these reasons the mechanical form of stimulation is inappropriate to the investigation. The three most perfect modes of stimulation which I have been able to render practicable are, then, the thermal, the electrical, and the stimulus of light. The action of the last will be described in detail in another chapter, and we shall for the present confine our attention to the first two.

Electro-thermic stimulator.— Thermal stimulus may be applied very easily by touching the plant with a hot wire, but it is difficult by this means to ensure the uniformity of successive stimuli, inasmuch as the wire cannot be heated repeatedly to the same temperature, or made to touch the same point, many times in succession, with an equally effective contact. This difficulty is removed by means of what I have named the ELECTRO-THERMIC STIMULATOR. This consists of a thin M-shaped wire of platinum, with thick copper leads. It is slipped over the petiole which carries the sensitive leaflets. By now sending through it a current of definite intensity and duration, we can raise its temperature to any point we wish, and thus secure the application of a known intensity of stimulus at will. The elasticity due to the peculiar form of the thermal stimulator gives a definite and constant pressure of contact (fig. 13).

The observer applies the stimulus with his left hand, by pressing a tapping -key which is interposed in With his right hand he records on the revolving drum the exact moment of this application. This mode of thermic stimulation is, as will be shown presently, very efficient. Electric stimulation. — I have been able, however, to employ a mode of stimulus still more perfect, that, namely, of the electrical discharge from a condenser. Other forms of electrical stimulation may be used, such as those given by means of constant or induction currents. But these are liable, not only to cause more or less permanent internal changes by polarisation, but also to induce fatigue of the tissue. It will be shown in a later chapter that, on making the circuit, excitation takes place at the point where the current leaves the tissue — that is to say, at the kathode — and not at the anode, or point of entrance. By appropriate connections shown in the diagram (fig. 14), the point to be excited can be made

kathode during ' charge,' when the key is pressed. When the key is released, the circuit is ' discharged,' and the current flows in the opposite direction. The given point 1$ is, as has been said, excited by being made kathode at the moment of charge. The immediately succeeding discharge produces no exciting effect, but it wipes off any residual polarisation effect caused by charge. The plant-tissue is thus maintained in as completely normal a Fig. 14. Diagram of Connections for Stimulation by Condenser Discharge

Pressure of key K charges the condenser c through the plant. Release of key brings it in contact with M, discharging the condenser through the plant. L, responding leaflet attached to recording lever by thread s. condition as possible. The excitation produced in the plant by current to or from the condenser, I shall, for simplicity, designate as ' stimulation by condenser discharge.' In the course of the present chapter we shall study the response of the leaf of Mimosa^ shown by its fall, and also that of other sensitive plants, exhibited by the closure of the leaflets, as in the case of Biophytum sensitivum. One difficulty encountered in obtaining successive responses, in these latter cases, was due to the fact that the responding

leaflets, after each downward response, would sometimes remain persistently closed, for an indefinite period, thus preventing the continuation of the experiment. In cases where the leaflets are completely closed, one naturally regards the position as one of fatigue, or complete insensitiveness, because no further mechanical response is then obtainable. This depressed position, however, may not be indicative of total want of sensibility, for the apparent absence of response may really be due to the fact that further closure of the leaflets is a mechanical impossibility. We may consider an analogous instance in the case of animal tissues, muscle floating in mercury for example. The tissue remains persistently contracted after a single stimulus, and further response is impossible. But if, again, the muscle be stimulated while under tension, it responds to each stimulation, the process of recovery being aided by the external tension.

Practical importance of counterpoise. — Acting on this idea, it appeared to me that if we applied an external tension, the restoration of the leaflet to the natural outspread position might be helped, and the difficulty solved of obtaining the uniform repetition of effects of successive stimuli at brief and regular intervals of time. I therefore placed a small sliding counterpoise on that arm of the lever which was not attached to the leaflet This was found to fulfil its purpose. For in observing the effects of successive stimuli on different leaflets, I found that while neighbouring leaflets, not under tension, closed up after a few stimulations, and gave no further response, the leaflet which was attached to the lever, and which was under some slight tension, recovered its normal outspread position in the course of three to five minutes, and continued to respond in a normal manner to a long series of successive stimuli.

We shall now proceed to observe the actual responses obtained. The object here is not to investigate the peculiar or specific reaction of any one 'sensitive plant in particular, but the effects found universally among motile plant-organs. The occurrence of such effects in plants exhibiting all degrees of mechanical sensibility — from those in which it is shown in an extreme degree, to others again in which it is apparently almost non-existent — will be demonstrated in this and succeeding chapters,

In order to study the responsive movements of plants, we may take either the leaflets or the main petiole of Mimosa pudica. The leaflets, however, in this case are so excessively sensitive that even the contact for experimental adjustment is sufficient to produce a closure from which they do not recover for a considerable time. The pulvinus of the main petiole, on the other hand, is considerably less sensitive. Of intermediate sensibility are the leaflets of Biophytum sensitivum, which on the whole furnish the most suitable specimens for the general purposes of these experiments. This plant, which is known to be sensitive, grows in a wild state near Calcutta, and is so common as to be considered a weed. It is a low-growing herb, with simply pinnate leaves, each bearing from ten to sixteen pairs of leaflets. A better specimen could hardly perhaps be found for the exhibition of some of the most important characteristics of mechanical response. It is not, under ordinary conditions, excessively sensitive. A gentle touch does not, as a rule, produce the closing effect, but under specially favourable circumstances its sensitiveness may equal, if not surpass, that of the Mimosa leaflets. The closing of the leaflets takes place not upwards as in Mimosa^ but in the downward direction. I shall presently give details of the response obtained with Biophytum. But as this plant is not universally obtainable, and as it flourishes only for a short season, during and after our tropical rains, it may be best first to give an account of experiments made on the more generally accessible leaf of Mimosa.

Response of Mimosa. — As the responsive movement of the leaf of Mimosa is of considerable extent, no magnification is necessary for the record. Indeed, on the contrary, for the illustrations in the present work, the records had frequently to be taken on a reduced scale. This was accomplished by attaching the leaf to the long arm of the recording Optic Lever, and shortening the distance of the recording surface. The records given in figs. 15 and 16 were automatically obtained by the impression of the moving spot of light on a sensitive film wrapped about the recording drum. The leaf was excited by a single strong induction-shock. In order to obtain the complete curve of response and recovery — the double process being accomplished in the course of about seven minutes— the first record was taken on a slowly moving drum. For the detailed study of the characteristic timerelations of the first part of the curve, again, two more records were obtained, one with a moderate (fig. 15) and the

Fig. 15. Photographic Record of Response and Recovery of Mimosa, taken on a slowly moving drum. Record shows actual movement reduced to one-third. Fig. 16. Photographic Record of Response in different specimen, taken on a faster-moving drum showing only first part of the curve. Each division of time-scale = -5 second. other with a rapid speed of drum (fig. no). The last of these enables us to obtain time-measurements which are accurate to less than -^ of a second. The method by

which these rapid records are obtained will be described in Chapter XXII. From records obtained on a fast-moving drum, with a fairly average specimen of Mimosa, it is found that the leaf does not respond to stimulus immediately, there being a latent period of -f-^ of a second before it begins to move. The maximum fall is attained in the course of 2 seconds after the shock. After attaining the position of maximum depression, the leaf remains in its contracted position for a further period of about thirty seconds. It then begins slowly to recover, and perfect recovery takes place in the further course of six minutes. The record given was obtained from the leaf of a plant which was one year old, and in the summer season. It will be remembered that these responsive curves are modified by the physiological condition of the plant ; thus, for example, the time taken by the leaf of a vigorous young plant for recovery may be as short as four minutes, whereas an older specimen in winter may require as long a period as eighteen minutes. We may thus obtain from the record an idea of the physiological condition of the specimen.

By means of photography the taking of the record is made extremely simple, but there are certain disadvantages inseparable from this method, which render the devising of other means essential. For example, the motile sensibility of plants like Mimosa and Biophytum is profoundly modified in darkness. In the case of the records given, the plants have been kept outside in the light, and brought in immediately before experiment. But even then, after remaining in the dark for half an hour or so, the leaves of Mimosa become abnormally erected, till it can hardly be believed that the plant is sensitive, for it often becomes irresponsive to the hardest blow. Biophytum leaflets, again, in the same circumstances undergo closure. For these reasons, long-continued experiments in a dark room are an impossibility. Various sensitive plants, again, flourish only for a short-lived season, and during that period some hundreds of experiments have to be carried out.

This necessitates some method of record more expeditious than that of photography. Fortunately, the responsive movements of these sensitive organs are relatively slow, usually requiring several minutes for completion. And it is quite easy to follow the excursion of the responding spot of light, with the recording pen, on a horizontal drum. There are some few special investigations, such as those on exceedingly short latent periods, in which automatic records by photography are a necessity, but for the majority of the records the second method is all that is required. By the latter means, moreover, we overcome the serious difficulty occasioned by the variation of sensibility which the plant undergoes when kept long in a dark chamber. When the second method is employed, the specimen may be placed in a well-lighted and well-ventilated room, and under these conditions it is found to maintain its sensitiveness unchanged for a considerable length of time. The fact that the spot of light reflected on the drum becomes inconspicuous in the surrounding daylight is overcome by placing in front of the recording drum a special hood with a long horizontal slit. The back of this hood curves over the head of the observer, and the spot of light then appears very bright.

Response of Biophytum. — I shall next deal with the responses obtained from Biophytum. In fig. \J are given two successive responses to two successive thermal stimuli. It will be noticed how uniform these responses are. The upcurve represents the fall of the leaflets, and the subsequent down-curve of the response exhibits its gradual return to the normal outspread horizontal position. An abnormal erectile twitch will be noticed at the beginning of each of these responses. This effect is usually present when a stimulus of whatever nature is applied at a distance from the responding leaflet. Its cause will be explained later. It should be stated here that stimulus was in this case applied at a distance of 35 mm. from the responsive leaflet, and that the true excitatory reaction, by the depression of the leaflet, took place fifteen seconds after the application. In

other words, the excitation travelled the intervening distance with a speed of 2-3 mm. per second. The abnormal erectile Stimulus was applied at some distance from the responding leaflet. Thick dot represents moment of application oi stimulus. Fig. 18. Response of Biophytum to Electric Stimulation Stimulus was applied directly on the pulvinus. Ordinate represents absolute effect, however — due, as will be explained later, to hydrostatic disturbance — took place almost instantaneously.

The next figure (fig. 18) gives successive responses of Biophylum to condenser discharge, the pulvinus of the leaflet being directly excited. It will be noticed that in this case of direct stimulation, no abnormal erectile twitch is present. From the magnification of the record the absolute value of the movement is known, and in the present case it was r88 mm. The force exerted by the leaflet during its responsive movement was found equivalent to that exerted by the weight of 17 milligrammes. The total work performed by the leaflet during each responsive movement is therefore nearly equal to 1,600 millimetre-milligrams.

Effect of load. — In order to observe the effect of load on the response-curve, I added a slight additional counterpoise to the other arm of the lever. The record (fig. 19, d) shows the response-curve when the acting load is the weight of the lever ; (b) shows the effect of the additional load. It will be seen that while the height of the responses was diminished, yet the period of recovery was very much reduced, from five minutes to FlG- l9- Effect of L?*d

Isometric record. — The method of observing response employed in the foregoing results was that of recording the movement of the leaf. Similar methods are known in Animal Physiology as isotonic. There is, however, an interesting method corresponding to that known in Animal Physiology as the isometric — where, in obtaining records of responses, actual movement is almost abolished. The contraction of the more excitable half of the pulvinus exerts a certain tension, or pull. The object, under the isometric method of experiment, is to obtain records of varying responsive tensions of excited tissues, the physical movement being at the same time restrained. This I have been able to accomplish,

in the case of plant response, by the construction of a spiral spring-recorder, the movement of whose index is approximately proportional to the tension. The recorder is constructed of a fine flattened spiral spring. Springs of this description have the peculiarity that, when they are stretched by tension, the free end of the spiral rotates round the axis of the spring. A slight rotation may be magnified by means of a mirror and reflected spot of light (fig. 20). This arrangement is specially appropriate to the leaf-stalk of Mimosa, where the pull exerted by the excited leaf is considerable. Fig. 21 gives the isometric response of Mimosa.

Minimally effective stimulus in Biophytum.— It is well to mention here that at least in the case of Biophytum the minimal intensity of stimulus necessary to cause response is very well defined. With a certain specimen for example, when the. plant was excited by the discharge from a -oi microfarad condenser, charged to seven volts, there was no response. But when the condenser was charged wl, f"g °!u COndenser (o ■*» and seven volts alternately would ,„ the one ease produce response, and In the other none. If now, by the action of an external agency, the

practically prevented from moving. The tension exerted by excited leaf causes rotation of index or mirror. excitability of the tissue be increased, the seven-volt condenser charge, before inadequate, will become adequate. Conversely, if by the action of an external agent the excitability of the tissue be depressed, the nine-volt charge, which was formerly effective, will become now ineffective. I find, for instance, that lowering of the temperature will, by increasing molecular sluggishness, reduce excitability. Hence a minimally effective stimulus becomes ineffective when the tissue is cooled. Conversely, a rise of temperature produces the reverse effect, namely, increase of excitability. This was seen in a particular experiment with Biofthytum, where the minimally effective stimulus necessary at 300 C. was found to be reduced to two-thirds when the temperature was raised to 350 C.

Having thus obtained a reliable stimulus, whose value may be measured with precision, and which is capable of being repeated, and having also discovered an arrangement by which the effect of a given stimulus is invariably exhibited by a uniform response-record, we are now in a position to attack various physiological problems, as regards the influence of given external agencies on the conductivity and excitability of the plant-tissue. A differential response, causing lateral movement, is given by an organ in which the excitability of one half is different from that of the other ; and the movement takes place in a direction perpendicular to the plane of separation of the two halves. Such responses are characteristic of dorsiventral plant-organs.

In the responses of the sensitive organs of plants we notice : a short latent period ; a period during which the excitatory movement attains its maximum ; and a period of slow recovery. When the stimulus is applied at a distance, a preliminary abnormal erectile twitch is occasionally observed, which is due to hydrostatic disturbance. The true excitatory response takes place later. Besides the isotonic response, obtained by recording the actual movements of the excited leaf, it is also possible to record the isometric response where the movement is restrained, and the variation of tension caused by the contraction of tissue is alone recorded.

The intensity of a minimally effective stimulus in the case of Biophytum is definite. This value undergoes appropriate variation with the variation of excitability of the organ. ON THE UNIVERSALITY OF SENSITIVENESS IN PLANTS AS DEMONSTRATED BY MEANS OF ELECTRICAL RESPONSE Arbitrary classification of plants into sensitive and ordinary — Method of electromotive variation for detecting state of excitation — Hydraulic model— Excitation of vegetable tissue, like that of animal tissue, induces galvanometric negativity — Methods of direct and transmitted excitation — Electrical and mechanical response alike record molecular derangement .and recovery- Similarities in simultaneous record of mechanical and electrical response — True excitation has a concomitant negative turgidity-variation, negative mechanical response or fall, and galvanometric negativity — These are true physiological responses, and are abolished at death — Abnormal positive mechanical and electrical responses brought about by positive turgidityvariation - Direct and indirect effects of stimulation —Discrimination of differences of excitability by electric test — Excitability of planttissues in general — Responsive power characteristic of matter.