Plant Response as a Means of Physiological Investigation

' I have shown that galvanometric negativity is an unmistakable indication of the excitatory contraction of a vegetable tissue. Employing this test, I find that all living vegetable tissues are excitable. But the mechanical contraction produced will, other things being equal, be very marked only in long prosenchymatous tissues, and in a mass of parenchyma relatively less marked. anther-tube, in consequence of the general shortening. After a few minutes the filaments again elongate, returning to their original arched position. They are now once more irritable. Again, if one of the filaments be freed, and stimulation be applied to its outer surface, this convex surface becomes concave, returning to the original convex form during recovery. But if the inside of the filament be touched the reverse action takes place, that is to say, the inner side now becomes strongly concave. On excitation, the expelled water passes from the excited cells into the intercellular spaces. This excitatorily expelled water was observed by Pfeffer to well forth from the cut end of the filament. He also studied the contractile shortening, using a magnification of one to two hundred diameters, and found it to amount in various cases to from 8 to 22 per cent, of the original length.

It will be seen that in this case of Cynerece we find those conditions of extensibility and elasticity which are so characteristic of muscle, to be present, though not in an extreme degree. In muscles, contraction is brought about by redistribution of fluid, as between the constituent isotropic and anisotropic elements. In Cynerece, also, the phenomenon is not altogether different, for here it is known to be brought about by a redistribution of water as between the cells and intercellular spaces. It will be well to remember here that the effect of stimulus acting on an organ from without is, in general, to force the expelled water inwards, whence it may be driven, through intercellular spaces and fibro-vascular elements, out of the excited region, into the interior, or the rest, of the plant. On the cessation of stimulus, the water, thus expelled under tension, returns into the contracted cells, and this fact aids the process of recovery.

I have already demonstrated in the beginning of this chapter the contraction and concavity of the excited side of an organ, in response to unilateral stimulus. The responsive concavity of either side of the filament of Cynerece when excited, as observed by various investigators, is simply ar instance of this. The filaments of Cynerece cannot be regarded as, strictly speaking, radial organs, for their tangential diameters are nearly twice as much as the radial, hence their response cannot be considered as entirely unaffected by differential action on the two sides.

I shall now proceed to demonstrate that the radial organs of plants exhibit response by longitudinal contraction, just as muscle is seen to contract under stimulus, and that such contraction, so far from being distinctive of any specific plant, or of any organ of such plant, is characteristic of all radial organs of plants in general. It will also be shown that the lateral response of anisotropic organs is to be regarded as an instance of differential longitudinal contraction.

The Kunchangraph.— But it is here necessary to give a full account of the experimental arrangements by means of which this demonstration has been rendered possible, and to show that the results thereby obtained are reliable, consistent, and capable of the highest quantitative exactitude. In this ' Kunchangraph,' l which records the contractile response of the plant, as the Myograph that of the animal, we have, first, the plant chamber proper, which is made small, in order that the conditions, and variations of conditions, whose effects are to be studied, may be easily and rapidly changed. The lower end of the organ, securely held by means of a cork, is immersed in a small test-tube containing water, and fixed in the middle of the base-board. The upper end of the organ is connected with one arm of the Optic Lever by means of a thread. The fulcrum-rod, carrying the reflecting mirror, projects outside the chamber. A thin glass cylinder, not shown in the figure, may be used to cover the projected portion of the fulcrum-rod, thus protecting the mirror from the disturbance caused by air-currents.

One important condition which I find essential to the maintenance of uniformity of sensitiveness is that the plant shall be surrounded by a moist atmosphere, whose humidity is constant. An air-bag is kept under suitable pressure, and 1 From the Sanskrit kunchan = contraction. The u is here pronounced as in /////. air, bubbling through water, is made to enter the chamber through an entrance-pipe. The exit-pipe is connected with an aspirator. By proper manipulation of the stop-cocks of the air-bag and the aspirator, a gentle stream of humid air is kept in constant circulation through the chamber. A modification of this arrangement enables us to study the effect of various gases and vapours on the excitability of the organ. A series of responses is first taken, under normal conditions, that is to say, when the plant is surrounded simply by a moist atmosphere. By now turning a three-way tap in a given direction, the water-vapour can be made to pass through a vessel filled with a given gas, before reaching the plant chamber. Or the pure gas can be introduced alone. The series of responses now obtained shows both the preliminary and the permanent effects of the gas. For it is now easy, by means of the three-way cock, to shut off the gas, and to re-establish the first or normal condition. The responses next given afford an indication of the after-effect of the gaseous reagent. It will be seen that by this means the effects of various gases and vapours can be studied with the greatest ease (fig. 35).

The next point to be dealt with is that of the mode of application of a uniform or graduated stimulus. For electrical stimulation we may use induction currents. For this purpose, a sliding induction coil of Du Bois-Reymond's pattern is used. The intensity of the shock is here graduated, by bringing the primary coil nearer and nearer to the secondary. Non-polarisable electrodes make suitable connections with the upper and lower ends of the organ. Electrical stimulation is often preferable on the whole, but unless vigorous specimens are used, it is apt when strong to induce fatigue. This may be avoided by the use of moderate stimulus and high magnification. Or we may, if desired, use thermal stimulus, which is effected by means of heat generated in a continuous length of thin German-silver wire which surrounds the whole length of the specimen, in the form of a cylindrical cage. This wire cylinder is fixed appropriately

The plant enclosed in semi-cylindrical wire heating cage, H, seen open. The plant is attached to the Optic Lever. Light proceeding from focussing tube, L, after reflection from optical mirror, M, falls on the recording drum, d. Stimulation is periodically effected on closure of electrical circuit, containing storagebattery, s, by the rotating rod, r. Air from bag not shown is passed through water-vessel to right, and circulated through plant chamber. The vessel to left is the aspirator. The middle vessel contains ether or other chemical substance, which is made to displace air in plant chamber, by manipulation of stop-cock, g, the clock-governor.

in wooden or ebonite forms, which are made in halves and hinged, so that one half of the cylinder may be swung back in order to afford easy access to the specimen, for the purpose of adjustment. When closed, the wire is in complete circuit with the electrodes outside. By sending through this wire a strong current of short duration, the sudden rise of temperature generated in the chamber causes the stimulation of the tissue. This heat is quickly dissipated, again, by the stream of air charged with vapour, which is in constant circulation. The effectiveness of stimulation will depend on the range, and also on the suddenness, of the temperaturevariation. The required thermal stimulus is thus most easily effected by electrical means, the degree of rise of temperature being determined by the strength of the current acting on the heating circuit, and the requisite suddenness of variation being the result of temporary completions of the circuit of definite and short duration. This mode of stimulation, I shall, for convenience, designate as stimulation by thermal shocks.

It is very difficult, using only the hand, to attain the necessary precision in making these brief and equal completions of the circuit several times in succession. Hence, the stimulus not being strictly uniform, the responses are apt to become unequal. I have overcome this difficulty, however, by the construction of a closing key regulated by clock-work, which enables successive stimuli, of equal intensity and duration, to be applied automatically, at predetermined intervals. This is accomplished by means of a clockwork arrangement, which enables uniform and successive electrical or thermal shocks to be applied, while at the same time the intervening periods between successive shocks may be so adjusted as to allow for complete recovery. A radial arm, carried on the axle-rod of the clock, at each complete revolution strikes against a balanced key, which completes the electric circuit. The intervals of successive stimulation may be determined by regulating the speed of the clock. This may be done by suitably inclining the

blades of the air-vane governor (fig. 36). The diagram shows the mode of making successive closures of the electric circuit for giving thermal shocks. And the same arrangement serves to close the primary of the induction coil. The duration of stimulus, depending upon that of the closure of circuit, may be adjusted by varying the length of the radial arm. The effective intensity of stimulation may be increased by applying three or four shocks in rapid

Fig. 36. Diagrammatic Representation of Apparatus for Periodic Stimulation of Plant H, the wire cylinder made in hinged halves, periodically heated when the electric circuit is closed by tilting over of balanced key, K, when pressed by rod, R. v, air-vane of clock, by which period of rotation is adjusted. succession, instead of one. For this purpose the radial arm carries at its end a small plate, of which the margin is divided into three or four teeth as the case may be. It is thus possible to produce a stimulation which consists of the requisite number of summated shocks. Or, instead of the clock, we might, for the purpose of producing brief and definite closures, have a metronome. But this is a less perfect arrangement than that of the clock.

By wrapping a sensitive film round the recording drum, all these response-records may be obtained photographically. Thus the whole process of stimulation and its record may be rendered automatic. The records given in this and succeeding chapters have been obtained sometimes by photography, and sometimes by employing the simpler process of following the spot of light with a recording pen. From a knowledge of the magnification produced by the Optic Lever, and the height of the responses, it is easy to calculate the actual contraction produced by the stimulus. From the length of the specimen experimented on we can also determine the coefficient of responsive contraction — that is to say, the absolute contraction for unit length. When the magnification of the lever, the length of various specimens, and the strength of the stimulus are all kept constant, then the heights of the responses in different cases will be found to afford us a measure of the mechanical excitabilities of the different specimens.

Demonstration of universality of excitatory longitudinal contraction in radial organs.— I shall now describe my experiments on the longitudinal contraction of various radial organs. The first of these was performed on a straight radial internode of Cuscuta, which was attached to the recording Optical Lever in the usual manner. Tetanising shocks were given for twenty-five seconds at a time, from an induction coil, and the successive responses were obtained, at intervals of two minutes (fig. 37). The contractile effect persisted, even after the cessation of stimulation, for a period of five seconds, after which there was recovery, which was completed in a period of ninety seconds. It will be seen from the record that the successive responses were uniform.

I obtained similar responses from the root of a watergrowing plant of the Bindweed family. Successive responses were obtained at intervals of two minutes, the stimulus in each case consisting of tetanising shocks of forty-five seconds. The maximum contraction was attained fifteen seconds after the cessation of stimulus ; and recovery was completed after a further period of one minute. In this case the responses exhibited fatigue. It may be stated here that, speaking generally, the period required for recovery is dependent on the strength of stimulus. With moderate intensity of stimulation, recovery is complete within a comparatively short period. But it is protracted, or indefinitely delayed, when the stimulus is strong. Again, if successive stimuli be applied, before recovery is complete, the responses will be found to be additive.

In order to convey some idea of the amount of contraction produced by stimulus, I shall here give a detailed account of an experiment on longitudinal contraction, the specimen Fig. 37. Response of Stem of Cuscuta to Electric Stimulation used being a young stem of the species of Bindweed {Convolvulus) already referred to. The length experimented on was 5 cm. On passing through this tetanising shocks of five seconds' duration, a maximum contraction was found to occur in the course of two minutes. The magnification used for record was fifty times, and the extent of contractile response recorded was 7*5 cm. Hence, the actual contraction was 1*5 mm., in a stem whose length was 50 mm. The contraction produced is thus 3 per cent, of the original length.

Similar contractile response may be obtained with other forms of stimulation, and I shall now describe that induced by thermal stimulus, the specimen used being the radial style of Datura alba. Experimenting1 with the style has the special theoretical advantage that, owing to the soft nature of the tissue, the effect recorded is purely of longitudinal contraction. In specimens which have not this characteristic to the same extent, and which may be anisotropic, like the filaments of Cynerece, the contraction is not always purely longitudinal. There is a tendency, owing to differential contraction, to the production of curvature. For these reasons, a limp and thread-like style fulfils the ideal requirements of an experiment for obtaining true longitudinal contraction. With this specimen of Datura I applied thermal stimulus at intervals of Fig. 38. Phototwo minutes, in the manner already degraphic Record of scribed. The records show how extremely

of Datura alba to uniform the responses are (fig. 38). The Thermal Stimulasame longitudinal contraction may also be obtained from plants other than phanerogams. On applying electrical stimulus to the stalk of the fungus Agaricus I obtained a contraction of 2 per cent, of the original length. Remarkable extent of contraction in coronal filaments of Passiflora. — There are some plants, again, in which the extent of the excitatory contraction is very great. For instance, the filamentous corona of Passiflora quadrangularis often gives a contraction of as much as 20 per cent.

It will thus be seen that not only is the phenomenon of longitudinal excitatory contraction present in all plants, but that such excitatory movements in some which are supposed to be insensitive, rival in extent those of the typically sensitive filaments of Cynerece, which are said to exhibit a contraction of from 8 to 22 per cent. In order to obtain a suitable record a magnification of only twenty to thirty times is necessary in the case of the highly excitable tissue of the corona of Passiflora. In less excitable specimens, a magnification of 100 would be enough. The advantage of relatively high magnifications in general

lies in the fact that they necessitate only moderate intensities of stimulation, which have the advantage of not fatiguing the tissue. Modification of excitatory contraction by physiological conditions. — That these contractions are the expressions of true excitatory response is proved by the fact that they are modified by whatever affects the physiological condition of the tissue. Thus, for example, they undergo a temporary abolition under the action of anaesthetics, and a permanent abolition under the action of poisons. This will be demonstrated in more detail in a later chapter. They also exhibit very interesting modifications, according to the age of the specimen and the season of the year, as might theoretically have been expected. In experiments. on this subject, undertaken with the filamentous corona of Passiflora, stimulation was produced by tetanising electric shocks, and the maximum contraction was measured by means of a micrometer. The following results show, in condensed form, the effect of age on excitatory contraction.

Table showing Effect of Age on Excitatory Contraction (Coronal filaments of Passiflora </.) [Length of specimens, 21 mm. Time, February, i.e. end of winter.] It will thus be seen that when the physiological activity of the specimen is at its greatest, that is to say, just before the opening of the flower, the excitatory contraction is also at its maximum. In order next to determine the effect of season on the excitatory contraction, we shall compare the mean percentage of contraction in winter with that obtained in spring, in the month of April. The average contraction in winter may be taken as J'6 per cent. But in spring I obtained (i) with a specimen 12 mm. long, a contraction of 17 mm., i.e. 14 per cent. ; and (2) with a second specimen 10 mm. long, a contraction of 2 mm., i.e. of 20 per cent. The mean of these, 17 per cent, representing the contraction in spring, is thus seen to be about two and a half times that obtained in winter.

We have now ascertained the universal occurrence of longitudinal contraction in the organs of plants, and we have seen that lateral response cannot take place under diffuse stimulation in a strictly radial organ, owing to the antagonistic character of the equal and simultaneous responsive contractions on diametrically opposite sides. Lateral response, however, as we have seen, will take place in a radial organ when stimulus is not diffuse, but unilateral. Such lateral response is only possible under diffuse stimulus, when the excitability of the two opposite halves is different, that is to say, when the organ is anisotropic. In such cases, as in the petioles of leaves, for example, the very striking lateral movement is simply the result of differential longitudinal contraction. The differentiation of the upper and lower halves is anatomically evident in the case of dorsiventral organs. But the anisotropy may often be undistinguishable to the eye. For an organ, originally radial, may become molecularly bilateral, owing to the unequal action of external forces on diametrically opposite sides. It will be shown in the next chapter that such molecular differentiation gives rise to physiological differentiation, and there I shall be able to trace a continuity between the longitudinal response of radial organs and the lateral response of dorsiventral organs through intermediate types.

In a radial organ, unilateral stimulation causes contraction, and consequent concavity of the acted side. Under diffuse stimulation there is no resultant lateral response, owing to the balanced and mutually antagonistic character of the contractions. A radial organ under diffuse stimulation exhibits longitudinal contraction. Longitudinal contractions are observed in the radial organs of all plants. Such responsive contractions as seen in some ' ordinary ' plants are strictly comparable in extent to those which are known to occur in the sensitive filaments of Cynerece.

These excitatory responses are modified by all those agencies which affect the physiological condition of the plant. Molecular anisotropy artificially induced by one-sided cooling — Cooled side less responsive — Diffuse stimulation causes concavity of the uncooled, that being relatively the more excitable — Local fatigue diminishes excitability — Diffuse stimulation now causes concavity of the unstrained side — Similar anisotropy induced in plagiotropic organs, by unilateral action of light — The lower or shaded side of such organs relatively more excitable — Diffuse stimulation causes current of response from lover to upper, and also concavity of lower half — Responses of plagiotropic Cucurbita and Convolvulus — Differences in excitabilities of outer and inner surfaces of tubular organ — Complex response due to successive excitations of two antagonistic halves of an anisotropic organ — Response of spiral tendrils by uncurling — Response in certain cases by contraction of the spiral or curling— Writhing movement in spiral tendril under strong stimulation.

We have seen that in a radial organ, owing to balanced actions, there is no lateral response to diffuse stimulus ; and that in dorsi-ventral organs, where there is pronounced anisotropy— as seen in anatomical differentiation — lateral movement is produced by means of differential action. I am now about to demonstrate the fact that these phenomena are not sharply divided, but merge gradually one into the other, through intermediate types. Molecular anisotropy induced by unilateral application of cold or of excessive stimulation. — If we take a hollow radial petiole of Gourd {Cucurbita maxima) growing erect, we shall find, on application of diffuse stimulus, that it gives no responsive curvature, but exhibits the simple longitudinal contraction of a radial organ. We may now take this petiole, and split it into two equal halves, throughout almost its entire length. We have now a single specimen bifurcated, the forked divisions being equal in every respect. One fork,

say A, is now immersed in ice-water, the other being dipped in water at the ordinary temperature. The two halves are next taken out of the water and bound together, from their free ends upwards, so as to make once more a single tube. The petiole is now held by the uncut part vertically, with a long index projecting downwards from its lower end. The petiole was, as will be remembered, originally radial; but now, by the local application of cold to one half, a certain molecular differentiation has been induced, and the particles in the cooled, or A half, are now therefore more sluggish and irresponsive than those of the B side. This induced molecular differentiation, moreover, is invisible to the eye, and had we not observed the process by which it was brought about, it would have been impossible, from mere visual inspection, to know which side had been subjected to cold. The application of diffuse stimulus, however, reveals it at once ; for on passing electrical shocks along the length of the specimen, the relatively more excitable, or uncooled B half, becomes concave. It is clear, then, that at this moment the B half is the more excitable, and stimulus acts on it preferentially. But after long-continued stimulation, the 15 half becomes overstrained, and its excitability undergoes diminution or fatigue. At the same time, by means of repeated shocks, the sluggishness of the A half has been gradually made to disappear ; that side now regains its excitability; and, the excitation of A becoming thus relatively greater, the curvature of the specimen is reversed.

I have said that excitability is diminished under the molecular strain caused by over-stimulation. I shall now demonstrate this by another and independent experiment. A new specimen, slit like the last, is taken, and one half, say A, is alone subjected to strong excitation, by sending electric shocks along its length. The two halves are again brought together, and on now subjecting the whole petiole to electric stimulation, it is found that the fresh, or previously unexcited, half is that which becomes concave, thus proving that the fresh half is the more excitable, and that strong or

long-continued stimulation diminishes the excitability of a tissue. From these two experiments it will be seen that loss of excitability may be produced in, amongst others, two different ways. First, there is the molecular sluggishness, induced, as we have seen, by cooling, which is, however, only temporary, the original sensitiveness being restored on warming. Secondly, we may have loss of sensibility due to overstrain, owing to strong or long-continued stimulation. This strain-effect, with its attendant loss of excitability, may, if excessive, prove more or less permanent. It will thus be seen that in consequence of unilateral stimulation, a molecular differentiation is produced, in consequence of which an organ originally radial becomes physiologically anisotropic. The unstimulated portion of the organ is now the relatively more excitable, and on diffuse stimulation becomes concave.

Molecular anisotropy induced under natural conditions.— We shall next observe the induction of such molecular anisotropy under natural conditions. The stem of a young Gourd is at first erect and strictly radial ; but later it bends over, and then assumes a creeping habit. Under these conditions, its upper 'surface is subjected to the unilateral action of vertical light, the lower half being shaded and protected. The organ is no longer radial, then, but plagiotropic, and from what has been said already, we shall expect its upper or exposed surface, in consequence of the prolonged action of stimulus of sunlight, to be less excitable than the lower or shaded surface. This inference I have been able to verify by means of the electric mode of investigation. I find that on simultaneously exciting both sides of such a stem, a current of response flows from the lower to the upper surface ; hence it will be seen, according to the third law of electrical response, as enunciated at the end of Chapter III., that this lower side is the more excitable, and ought to become concave under diffuse stimulation.

Such induced concavity in response to diffuse stimulation I have found in the case of various plagiotropic stems, for example, in those of Cucurbita and Convolvulus. In order to demonstrate the greater contraction of the shaded side — which is seen as responsive curvature — while eliminating the effect of gravity, I have employed two different modes of experiment. In the first, stems are held with their tips vertically downwards, and electric shocks are passed through them ; a curvature is then produced by the greater contraction of the shaded side, in consequence of which the free end of the stem is lifted up against the force of gravity. The second method consists in supporting the stem horizontally in such a way that the plane which divides the previously shaded and unshaded sides is vertical : on strong stimulation, the stem now moves in the horizontal plane, in a definite direction which is determined by the induced concavity of the shaded and more excitable side. Here we see the plagiotropic stem behaving like the pulvinus of Mimosa, the more excitable side becoming concave under diffuse stimulation in both.