Plant Response as a Means of Physiological Investigation

Darwin's theory of modified circumnutation. — Before describing the response, it is necessary to say a few words regarding Darwin's view of heliotropic movements as a modified form of circumnutation. According to this, the already existing movement (of circumnutation) had only to be increased in some one direction, and lessened or stopped in others, in order to become heliotropic or ap-heliotropic, as the case might be ; l but, in order to prove conclusively that heliotropic curvatures were caused by the modification of the pre-existing movements, it would be necessary to show that they did not take place in those organs from which circumnutation was absent, and this would constitute the crucial test of the theory. The difficulty of obtaining such a specimen is, however, so great, that Darwin, although he noted the point, was unable to apply the test.

I have shown elsewhere that circumnutation is only a particular manifestation of that multiple or autonomous response of plants which is due to an excess of energy, previously absorbed. In order to obtain a plant-organ completely at standstill, therefore, it would be necessary to find a specimen in which there was no such excess of latent energy. The fact that circumnutation was absent could then be ascertained by means of the high magnification obtainable from the recording apparatus which I have already employed. Another difficulty lay in the fact that the use of light, however feeble, for purposes of observation, would be apt of itself

to give sufficient stimulus to initiate multiple responses which had not been present before. This might have appeared incredible had I not, in the course of my experiments, had reason to know how extraordinarily sensitive plants may become to the influence of light, instances of which will be found in the investigations presently to be described. In view, indeed, of the vitiation of results which might be caused in this way, I was compelled to devise special means for obviating the use of any light whatsoever for the observation of responsive curvature.

Response of the terminal leaflet of Desmodium. — In order, then, to determine the important question of whether or not light will produce heliotropic movement in a plant devoid of circumnutation, I acted on the idea that the withdrawal of superfluous energy was the essential preliminary condition, and chose for my purpose, as already said; the large terminal leaflet of a specimen of Desmodium gyrans in which all movement had come to a stop — the plant having been exhausted by flowering, and by the unfavourableness of the season, which was winter. And further, in order that there should be no storage of energy derived from light, I kept this plant for one day in a dark room. To eliminate the necessity of using light for purposes of observation, I attached the leaflet by cocoon fibre to the arm of the Optic Lever Recorder. The plant itself was enclosed in a dark box, and thus protected from any access of light. Through a trapdoor in the box, light for stimulation could be thrown down on the leaf at the desired moment. The preliminary absence of any autonomous movement in the plant was seen by the quiescence of the spot of light reflected from the mirror attached to the recording lever.

I now subjected the terminal leaflet of the selected specimen to light from a candle, this being thrown down on the leaflet vertically by means of a mirror, the effective distance of the candle from the leaf being twenty centimetres. The leaflet, which had previously been quiescent, began to respond after a latent period of ten seconds, and during the course of an exposure of twenty minutes executed five complete oscillations (fig. 237). The notable points in this record are : (1) that a perfectly quiescent organ is made to give multiple response by the stimulus of light ; (2) that molecular sluggishness appears to be gradually removed by the continuous absorption of energy, and the successive responses exhibit an enhanced or ' staircase ' effect ; and (3) that from the tendency of the series of curves to tilt towards the light, the organ is seen to exhibit a resultant positive movement.

It will thus be seen that we have here not a modification of an existing movement, but a series of multiple responses, with a trend in a particular direction, that is to say towards the light, constituting a positive heliotropic movement. In a growing organ also, which was previously devoid of circumnutation, we shall be able to observe the induction of a similar movement. As in the case of the movements of growth, so also in those of heliotropism, we are often able to detect multiple constituent pulsations, especially at the commencement of response when stimulus is moderate. It will be understood here that, under the unilateral contraction induced in the excited side of the organ by the stimulus of light, a hydrostatic disturbance is set up, the expelled water being forced to the opposite side — a state of things calculated to show pulsation in a marked degree. The final resultant curvature, then, as we have seen under the action of other forms of unilateral stimulus also (p. 521), represents the joint effects of the concavity of the proximal and the convexity of the distal sides. When light acts on the organ continuously for some time, the

Fig. 237. Multiple Response to Light of Terminal Leaflet of Desmodium The moment of application is marked by x . The arrow shows the direction of light, i.e. from above. The numbers in the abscissa represent time in minutes. multiple responses become more frequent by the increased absorption of energy, and their individuality is lost. The extreme sensitiveness of some plant-organs to light. — I shall now say a few words about the extraordinary sensitiveness of some plants to the stimulus of light. Darwin gives a striking example of this in a case where the cotyledons of Phalaris canariensis, after three hours of continuous exposure to a small lamp at a distance of twelve feet, became doubtfully curved towards the light, and after seven hours and forty minutes from the first exposure were plainly, though slightly, curved towards the lamp. The candle-power of the lamp is not given, but it may be taken to be about four candles. Reducing this to the standard distance of one metre, we find four candles at a distance of twelve feet (four metres approximately) to be equal to a quarter candle at a distance of one metre. If three hours' exposure induced a doubtful curvature, then the smallest amount of light to be effective must have been I or 75 candle-hour ; the candle-hour giving an indication of the quantity of light that had to be absorbed by the plant in order to induce a movement that was just perceptible.

Now, with the terminal leaflet of Desmodium, exposure to the light of a candle at a distance of 20 cm. for ten seconds was sufficient to initiate responsive movement. This when reduced to standard conditions is equivalent to '07 candlehour. In other words, we find, as far as these two experiments can determine the point, that the terminal leaflet of Desmodium in this experiment was at least ten times more sensitive than Darwin's cotyledons of Phalaris canariensis.

Darwin, however, mentions another instance which is more like the sensitiveness of which I have just given an example. He has been using a small wax taper, in order to observe the cotyledons of Phalaris ; he used this light for one or two minutes at each observation, and observed the seedlings seventeen times in the course of the day, in consequence of which he found that zigzag responsive movements had been induced. Desmodium which exhibited such remarkable sensitiveness had been specially chosen, as being in the least favourable tonic condition. And yet I could not even strike a match near this plant without inducing responsive movements. This will indicate the extreme sensitiveness of certain plants to light, and show that it is necessary to make our observations of induced movements without its aid. The manner in which this was done will be described presently.

Merging of multiple in continuous response. — We found that light acting from above on the terminal leaflet of Desmodium gyrans gave rise to multiple responses. As the light was acting constantly on the upper half, there was a cumulative contractile effect on that half. The consequence of this was a trend of the series of curves towards the light, or an incipient positive heliotropic movement. I shall now proceed to show how these constituent multiple movements may often, if not always, merge into one continuous movement.

For this experiment I used the same leaflet as in the last, the only difference being that I now applied the strong stimulus of sunlight from above. The response induced is seen in fig. 238. During the first impact of the stimulus a pulsatory movement may sometimes be observed. But the response soon becomes a continuous movement upwards. Generally speaking, the constituent multiple marks are to be seen under feeble or moderate stimulation, and a continuous movement when the stimulus is strong. In the present case, the average rate of movement of the tip of the leaf was almost 1*5 mm. per minute (fig. 238). On the stoppage of light there was persistence for some time of the after-effect of light. This was succeeded by recovery. The persistence

Fig. 238. Response of Terminal Leaflet of Desmodium to Strong Light from Above Abscissa gives time in minutes, and ordinate movement in millimetres. of the after-effect varies widely, depending on the condition of the tissue as well as the intensity of stimulus. Orientation induced by light— When the leaflet, with its sensitive motile organ, is exposed to strong sunlight, the heliotropic movement continues till the organ becomes parallel to the direction of light. The question now arises, in this as in other cases of heliotropic movement, why should the movement come to a stop when the organ reaches this parallel position ?

A partial answer to this question may be found in the fact that such movements depend upon the effective intensity of light which is absorbed, and this effective intensity is greatest at perpendicular incidence, and becomes reduced to nearly zero as the rays of light are rendered more and more oblique by the responsive movement of the organ. This consideration alone, however, would not wholly explain the orientation of the organ, parallel to the direction of light, for we know that the directive impulse caused by light persists for some time, and this would cause the organ to overshoot the parallel position. In order, therefore, to obtain a satisfactory explanation, I undertook the following experiment, which, as will be seen, completely meets the difficulties of the case.

I now took the same leaflet of Desmodium as was used in the previous experiments, and caused sunlight to strike the pulvinus vertically, from below upwards, by means of a suitably inclined mirror. I obtained, as will be seen (fig. 239), a continuous responsive movement downwards — i.e. towards the direction of the light. The average rate of movement of the tip of the leaf was in this case about 2*5 mm. per minute. This is somewhat greater than the upward rate of movement,

FiCx. 239. Response of Terminal Leaflet of Desmodium to Sunlight acting from Below The dotted portion of the curve represents the after-effect and recovery on the cessation of light. and is probably due to the fact that the excitability of the lower half of the pulvinus is slightly greater than that of the upper. We are now in a position to understand the reason of orientation ; for if we suppose light to be incident from a position slightly above the pulvinus, it will curve upwards, owing to positive heliotropism, till it has become parallel with the rays of light. Should there be any over-shooting of this position owing to after-effect, the pulvinus will then begin to curve downwards, because the light will now be acting from below. Permanent equilibrium can thus only be attained when the plant-organ has become parallel to the direction of light.

The perceptive region in the terminal leaflet of Desmodium. — In connection with the response of the Desmodium leaflet to light, it is interesting to note that the pulvinus is not only the responding, but also the perceptive region ; for, throwing the light on the leaflet alone, and protecting the pulvinus with an opaque shield of black paper, we find that no responsive movement takes place ; conversely, if the pulvinus alone be exposed, and the rest of the leaflet shaded, we observe the normal action.

Heliotropic response in radial organs. — Having observed the peculiarities of heliotropic movement in a pulvinated organ, I shall now describe the experimental arrangements for studying the same problem in non-pulvinated growing organs. It is understood that there is no essential difference between the two movements. They are both caused by the same contractile effect, due to the stimulus of light, the only difference being that, whereas in the pulvinated organ the recovery is complete, in the growing organ it remains more or less incomplete, the curvature being fixed by growth.

Magnetically controlled recorder. — The great difficulty which stands in the way of accurate investigation is the question of how to take a continuous time-record of the heliotropic curvature of the growing organ without being under the necessity of using light for purposes of observation, a procedure which, as we have seen, causes disturbance. The free end of a normally growing organ, when acted on, say by horizontal light, bends towards it. Thus the problem is to obtain a continuous time-record of this movement, from which the latent period, the actual rate of movement, the after-effect, and other related effects might be ascertained. I have been able to solve this difficulty by devising a mag-

Fig. 240. Diagrammatic Representation of the Magnetically Controlled i,l', the Optic Lever, to one arm of which the plant is attached by a thread ; m, the mirror, with small magnet, ns, attached behind. The lever is rotated to dotted position by heliotropic curvature of the plant, diagrammatically represented disproportionately magnified. netically controlled recorder, the principle of which will be understood from the accompanying diagram (fig. 240).

The principal part of this recording instrument consists of a magneto-metric arrangement. Attached to a long aluminium lever, LL', is a vertical T-piece, V. This T-piece, V, carries a reflecting mirror, behind which is a short magnet, ns. The whole arrangement is freely suspended by a silk thread. By means of a controlling magnet not shown in the figure, the suspended lever may be adjusted in any convenient azimuth. The free end of the growing plant is attached to one end of the lever, L, being on its own level, at a distance of, say, 20 cm. from the line of suspension, the attached thread being at right angles to the lever. There is a slight tension of the thread, due to the magnetic force of the controlling magnet, which tends to draw the lever away from the plant. This tension, however, is very slight. When the growing organ is acted on by light in a horizontal direction, parallel to the attached thread, and therefore at right angles to the lever, then if the action of light be to induce a positive heliotropic effect, the rotation of the suspended magnetic system, owing to the pull exerted by the curving organ, will be, when seen from the top, in the direction opposite to that of the hands of a watch.

If, however, the heliotropic effect be negative, the top of the growing organ will move in the opposite direction. But it has been said that there was a slight tension of the thread, owing to the action of the controlling magnet. This being now released, the lever will move to a proportionate extent in the same direction as the hands of a watch. A spot of light reflected from the mirror magnifies these movements, and a record of this moving spot of light on a revolving drum gives the response-curve. Instead of this magnetic control, it would also be possible to use, as the controlling force, the torsion of a fine metallic wire.

By using a shorter lever arm and increasing the distance of the revolving recording drum from the mirror, a wide range of magnification up to 500 times may easily be obtained. For ordinary purposes a magnification of ten times is all that is necessary. The movement of the spot of light is proportionate to the heliotropic movement of the plant, at least when the amount of that movement is not excessive. Thus, knowing the magnification produced by the system, and the rate of the revolving drum, we' can determine from the

j response-curves the absolute movement, and the rate of such movement. I now give a description of the complete apparatus actually used (fig. 241), consisting of the Recorder and the Heliotropic Chamber. In a dark chamber is placed the plant, attached to the Lever as explained before. A portion of the vertical piece with attached mirror, M, projects outside the chamber, It will be seen that by this arrangement the Fig. 241. Heliotropic Chamber and Magnetically Controlled Recorder

Heliotropic chamber seen in the middle of the apparatus. Guide-bars to right and left carry sliding lamps, l and l'. Exposure is given by pressing key, k, which raises shutter, s. The corresponding shutter to the right is not shown in the figure. M, mirror of the Optic Lever ; c, controlling magnet. plant can be completely protected from light, while its movements are at the same time recorded by the spot of light thrown from the mirror, M, upon the recording drum, without the possibility of its reaching the plant within the chamber.

The chamber carries two projecting graduated arms or guide-bars, one to the right and the other to the left, over which slides the holder for a candle, or incandescent, or Nernst's electric lamp, LL' ; but if a still stronger light be desired, sunlight may be reflected in the required direction by a mirror. From whatever source, the light can be made to strike the plant horizontally through the slit, which is usually covered with a sliding shutter, S. By manipulating a key, K, the shutter is raised. Thus exposure may be made at any moment, and continued for the length of time desired.

That part of the record which is made on the revolving drum before exposure begins, gives an indication of the quiescent condition of the plant. The moment and duration of exposure are found from corresponding marks made on the recording surface, at the instants of opening and closing the shutter. It will be seen that we have means of controlling the intensity of illumination within wide limits by (i) the use of different sources of light as enumerated above, and (2) variation of the distance of the source of light, at least when this is artificial.

Thus we are able to illuminate the plant from the right or left flanks, or from both simultaneously, and by lights of equal or of different intensities, at will. Again, by covering the slit with a second plate, provided with suitable apertures, we are enabled to subject any part of the plant, whether tip or growing region, or both, as desired, to the stimulus of light, and thus to determine the characteristic response of each. All these considerations will show the facilities afforded by this apparatus for carrying out a great number of diverse experiments, I shall, however, content myself here with the description of a few necessary examples.

Heliotropic response of hypocotyl of Sinapis.— As examples of radial organs, exhibiting the positive heliotropic effect, I took seedlings of Sinapis nigra. They were attached to the lever, as indicated above, and at least half an hour was permitted to elapse, in order to remove the last possible trace of excitation due to contact. The attainment of the quiescent condition was ascertained from the stationary position of the spot of light. In this experiment, on a seedling of Sinapis, light was allowed to strike the growing organ horizontally. The specimen was very sensitive, and the source of light employed at first was a candle placed at a distance of 20 cm. acting on the plant for three minutes. The responsive movement began within five seconds, and though the light was cut off, there were produced three multiple responses, after which the plant underwent a complete recovery and resumed its former position. Sunlight was next applied, and a continuous movement towards the light was induced (fig. 242).

x Application of candle-light for three minutes gave three multiple responses ; f application of sunlight gave rise to continuous response. Dotted line shows after-effect and recovery on cessation of light. In another case sunlight was applied for twelve minutes. The response commenced almost immediately on application, and the average rate of movement was 1 mm. per minute. In this particular case, the positive after-effect persisted for five minutes, even on the stoppage of light, after which there was a gradual recovery (fig. 243).

Recovery and theory of recti-petality. — On the cessation of stimulus, there is a more or less perfect recovery of the radial organ from its induced curvature. I have already demonstrated the fact that the growing organ acts as a diffused pulvinoid. We have just seen that the primary action of light is to induce similar motile effects in both pulvinated and growing organs. We have also seen that in both, on the cessation of stimulus, there is a tendency towards recovery. In the case of the growing organ, when stimulus is moderate, recovery is fairly complete ; but when the stimulus is very strong and long-continued, some part of the induced curvature is rendered permanent through fixation by growth.