Plant Response as a Means of Physiological Investigation

Antagonistic actions of internal energy and external stimulus. — We thus see that it is the internal energy of the plant — vaguely known as a favourable tonic condition — which actively determines the recovery of the organ. This will explain the fact which I have mentioned elsewhere, that in summer, when the internal energy is considerable, the leaf of Mimosa recovers from the effect of stimulus in about six minutes, whereas in winter, when the internal energy is low, the same process may take as long as eighteen minutes. We thus see that as regards mechanical response the external stimulus and internal energy act antagonistically. Local external stimulus induces a diminution of turgidity, while internal energy causes an increase of turgidity. Thus when the internal turgidity is very great it opposes the mechanical response to external stimulus. This we saw in the case of overturgid leaves of Mimosa, and in those of Artocarpus during the rainy season, which, though excited, did not exhibit mechanical response to stimulation (pp. 49, 58).

This will be clearly understood also from an attentive consideration of the experiment, the record of which is given in fig. 166. In that case, had the warm water which increased the internal activity been applied earlier at the root, that is to say during the application of external stimulus, the induced internal turgidity would then have been so great as to arrest the responsive down movement of the leaf. The amplitude of the response to external stimulus would thus have undergone diminution or even abolition.

We must remember, however, that the internal energy which maintains the normal turgid condition is itself the result of energy previously absorbed from external sources ; and if the plant be cut off from these sources of external energy, then its own tonic condition will fall below par. Thus the leaves of many plants are seen to droop when kept too long in darkness, and exposure to light makes them recover their natural position of normal turgidity. Hence in the case of a plant whose condition is sub-tonic the leaves may be made turgid by exposure to light ; but after the attainment of the normal tonic condition, exposure to strong light will bring about the proper contractile response to stimulus, with the characteristic external motile indication of diminished turgidity.

We have thus seen the various effects produced by the internal or latent energy of the plant. We have seen it bring about the ascent of sap by means of the increased activity of the plant-cells. It was seen to produce exudation pressure, and excretion of nectar from intact plants. We have traced it out to its appropriate expression by the lateral movements characteristic of positive turgidity-variation, in the case of anisotropic or dorsi-ventral organs. We have seen, too, how necessary it is to the production of recovery of an organ from the action of an external stimulus, the effects of local external stimulation, and of this internal activity, being opposed in character. Any increase of this internal energy is thus a factor tending to hasten the recovery of the organ from stimulation, and when it is sufficiently great, it may, by its antagonistic action, reduce the amplitude or even abolish response to external local stimulus. We have again seen this internal activity finding motile expression in the autonomous movements of leaflets of Desmodium ; but, for its mechanical exhibition, we need not confine our attention to the sensitive plants so called, for we shall find the same internal activity exhibited mechanically by all plants, in their rhythmic growth-responses, to be described in the following chapters.

The ascent of sap is fundamentally due to excitatory reaction, its uni-directioned flow being brought about by the passage, from point to point, of the co-ordinated excitatory reaction, propelling water forward. This rhythmic excitation is initiated in the intact plant at its root, by stimulus of contact with soil, the friction of the growing organ against rough surfaces, the excessive turgidity caused by the absorption of water, and possibly by the chemical stimulus of substances present in the soil. In the case of cut branches placed in water, the excessive turgidity at the cut end initiates rhythmic activity. Again, if stimulus be applied at the top instead of at the root, the direction of water-conduction may be reversed, along with the reversal of propagation of excitation.

The following facts show the intimate relation between the conduction of stimulus and conduction of water : (a) The movement of water takes place preferentially through the fibro-vascular elements, these being also the better conductors of excitation. (J?) The conduction of excitation along a plant is greater than across. The same is true of its power of transport of water. (c) Though conduction of excitation may take place either upwards or downwards, yet there is a preferential direction for such conduction. The same is true of the transport of water.

The same movement of water produced by the coordinated rhythmic activity of cells throughout the plant appears as either suctional or pressure movement, according to the point of view. When the removal of water from the plant is in any way arrested, a positive pressure is produced, owing to the excessive accumulation of water. When, on the other hand, the loss of water by transpiration is greater than the supply, a negative pressure will be observed.

The ascent of sap primarily due to cellular activity, may be secondarily aided by evaporation from the leaves, and the osmotic action of the concentrated cell sap in the leaves. Owing to the distribution of unequally active cells, an irregular variation of pressure is induced in the stem. The excitatory movement may be transmitted to a distance by conduction, or there may be conduction by ' relays.' An isolated mass of highly excitable tissue may thus be excited de novo.

The excretion of water and of nectar are phenomena of cellular activity, analogous to that which brings about the ascent of sap. The translocation of food-material is also probably due, at least in part, to excitatory reaction. The internal activity of the plant, causing increase of turgidity, may be detected mechanically by that erection of the leaf which is characteristic of the positive turgidityvariation. Any increase of internal activity is exhibited in dorsiventral organs, such as the petioles of Mimosa, Biophytum^ and Artocarpus, by the erection of the leaf. Thus, when the internal energy of the plant is increased by a rise of temperature, the leaves become erected. Conversely, under the action of cold, on account of the diminution of the latent energy, the opposite effect, or droop, is induced. This explains the drooping of various leaves during frost, and their subsequent erection when brought into a warmer atmosphere.

This internal energy is also an important factor in bringing about the recovery of an organ from the effect of external local stimulus. The effect of external local stimulus in causing the diminution of turgidity of an organ is thus antagonised by the internal activity, which causes an increase of turgidity. The internal energy, when sufficiently great, may thus hasten the recovery of the organ from the effect of stimulus. This increased internal energy may also reduce the amplitude, culminating in the total abolition of mechanical response, as seen in over-turgid Mimosa, or in Attocarpus during the rains.

The simple Growth-Recorder — The Balanced Growth-Recorder — Rhythmic growth-response— Growth-response and excitatory response — Law of direct and indirect effects of excitation — Positive turgidity-variation as indirect effect of excitation — Mechanical test — Significance of ' inner stimuli. ' One of the most characteristic manifestations of life is growth. The question then arises, whether this particular manifestation is to be regarded as a distinct and specific phenomenon, unlike all others, or whether it may be possible to trace a connection between it and those responsive reactions with which we are already familiar.

The occurrence, in response to stimulus, of numerous growth-curvatures, sometimes positive and sometimes negative in character, offers us again a problem of very great complexity. It is sometimes supposed that stimulus retards, and sometimes that it accelerates, growth. But it is difficult to understand how the same influence can produce opposite effects. Then, again, there is intruded upon the problem the unknown effect of ' inner stimuli.' From all these it will be seen that the subject of growth and growthmovements is one of extreme obscurity, and that the difficulties which baffle us can only be met satisfactorily if we are able to analyse and follow out, one by one, the various elements that enter into the problem.

We have seen in the Desmodium leaflet at standstill, and in that of Biophytum under ordinary circumstances, that when the latent energy is not excessive, we obtain a single movement in response to a single stimulus. When the sum total of the latent energy of the tissue, however, is above par, it is manifested in a rhythmic manner, by periodic variations of turgidity, bringing on responsive movements. It was also stated that responsive movements under the action of stimulus took place in ordinary young tissues, these movements being lateral when the tissue was anisotropic, and longitudinal when it was strictly radial. It would follow, then, that when the sum total of the latent energy in such tissues was above par, they might be expected — like the leaflet of Biophytum or Desmodium under similar conditions — to exhibit their rhythmic excitation by repeated lateral or longitudinal movements. I shall now proceed to show that in the case of these young tissues, under favourable circumstances, this multiple rhythmic excitation finds expression in the responsive movement known as growth.

In the majority of instances an organ is not absolutely radial ; hence, during growth, we obtain the lateral responsive movements which are known as circumnutation. In bilateral growing organs, these movements are to and fro, in strict parallelism to the to and fro movements of the leaflet of Biophytum. In such instances the axis of bilaterality is fixed, and the responsive movement takes place in a definite plane. In the leaflet of Desmodium also rectilinear movements are often observed ; but as a rule, owing to the revolution of the bilateral axis, the movement of the leaflet is circular or elliptical, and in the case of growing organs, from the same cause, circular or elliptical movements of nutation are common. The ideally simple and most interesting example of this multiple rhythmic activity is seen, however, in the growth-movements of radial organs, these being longitudinal ; for there is not in this case that complication which arises from the gradual shifting of the bilateral axis seen in the growth of anisotropic organs.

In order to prove the identity of these rhythmic growthmovements with multiple response, we have to show (i) that such rhythm is a characteristic of growth; (2) that each pulsatory growth-movement of the series exhibits all the characteristics of true response ; (3) that the series itself is characterised by the same cyclic variation which we have observed in the case of multiple response ; (4) that just as the application of appropriate stimulus renews pulsation in a Desmodium at standstill, so, in a plant with growth at standstill, appropriate stimulation renews pulsatory growth ; and, lastly (5), that the modifying influence of external agents is similar in both cases.

From a series of observations, taken at intervals of several minutes, on Spyrogyra princeps, Hofmeister found that growth undergoes fluctuation, the first and second maximal points in his series of observations being separated by an interval of forty-four minutes, and the second and third by an interval of ninety-five minutes. Such experiments, however, have laboured under the great disadvantage of discontinuity, and in order to overcome this I undertook to devise some apparatus by whose means growth-pulsations might be recorded continuously, in such a way as to give not only the period, but also the individual peculiarities of each pulsation. And I may here forestall matters to say that by such means I have been able to detect longitudinal pulsations two hundred times as quick as those observed by Hofmeister.

Conditions to be kept in view.— Before attempting to demonstrate the pulsatory character of growth-movements, however, I shall point out certain facts which it is essential to remember. We have seen that under rapidly succeeding excitations, the separate responsive effects become merged, and a response is produced, which is apparently continuous, although the stimuli themselves were discontinuous. This is seen, for instance, in the first part of the tetanic curve (figs. 49, 50). But when once the maximum responsive effect is produced, as there can be no further additive effect, the subsequent responses show themselves in a series of fluctuations of the top of the tetanic curve. In that form of response, which we are now considering, as there is no maximum limit, the additive effect of growth continues indefinitely. It is thus clear that when rhythmic excitation is very rapid, it may produce a growth-movement which

appears to be continuous. But when this rapidity is not excessive, it should be possible, by employing sufficient magnification and a suitably quick rate of movement of the recording surface, to display its actual pulsatory character. The sensitiveness of this mode of detection becomes again very much increased if we employ the method of balance, or compensation, which will be described presently. We thus require a high magnification, and some means of continuous record.

A high magnification may be produced by microscopic optical projection, but this labours under the great disadvantage that the specimen is subjected to the strong and unilateral stimulus of light, by which its normal growthmovements are greatly modified. The ordinary auxanometric method, again, cannot be employed, (i) because the magnification produced is not sufficiently great ; and (2) because the inertia of the wheel, and the unavoidable friction of the apparatus, themselves combine to obliterate the quick pulsations of the growth-response.

The Crescograph. — All these difficulties were overcome by the use of my Optical Lever, for making growthrecords. The lever is made extremely light, and the fulcrumrod rests on agate planes. When the tip of the growing organ is attached by a thread to the short arm of the Lever, the length of the latter being *5 cm., and when the recording surface is at a distance of 2-5 metres, a magnification of 1 ,000 times is obtained. This is in most cases more than sufficient. But, when necessary, a magnification of 10,000 times can easily be secured. In order to avoid any disturbance due to vibration in the room, the apparatus is supported on a steady bracket, fixed on the wall. Using these ordinary precautions, records are obtained with this instrument which are absolutely free from external disturbance.

The Balanced Crescograph. — In records of growth we obtain a sloping curve whose abscissa represents time ; and ordinate, the elongation produced in the organ during that time. A variation of growth will produce a variation in the slope of the curve. When this variation of growth, however, is slight, the variation of slope of the curve is so small as not to be detected. It was in order to detect and measure such small variations that I devised the Method of Balance, in which the average rate of growth is represented by the horizontal line of balance, any fluctuation, even the slightest, appearing as a deviation from this horizontal. The influence of various agencies, again, may be displayed in a marked manner, by using this method ; for in such cases we do not so much require the rate of growth itself, as the variation — i.e. acceleration or retardation-- in the normal rate, which is induced by one agent or another.

The principle of the Method of Balance consists in making the spot of light — which is moving in response to growth — become stationary, by subjecting it to a compensating movement. An example will make this clear. We shall suppose the average rate of growth to be V2 mm. per hour. This will cause an excursion of the moving spot of light, from, say, left to right, through 1,200 mm. by the end of the hour, in that case where the magnification is 1,000. Had the growth been uniform, this would have meant a movement of 20 mm. per minute. But if not uniform, the rate might sometimes have risen above, and at others fallen below, this average. If now we subject the spot of light to a uniform compensating movement, such as by itself would have made it move from right to left of the recording surface, to the extent of 1,200 mm. by the end of the hour, we shall find that, being acted on by these two opposite movements, of growth and compensation, the spot will remain approximately on a single base line of compensation. The fluctuations, or variations, which have occurred in this average rate of growth, will, however, be recorded as deviations to one side or other of this mean neutral line. Thus it will be seen that the slightest deviation from a uniform rate of growth, will be found displayed by the record of the moving spot of light. We are further enabled, from our

knowledge of the speed of the recording-drum, and the balancing rate, and from an inspection of the curve itself, to determine not only the periodicities, but also the absolute value of the rate of variation of growth, at any given moment. The compensating movement to which I have referred is effected by means of an hydraulic device. The spot of light from the Optical Lever falls upon a mirror attached to a second lever, or to a rotating wheel. The arm of the lever, or a thread which is passed round the wheel, is attached to a float on the surface of a cylinder of water. Water is escaping from this cylinder, by means of a syphon arrangement, at a rate which can be adjusted with the greatest nicety. The float can thus be made to descend at any speed that is desired, this descent producing a rotation of the second lever or of the wheel. We have, then, two mirrors, of which one is rotated in one direction by the growth-movement of the plant, and the second in the opposite direction by the descent of the float. A spot of light reflected on the two mirrors will thus remain stationary when the precise balance is effected, by proper regulation of the escape of water from the cylinder (fig. 167).

Fig. 167. Diagrammatic Representation of Balanced Crescograph p, plant attached to Optic Lever, l, with mirror, M, attached to fulcrum -rod, resting on knifeedges, A and a' ; \J, lever attached to float, F F, with second mirror, M, attached to its fulcrum-rod ; B, balancing wheel adjusting differences of level of the two limbs of syphon, s. This outflow of water is roughly adjusted by opening the stop-cock, to a greater or less extent. The finer adjustment is then effected by the suitable variation of the difference of level in the two limbs of the syphon. One end is connected with a flexible india-rubber tube, which is attached to a string passing over a pulley, and fixed to the adjustable wheel B, attached to the observer's table. Rotation of the wheel in one direction will depress this end of the syphon, so increasing the flow, and in the other direction will raise it, so diminishing the escape of water. It will be noticed that the rate of outflow of the water is not in any way affected by the variation of level of the water in the cylinder. It simply depends on the difference of level between the two ends of the syphon. The rate of descent of the float is thus regulated with the utmost nicety, till an absolute balance is obtained. The observer recognises the condition of balance when there is no drifting of the spot of light on the recording drum. The adjusting wheel is now fixed at this position of balance. If any agent should induce an acceleration of growth, the balance is disturbed, and the spot of light moves, say, to the right, or in a positive direction. Any agent which induces retardation will, on the other hand, cause a deflection of the spot of light in a negative direction. Should there be any natural fluctuations in the growth, the oscillation of the spot of light will give an indication of the fact.

The recqrd is made in the usual manner on a revolving drum. Fig. 168 illustrates the complete apparatus, which enables us to obtain a record under balanced — or by closing the stop-cock of the syphon, also under unbalanced — conditions. The wheel is graduated, and the absolute value of the compensatory movement, at any position of the circular scale, can be previously calibrated, by fixing the plant-mirror, and observing the extent of movement of the spot of light on the drum, due to the subsidence of the float in a given time.

For the exhibition of pure longitudinal growth-response, the most perfect specimens are the growing radial styles and stamens of flowers. There are also other organs, which are more or less strictly radial, such as the peduncle and the hypocotyl, But in these latter cases care should be taken that the specimen have been so grown, that its different sides have been subjected to uniform conditions of light or darkness ; for one-sided illumination tends to produce anisotropy,

Fig. i 68. Complete Apparatus for Crescographic Record under Ordinary and Balanced Conditions With specimens of all the types mentioned, I have obtained multiple growth-responses when the constituent pulsations were not too rapid. In some cases, indeed, the effects were so marked that they did not even require a balancing arrangement to render them conspicuous. As an instance of this, I shall give a record of the growth-response of a vigorously growing peduncle of Crocus which brings out in an interesting manner the mechanics of growth (fig. 169).

Rhythmic growth-response.— We saw that when the sum total of energy is above par, a tissue becomes selfexcitatory in a multiple or rhythmic manner, giving rise to periodic turgidity-variations. There may thus be responsive pulsations of increased turgidity, each followed by slow recovery from such excess. With each such pulse, a transient elongation of the growing tissue will be produced, and the succeeding slow recovery will be more or less incomplete. This incompleteness is due to the deposit of material which fixes growth. The irreversible or permanent growth-effect produced by each pulsation, will thus be measured by the responsive elongation minus the recovery. This is well illustrated in fig. 169, where

The ordinate represents the extent of responsive elongations in mm. ; the abscissa, time in seconds. three separate sets of responses are given, taken from a single specimen in the course of the day. Growth, as will be shown, is not uniform throughout the day, but exhibits variation, in consequence of changing conditions, such as that of temperature. But for a short interval of time, the rate of growth under a constant environment, it may be taken as uniform. In the present

instance the maximum rate was as high as '0035 and the minimum as low as *ooio mm. per minute. Confining our attention to the uppermost of these series (c\ we find that the responsive elongation is very quick, and the recovery slow and incomplete. The average period of a single pulse is twenty seconds. The results of series (c) are given in the following table: It will be seen from this table that the total growth in eighty seconds is '0047 mm., giving an average rate of growth of '003 5 mm. per minute. Had a magnifying arrangement not been used, this average rate of growth, shown by the dotted base line, would have appeared as continuous growth. By the magnification of the responsive curve, however, we are enabled to see that such a rate is in reality made up of numerous fluctuating growths of which it is an average.