Plant Response as a Means of Physiological Investigation

acceleration that precedes it. Incidentally, we see here the difference between the temperature-effect per se, and the stimulating effect of sudden variations of temperature, constituting thermal shocks. In the simple case we have just studied, where the whole amount of incident stimulus was expressed in work external and internal, without any loss from molecular friction, the sum total of the two forms of response was found approximately the same under a constant stimulus ; but in other cases, where a certain amount of energy is wasted in overcoming molecular sluggishness, the results will be slightly different, for at a temperature below the optimum a portion of the stimulus will be wasted in overcoming such sluggishness, whereas near the optimum temperature the loss entailed on this account will be very slight. Hence, the sum of direct and indirect responses, near the optimum, will in such cases be somewhat greater than at a temperature several degrees lower. I give below a table which shows at a glance the direct effect and the indirect after-effect, obtained at the three temperatures of 30° C, 35° C, and 37° C. respectively, with three different specimens, one of which was a rice-seedling {Oryza sativa), and the two others flower-buds of Crinum Lily. Each response given is the mean of three.

Table showing Direct and Indirect Effects at Three Different Temperatures It will be seen from the readings given by specimens 1 and 2, that up to the optimum the total response is approximately constant, while at n° C. there is no expression of energy held latent. The condition of specimen 3 having been at starting somewhat sub-tonic, the total effect is heightened at the optimum, for reasons which have been explained. It has already been said that the proportion of stimulus held latent will be greater with the degree of subtonicity of the plant. That this was the condition of specimen 3, then, is demonstrated, not only by the increase of the total effect at the optimum temperature, but also by the fact that at 300 C. so large a proportion of the stimulus as almost exactly one-half is held latent. At 370 C. here, as in the other cases, there was no latent component.

Is the change induced by stimulus always of an explosive character? — It has generally been supposed that stimulus causes response by an explosive chemical change. According to this theory, the stimulus acts as upon a trigger, to release suddenly a large amount of energy previously held latent in the tissue. The response is thus assumed to be always disproportionately larger than the stimulus, and to be brought about by chemical degradation, or dissimilation of the living tissue. The tissue, thus reduced below par, is supposed to be restored by the process of assimilation.

In Chapter X., however — on Theories concerning Different Types of Response — I adduced considerations, showing that there are cases of responsive phenomena in which this description would not hold good ; that is to say, there are instances in which the response cannot be due to a chemical down change of explosive character. Nor is it always true that response is disproportionally larger than stimulus. The series of experiments which has just been described offers conclusive evidence on this point, of a quantitative character. Further, if the theory of an explosive down change had been the real explanation of response in general, then it is clear that in the case of the response induced by stimulus in growing organs, recovery would have taken place slowly, and

would have culminated in the restoration of the original rate of growth ; but we actually find, on the contrary, that, immediately following the responsive retardation, there is an acceleration of growth above the normal, and the true recovery, or restoration of the normal rate, takes place only after this. Thus there is here, instead of a run down, an actual increase, of the energy of the system. Again, from this constancy of the sum of the immediate and the after-effects of stimulus, we can see that, as in an inorganic system, so also in a living organism, the law of the Conservation of Energy holds good. For while at the optimum point the entire stimulus finds expression in direct response (stimulus being here equal to response), below the optimum the direct response is less than the stimulus, the missing fraction being left to find expression in the negative after-effect.

Relation between stimulus and response in different tonic conditions. — The experiments which I have described were carried out under the thermal form of stimulation, which is, as already explained, the most satisfactory in practice. We shall hereafter come across instances of the after-effect of accelerated growth, as caused by stimulus of light. And a similar after-effect has already been seen to be caused by electrical stimulus (p. 436). But for the clear demonstration of this particular effect, electrical stimulus is not very suitable, inasmuch as it is apt to induce fatigue and, through electrical polarisation, a certain amount of tissue-change. I shall, however, describe an experiment, using this mode of stimulation, which, by its responsive indications, will demonstrate certain differences in the internal conditions of tissues, according as they are sub- or super-tonic.

I have shown that the general excitability of the tissue at 300 C. is superficially the same as that at 370 C. or thereabouts ; that is to say, the direct responses at these two temperatures are in some cases approximately the same ; but we have seen that there is a difference of molecular condition at these two points, for at 300 C. the tissue is capable of holding a portion of the incident stimulus latent, by which its molecular mobility becomes enhanced, whereas at 3 7° C. there is already the fullest molecular mobility. If now*we apply at 300 C. stimuli which increase, say, in arithmetical progression, we see that by the very reception of the increasing stimuli the tissue is made to approach more and more closely to the optimum condition, at which point, as we have seen, the whole of the stimulus is given up immediately, in the form of direct response. Hence the curve showing the relation between stimulus and response in a tissue that is in a condition below the optimum will be steep, and somewhat convex to the abscissa which represents the stimulus ; but at 37° C, when no molecular sluggishness has to be overcome, we may expect the response to increase proportionately with the stimulus ; that is to say, the curve showing the relation between stimulus and response will now tend to be a straight line.

In order to apply electrical stimulation whose intensity was increased by known amounts, I used an induction coil, the primary coil of which was completely within the secondary. The ordinary method of Du Bois-Reymond's sliding coil was not very suitable in this case, because the increasing intensity obtained by sliding the coil inwards is merely qualitative. The required definite increase of induction-shock I secured by suitable augmentations in the value of the current that flowed round the primary coil. In order to determine these values, a preliminary experiment was carried out. A storage battery was in circuit with the primary coil, which had interposed in it also an ammeter and a rheostat. By increasing the resistance, a moderate current, say C, read by the ammeter, was adjusted to flow round the circuit. The secondary circuit contained a ballistic galvanometer, which by its throw indicated the intensity of the induced current at make or break of the primary circuit. When the first current, C, had given an induced current, which caused a deflection of, say, 30, it was increased to C„ when the deflection due to induction was found to be 50 ; and lastly a third value, or C;;, was found, whose induction-effect was 70. In this manner,

induction-shocks, increasing in arithmetical progression as 3 to 5 to 7, were determined. Tetanic shocks of the described intensity — 3, 5, 7 — the total application of each group being a period of 5", were now given, response being taken after each. In this manner two series of responses were obtained at the temperatures of 300 C. and 370 C. At both temperatures with the stimulus-intensity of 3, the responses given by this particular specimen were the same. But while at 370 C. the lower of the two curves, showing the relation between stimulus and response, is a straight line, that at 300 C, the upper of the two, is seen to ascend more steeply and to exhibit convexity to the abscissa (fig. 186).

After-effect.— The phenomenon which is generally referred to as the after-effect is characterised by so many complexities as to have been regarded as highly perplexing. After the foregoing analysis, however, by which it has been resolved into two elements, much of this obscurity will be found to have disappeared. We saw that the first effect induced by a very strong or long-continued external stimulus consisted in bringing about the continuation of the direct effect itself, its persistence depending on the intensity and duration of that stimulation. And

this we have distinguished as the positive after-effect. A component part of the external stimulus, however, as we also saw, becomes latent, thus increasing the internal energy ; and the expression due to this element — the negative after-effect— is opposite in sign to the effect of direct stimulus and its positive Fig. 186. Curve showing the Relation between Stimulus and Response in the same Organ, under the two different Tonic Conditions of 300 C. (upper curve) and 370 C. (lower curve)

The abscissa represents the intensity of stimulus, and the ordinate the extent of response. The response was the same in both cases when the stimulus-intensity was 3 ; but later, the curve for 300 C. is seen to rise more steeply than that at 370 C, exhibiting at the same time convexity to the abscissa. after-effect. Thus a plant under natural conditions, acted upon by different stimuli, gives to each stimulus direct response, direct after-response, and indirect response. But as all these individual stimuli do not act, or cease to act, simultaneously, we can easily understand the infinite complexity of the combinations which take place between the direct and indirect effects of stimuli of unlike forms, whose maxima, instead of being coincident, are superposed on each other, with various differences of phase.

Factors which determine periodic after-effects.— We shall now proceed to enumerate some of the most important stimulating factors instrumental in modifying the response of growth. (i) Stimulus of light. — If we take the average rate of growth during the twenty-four hours as the normal, then the direct effect of this stimulus will appear as a retardation of that normal rate of growth, and, after the long-continued action of the whole day's illumination, this retardation may persist for a time as the positive after-effect. Later, however, on account of the stimulus which has been absorbed and held latent, we shall observe an acceleration of growth above the normal, or a negative after-effect. The persistence, again, of this negative after-effect will depend on the amount of the energy held latent by the tissue. The diurnal sequence of light and darkness will, after long repetition, impress itself upon the organism, and, other factors remaining constant, will find expression as periodic retardation and acceleration of growth during day and night ; such periodicity continuing to show itself, for some time, even when the plant is kept in continuous darkness.

(2) Temperature. — The effect of temperature up to the optimum point will, by increasing the internal energy, prove favourable to growth. Thus, the temperature during daylight will usually be favourable, with the exception of the tropical noon, when it may be excessive. A certain amount of heat, again, may be stored up in the plant to give the aftereffect. At night, if the fall of temperature be very great, there will be, relatively to this factor, a retardation of growth.

(3) Chemical stimulus. — This we see supplied by the salts taken up by the roots, and by the process of photo-synthesis in the leaves. The amount of the former supply is dependent not only on the richness of the soil, but also on the suctional activity of the plant ; and the latter on the effective intensity of light. (4) Turgidity. — The turgid condition of the plant will depend, firstly, on the supply of water ; secondly, on the suctional activity ; and thirdly, on the relative absence of a loss of water by transpiration. We have seen how watering the roots of the plant will cause an immediate response of enhanced growth, and the seasonal periodicity induced by this cause may be observed in a very striking manner in tropical countries.

The suctional activity, depending as it does on the internal energy of the plant, will tend to be augmented by an advantageous temperature, and by the after-effect of the absorbed stimulus of light ; but the turgid condition will be reduced by active transpiration, which is relatively greater in the daytime. We thus see howr numerous are the factors which cooperate to bring about periodic fluctuations in the rate of growth during every twenty-four hours. Of all these factors, the alternation of day and night is the most pronounced in its action. The curve of growth, then, will exhibit not only a large wave of alternation due to the diurnal period, but also a number of sub-waves. And even beyond these, superposed upon them, we may expect, when the magnification is sufficiently great, to observe systems of still smaller wavelets, caused by the rhythmicity of growth.

Continuous photographic record of the pulsations of Desmodium. — Before describing the periodic diurnal variation of that autonomous response which we know as growth, it occurred to me that a continuous record of another form of autonomous response — that is to say, of the rhythmic pulsations of Desmodium — might prove interesting. I was fortunate enough to succeed in obtaining a very good record of these pulsations during a period of twelve hours, by means of photography. The record began at 6 P.M. and ended at 6 A.M. During this time there were no fewer than 180 constituent, pulses, and it will be noticed that these again fall into groupings, whose average period is a little over an hour, there being about ten such groups in the course of twelve hours (fig. 187).

Returning now to the question of periodic growth-fluctuations, Sachs and others have measured the different rates of growth at various hours within the twenty-four, and from the data thus obtained have constructed curves which showed Fig. 187. Continuous Photographic Record of Autonomous Pulsation or Desmodium gyrans from 6 p.m. to 6 a.m. the periodicity of the rate of growth. These curves exhibit the daily period in a marked manner ; but the subordinate waves are more or less obliterated, in consequence of the fact that the data from which the curves were constructed were obtained from discontinuous observations. The curves thus deduced show marked differences also, according as their points were determined frequently or at long intervals. Record of periodic variation of rate of growth. — For this reason it appeared to me important to devise means by which, not the growth, but the variations, of its rate might be automatically recorded, directly and continuously, for any

length of time. The curve thus obtained ought instantaneously to mark the fluctuations of rate throughout the period in question. This I have been able to accomplish by means of a modified Method of Balance. From the description of that method already given, it will be understood that after the establishment of the average balance, if the rate increases, the curve will move upwards. When, after this, the rate of growth returns to the normal, the balance will be re-established, and the curve become again horizontal ; but if the growth at any time should fall below the average, the curve will descend. In this way periodic fluctuations in the rate of growth may be recorded.

For the purposes of the modified method of record, the compensating arrangement used for balance has to be somewhat altered. In recording these long periodic changes, we have fluctuations of larger amplitude than those of autonomous pulsation. The spot of light which is thrown from the mirror of the experimental Optic Lever upon the second, or order to overcome this difficulty I mount the plant on the float itself, and adjust the outflow of water from the cylinder, so that the upward growth of the plant is, at a given moment, exactly compensated by the descent of the float supporting it. Deviations above or below the balanced rate of growth may then be followed with a recording pen,

or will record themselves on a sensitised photographic film wrapped round the revolving drum. In carrying out such a continuous record, certain precautions are necessary. Owing to transpiration, the float on which the plant is mounted will become lighter, causing an ascensional movement of the record. In order to obviate this, it is only necessary (i) to weight the float to such an extent that the variation caused by transpiration is negligible ; and (2), in addition to this, the diameter of the cylindrical float may be so increased as to reduce still further the ascensional movement due to loss of weight by transpiration. By these means the error from this source may be reduced

to any extent desired. In the figure of the apparatus which is here given (fig. 188), the specimen was a young seedling of Oryza sativa, in v/hich transpiration was relatively little. I shall presently give photographic records obtained in the manner described. Continuous photographic record of periodic variations of transpiration. — By a somewhat similar method we are enabled to determine the periodic variation of the rate of transpiration. In this case, the plant is mounted with its roots in a test-tube, which acts like a float, and is partially filled with water, but not so full as to make it sink. The test-tube containing the plant is attached to one arm of the Optic Lever, and the outflow from the outer cylinder so adjusted that the ascensional movement of the test-tube, due to its loss of weight by transpiration, is exactly balanced by the subsidence of the water-level of the water that buoys it up. In order

Eig. 189. Photographic Record showing Variation of Rate of Transpiration in Cucurbita, from 3 P.M. to 12 P.M. The rate is seen to undergo enhancement till about 11.30 p.m., after which there is a rapid fall of the rate of transpiration. to prevent the loss of water from the cylinder by evaporation, a film of oil covers the surface. In this way I obtained the accompanying record of variation of rate of transpiration in a young specimen of Cucurbita, from 3 P.M. to 12 P.M. (fig. 189). It will be noticed that in this case there is an enhancement of transpiration which continues with a single fluctuation till past 1 1 P.M., after which there is a sudden depression of the rate.

Continuous photographic record of the diurnal variation of the rate^of growth. — I shall first give a photographic record (fig. 190) taken from a seedling of Oryza sativa, only Fig. 190. Continuous Photographic Record of Variation of Rate of Growth in Four Days' Old Seedling of Oryza sativa, from 3 p.m. till 9 a.m., that is during Eighteen Hours four days old. The diurnal periodicity has already, it will be seen, become fairly impressed, though it is not yet sufficiently powerful to mask, to any great extent, the subsidiary periodicities induced by other factors. The record, it must be remembered, was taken in continuous darkness, being commenced at 3 P.M., when it was balanced. From this time to 9 P.M. there were three pulsations. From 6 till after 8 P.M. there was depression of the average rate of growth, after which it rose somewhat rapidly till 12.30 A.M., exhibiting during that period two groups of two pulsations each. There was now a quick fall for the next half-hour, and after this

the growth-rate rose more or less continuously till 8 A.M. The growth-rate then began to fall during the course of the day. The second record was taken with a seedling of Tamarindus indica fourteen days old, the diurnal periodicity being thus deeply impressed. It was placed in the dark room, mounted on the float, and the balanced record begun at 3 P.M. It will be seen that, as the positive after-effect of the day's illumination, there was a depression of the rate

Fig. 191. Continuous Photographic Record of Variation of Rate ot Growth in Seedling of Tamarindus indica, a Fortnight Old, from 3 p.m. to 3 A.M. Owing to positive after-effect of daylight, there is a depression of rate of growth, although the seedling was now placed in a dark room. In the evening, however, the rate began to rise. of growth, though the plant was kept in the dark. This persisted for two hours, till 5 P.M., after which the rate showed increase, there being three pulsations before the end of the record, at 3 A.M. (fig. 191).

Annual rings of wood and seasonal periodicity. — The different growths of wood in spring and autumn, leading to the formations known as ' annual rings,' constitute a phenomenon of growth not yet fully explained. I may here point out an important factor in connection with this subject. It has already been demonstrated that growth is a phenomenon of excitatory reaction, being at its maximum when the excitability of the tissue is greatest. Thus varying expressions of growth at the two seasons, as seen in the production of different sized cells during spring and autumn, would appear natural, if they could be correlated to differences of excitability, characteristic of those seasons. Now all modes of testing degrees of excitability lead to the conclusion that while it is very great in spring and summer, it is very much enfeebled in autumn and winter. Thus, in the latter season, contractility under stimulation, velocity of transmission of excitation, and the electrical response of a tissue, are all found to undergo a marked diminution, as compared with spring and summer.

On the cessation of strong stimulus the responsive movement continues for a time in the same direction. This is the positive after-effect. A portion of the incident stimulus is absorbed and held latent, thus increasing the latent energy of the plant. On the cessation of stimulus this latent component, either immediately or after a time, finds expression in an opposite responsive movement. This is the negative after-effect In the case of growth-response, the positive after-effect consists in the persistence for a time of the retardation of growth, and the negative after-effect exhibits itself as an acceleration of the rate of growth above the normal.

With moderate stimulus and under normal conditions the sum of the direct effect and the negative after-effect (due to the latent component) remains constant up to the optimum ; that is to say, the sum of the external work (direct effect) and internal work (negative after-effect) done by the stimulus is the same. The direct and the negative after-effect of stimulus are thus complementary. At the exact optimum almost the whole stimulus will find expression in direct response, there being little or no

latent component. In a sub-tonic condition, on the other hand, a greater proportion of the stimulus is temporarily held latent, and expresses itself as the negative after-effect, the direct responses being here correspondingly diminished. Above the optimum there is no latent component, and the general receptivity of the organ shows great diminution. From the constancy of the sum of the direct and indirect effects it is demonstrated that, with regard to some forms of response at least, response is not disproportionately greater than stimulus. Thus the theory that response must always be due to an explosive chemical change does not hold good.