Plant Response as a Means of Physiological Investigation

I next attempted to obtain a series of responses, when the temperature of the space in which the specimen was placed was very gradually lowered. As recovery from the stimulus of electrical shock constitutes a very prolonged process, I had to use thermal stimulation. But this introduced the difficulty of itself raising to a certain extent the temperature of the space. I had been fortunate enough, however, to secure a few specimens of the style of Datura

which were extremely sensitive to cold, and thus, notwithstanding the disadvantages incidental to the experiment, I was able to obtain the very interesting records shown below. The experiment was carried out as follows : The specimen was mounted in the special plant chamber. By pioper manipulation of the stop-cocks it was possible to send at will through the chamber, first, air at the ordinary temperature — under which conditions the normal responses were taken — and, secondly, air which had been cooled by ice, reducing the temperature of the chamber by several degrees. The responses then obtained showed the effect of cooling. And, lastly, ordinary air was re-introduced, and the responses at this temperature again recorded. Fig. 77 shows that while FlG- 77- . Effect of Cold on 0 * ' Longitudinal Response

that, on the restoration of normal conditions, the responses increased in a staircase manner tending to return to their original value. Effect of rise of temperature : (a) on electrical response.— So much for the results of cooling ; we have now to study the effect of rise of temperature. And, first, I shall refer to observations made by means of electric responses. It may be said that the optimum degree of temperature for the excitability of the plant must be understood to vary with different species. In several cases, however, I have found it to lie at about 22° C. But it must be premised that this optimum of response refers to passive tissues only, that is to say, to those in which there is no growth. The optimum temperature for growth may be different. In the ordinary response of passive tissues, heat has only to bring about a condition favourable to mobility. In the case of growing tissues, however, something addi-

tional has to be effected, namely, the acceleration of growth. We may therefore expect that the optimum temperature for growth will be relatively higher than that of simple response. And this I generally find to be the case. When the temperature is much raised, the electrical response of a plant is seen to undergo diminution (fig. 78). It is to be noted that the temperatures referred to are dry, or atmospheric only. This explains why, even when the temperature was raised to 650 C, which is above the fatal point, response was still

FlG. 78. Effect of Rise of Temperature on Electrical Response observed. Owing to the relative nonconductivity of the plant, and the evaporation from its surface, the tissue does not actually attain the temperature of the surrounding air. When, however, it was subjected for some time to a watertemperature of 550 C. response disappeared, by the death of the specimen. (J?) On longitudinal mechanical response. — I give next (fig. 79) the effect of rise of temperature on longitudinal mechanical response. The specimen was a filament of the corona oi Pas si flora, the stimulus used being thermal. In order to subject the plant to definite temperature conditions, the adjustments were made by means of a subsidiary heating coil, placed inside the chamber. Any given temperature above the normal could thus be maintained constant, for the required length of time, by simple adjustment of the strength of the heating current. The results thus obtained in mechanical response

Fig. 79. Effect of Rise of Temperature on Longitudinal Contractile Re- sponse of Plant will be seen to be exactly parallel to those already given in electrical response. . The responses are seen to be very much diminished at 35°C, and almost to disappear at 55° C, which, as will be shown later, is near the death-point. Heat-rigor began to manifest itself a few degrees above this point, in the contracting of the specimen as a whole. This last phenomenon will be treated at greater length in a subsequent chapter. I have already alluded to the experiment in which, when the plant is killed by excessively high temperature, the response disappears altogether.

Effects of cyclic variation of temperature : (a) on electrical response. — I detected another very curious result Fig. 8o. Effect of Rising and Falling Temperature on the Electrical Response of Scotch Kale (stimulus constant) in the course of my investigations on the effect of temperature by means of electrical response. This was, a marked increase of sensitiveness, which often appears as the after-effect of a preceding cyclic variation of temperature. That is to say, if we take a series of responses while the temperature is rising, and afterwards a similar series while the temperature is falling, it is found that during the process of cooling, the responses are markedly enhanced in amplitude, as compared with those given at the corresponding temperatures during heating. This is seen in a very clear manner in fig. 80.

{b) On longitudinal mechanical response. — I have observed a very similar phenomenon in the longitudinal mechanical response of, for example, the coronal filaments of Passiflora. An ascending series of responses was taken at temperatures of 250, 350, and 450 C. They exhibited a regular decrease, as has already been explained. On now, however, taking records of responses during cooling, it was found that the response at 3 5° C. was SO per Fig. 81. Effect of Cyclic . V, ,lt ". if,

Rise and Fall of Tempecentgreater than it had been when the rature on Longitudinal temperature was ascending (fig. 81). response is found to be abolished when the plant is killed, by raising the temperature above a certain maximum point. The exact determination of this point has hitherto been a matter of great uncertainty. I shall in the next two chapters, however, explain several methods by which this investigation may be carried out with great precision.

The electrical and mechanical responses of plants undergo diminution under the influence of cold. The latent period of response is prolonged by lowering of temperature. responses, both electrical and mechanical, undergo a diminution. Prolonged exposure to excessive cold or heat brings on abolition of response. Owing to cold or heat-rigor this abolition may become permanent, indicating the death of the plant. Difficulty of determining exact moment of death — Various post-mortem symptoms afford no immediate indication — Ideal methods for determination of deathpoint — Realised in four different ways : {a) Determination by electrical method — (b) Determination by spasmodic lateral movement at moment of death — Experiments with Mimosa — Death-contraction a true physiological response — Continuity of fatigue and death — Death-point earlier in young tissues — Composite spasmodic movement — {b') Determination of death-point in tendril of Passiflora, by sudden movement of uncurling— (c) Determination of deathpoint by method of volumetric contraction of hollow organ, causing expulsion of contained water.

It is known that when the temperature to which it is subjected is raised above a certain maximum, a plant is killed. But it is very difficult to determine at what exact temperature this takes place. One reason of the difficulty lies in the fact that hitherto a sure criterion of death, which would give an immediate and reliable indication of its occurrence, has not been generally available. Its various symptoms, such as drooping, withering, discoloration, and the escape of coloured cell-sap, do not manifest themselves at the moment of death, but at some time indeterminately later. Even when a plant has been subjected to a temperature in excess of the fatal degree, it continues to appear fresh and living, and it is not till after some greater or less interval that the death-symptoms are seen. Various investigators have taken up different indications as the criteria of death, and this fact accounts for discordance in the results, which would already have been sufficiently uncertain even had a common standard been decided upon.

Exact methods of determination of death-point. -A good method for the determination of the death-point would consist in watching the waning of a given effect, characteristic of the living condition. Still better would be the discovery of some effect suddenly and strikingly manifested at death. But the ideal method would be found, if some effect could be detected which at the moment of death would undergo sudden reversal to its opposite. In this last case, there would not be even that minor degree of uncertainty which is incidental to the determination of the exact vanishing-point of a waning effect. I have been successful in devising four distinct means, by which the death-point might be detected with precision, and it will be shown that all these different modes of investigation enable results to be obtained which corroborate each other in a remarkable manner. The four means are : (a) the method of electrical response ; (J?) that method by which the point of death is determined from the occurrence of a spasmodic movement, in a dorsi-ventral or anisotropic organ ; (c) that method which depends on the sudden expulsion of water at the moment of death from a hollow organ, previously filled with liquid ; and (d) the method in which the death-point is determined from the sudden reversal of a thermo-mechanical response-curve. I shall, in the course of the present chapter, describe the first three of these, leaving the fourth method to be treated in the next chapter.

(a) Determination of the death-point by electrical response. — As regards the electrical method, I have shown elsewhere1 that the response of normal galvanometric negativity is characteristic of the living condition of a plant-tissue. When the plant is killed, by any means whatsoever, this normal response disappears. At the moment of death from rise of temperature, therefore, we shall have the abolition of the normal negative excitatory response. But at or beyond this point, on the other hand, we may have the positive response of hydrostatic disturbance replacing the true excitatory effect. By this electrical mode of investigation, I have been able to determine the death-points of different plants. In the following table, for

example, the specimens tested were radishes, and the experiment was conducted during the winter season in England. It would appear from the results given, that in these six cases response begins to be abolished at temperatures varying from 35° to 55° C. It will be shown later that the death-point depends on the season, being a few degrees lower in winter than in summer. Table showing Effect of High Temperature in Abolition of Response and Death of Plant

Teinperature sponse of specimen Temperature sponse of specimen (b) By observation of the spasmodic movement of lateral response. — I now turn to the second of the four methods which I have named, that in which a spasmodic lateral movement is looked for, in a dorsi-ventral or anisotropic organ, at the moment of death. It has been shown that when an electric shock of moderate intensity is applied to an anisotropic organ, say the leaf of Mimosa, response occurs, in consequence of molecular derangement, and recovery takes place on restoration of molecular equilibrium. If the shock, however, be excessive, response occurs, it is true, but there is no subsequent recovery, owing to the fact that the molecular derangement has passed beyond the point where restoration was possible. There is thus a permanent, irreversible ' set,' and the organ is now said to be killed.

ment is produced. It follows that if we could bring on very gradually those conditions which cause death, then, on arrival at the critical point, we might expect the irreversible molecular derangement to occur abruptly. If, further, throughout this process, the organ could be protected from stimulation, we might expect that this sudden molecular derangement would also be attended by a correspondingly sudden evidence of excitation, which would in this case, however, be at once the first indication of excitation and the last sign of life. This spasmodic movement we shall designate as the deathresponse.

As regards the protection of the experimental organ from accidental stimulation, it is to be remembered that excitation under ordinary circumstances depends upon some sudden variation of environmental conditions. A sudden change of temperature may thus act as a stimulating agent and produce depression of the leaf. But a gradual change will not act as a stimulus. The effect of such a gradual variation, on the contrary, as will be shown presently, is to produce no excitatory contraction whatsoever.

If now we take a specimen of Mimosa and place it suddenly in warm water, say at 3 5° C, a responsive collapse of the leaves will at once occur. But if the plant be placed in water at the ordinary temperature of the room, and the temperature gradually raised — say at a rate of i° per one minute and a half, or thereabouts — there will be no responsive downward movement whatsoever. On the contrary, owing to absorption of water by the organ, and also to the relaxing physiological action of heat, a delicate method of record will show a slight and continuous movement upwards. This proceeds till we reach a degree of temperature which proves to be the death-point. For example, in the case of a particular experiment in summer, with a young leaf of Mimosa, when the temperature of 590 C. was reached, there was a sudden spasmodic movement of the leaf downwards. This was, in fact, the death-throe of the plant. In winter, after a spell of cold weather, when the physiological condition

of the tissue was somewhat depressed, this spasmodic movement was found to take place at 530 C, which exactly agrees with that of radish, under similar conditions. That this was the true death-point of the Mimosa specimen was proved when, on trying the electrical test, it was found that the normal electrical response had disappeared. If, again, one branch of Mimosa on the intact plant be bent over, and subjected in the manner described to the deathtemperature, we find, on examination after a considerable lapse of time, that whereas the leaves of the rest of the plant are still fresh and healthy, reacting to stimulus, those of this branch may be seen, from their dried and shrivelled condition, to be quite dead.

Death-response a true physiological response. — It will be shown that this death-response is a true physiological response. Under normal conditions, it will be found to be extremely definite, even in different plants. But, under physiological modification, it varies appropriately with the season, age, condition of freshness or fatigue, and the action of chemical reagents. I shall first, then, demonstrate the effect of age on the death-point. Thus, on immersing a branch of Mimosa in water whose temperature is raised continuously, we find that the spasmodic movement of death occurs earlier in the young leaves than in the old. Young seedlings, again, have a lower death-point than mature plants. The following table gives results bearing on this fact. The death-points of different plants in the same season and of the same age are, however, so definite as to be almost like a physical constant. This will appear from the following tabular results, and also from results given in the next chapter. A fact which will be explained later must be stated here. In these experiments with continuously rising temperature, it is found that the first spasmodic movement downwards is succeeded by a later, upwards, by which time the temperature has risen a few degrees; The temperatures of both movements are given in the accompanying table, corresponding to their occurrence in two leaves, one old and

one young, of each plant tested. These experiments were carried out at the beginning of spring. Table showing Death-points in Old and Young Leaves of Different Specimens of Mimosa From these results it will be seen that there is a mean difference of 1-5° C. between the death-responses of old and young leaves. It would thus appear that the age of a cell must be the occasion of a certain amount of protoplasmic change, as manifested in the retardation of death-response. We may also infer that sudden change to unfavourable physiological circumstances — before the plant has accommodated itself to the changed condition — will tend to lower the death-point. This fact I found illustrated during the prevalence of an extraordinary wave of cold, which supervened recently, during the progress of these experiments. I then found that the mean death-point, in the case of various plants, was reduced by several degrees. In Mimosa it fell from the average of 59° C. to 55° C, i.e. as much as 40 C. We have thus seen that the physiological differentiation concomitant on protoplasmic change is attended by variation of deathpoint.

Explanation of the subsequent erection. — In animal tissues, the contraction produced by rigor mortis is succeeded by a relaxation. The contractile death-spasm in a plant is, similarly, followed by the relaxation seen in the subsequent erection of the leaf. There is another and very interesting point of view, from which we may see in this phenomenon the continuity of fatigue with death. In the curve of reversal, due to fatigue, in Mimosa, we saw that the first contraction, induced by strong and long-continued stimulation, passed into subsequent relaxation. In this latter state, the molecular condition was such that responsiveness was abolished. It was as if, in other words, the tissue had passed into a temporary state of death. It is true that, if the stimulation had not been excessive, the organ would recover its sensitiveness, after a period of rest. But this transient would pass imperceptibly into the permanent condition of death if, on the other hand, stimulation had been excessive. In that case, after the fatigue-reversal, the tissue would remain permanently irresponsive.

I have already said that the death-spasm is an instance of excitatory response to intense stimulation, and we should therefore expect the same kind of effect to be produced as is caused by excessive stimulation, that is to say, a preliminary contraction, followed by relaxation, after which there is no recovery. Again, we saw that in the fatigue-reversal of Mimosa, the subsequent erection of the leaf, mainly due as it was to relaxation, was possibly also aided by the later contraction of the less excitable upper half of the pulvinus. Similarly, it might be expected that the death-contraction of the less excitable upper, would take place slightly later than that of the lower, half of the pulvinus. We have also seen that the excitability of a tissue declines with age, and this decline would naturally be greater in the more excitable half. Thus the difference of excitability as between the two halves would at the same time tend to disappear. As, then, this spasmodic movement in dorsi-ventral organs is a true instance of differential excitatory response, it would appear that the younger the organ, the greater is the excitatory spasm caused by death, and in experimenting with Mimosa I have found that at the death-point hardly any spasmodic movement is shown by old leaves. These considerations will also explain

why older leaves give less motile indication than the young, in response to stimulus in general. (b') By observation of spasmodic movement of uncurling. — I shall now proceed to demonstrate that the deathmovement which we have seen in Mimosa is in its essentials characteristic of anisotropic response in general ; and this may be shown by taking a spiral tendril of Passiflora which has become anisotropic by curling. In order to detect and measure with ease the responsive movements of uncurling and curling the experiment is arranged as follows : a light index is attached to the tip of the spiral, and the whole is immersed co-axially in a glass cylinder filled with water. A strip of paper, marked with degrees, is wrapped round the outside of the cylinder on the plane of the index. The temperature of the water is now raised very gradually, and the responsive excursion of the index is read on the graduated circle formed by the paper.

It is to be borne in mind that the true excitatory response of the tendril is given by uncurling, which here corresponds to the fall of the leaf of Mimosa ; thus the movement of erection would be represented by that of curling. In the case of Mimosa we saw that the first effect during rise of temperature (due to absorption of water and relaxation) was slow and gradual erection. On the arrival of the deathpoint of the organ, however, this preliminary rise was succeeded by a sudden responsive fall. The subsequent relaxation of death then produced an opposite movement, of erection. Similarly, in the death-response of the spiral tendril of Passiflora, we observe parallel phases. There was first a slow and continuous movement of curling, during the preliminary stages of warming. But this movement ceased when a temperature of 570 C. was reached, and the tendril remained stationary for a time. At 590 C, however, there was produced a sudden excitatory response of death by an uncurling, executed with great rapidity, an angular movement of 360 degrees being described by the index during the course of the next few degrees of rise in temperature. With regard

to the short stationary period, it is to be borne in mind that the death-point depends on the age of the tissue, and in the tendril we have different parts in different stages of growth. Hence while the uncurling movement of death was being initiated in younger portions, older parts of the tendril were still moving in an opposite direction. The outcome of these antagonistic movements was a resultant pause, which only lasted for a little while, and was followed by the vigorous movement of uncurling, caused by the death-contraction of the whole tissue. After the completion of the uncurling movement, there followed the opposite, namely, the movement of post mortem relaxation. In a second experiment with a younger specimen of tendril, I obtained results almost identical. Here the uncurling response of death began at one degree of temperature earlier, namely at 5 8° C, and the index moved through 1 50 degrees of the circular scale.

On the subject of the death-contraction of the radial organs of ordinary plants, I shall speak in some detail in the next chapter, and shall there describe the perfected apparatus by which the thermo-mechanical response can be continuously recorded, the curve exhibiting the death -point with great precision. (c) By observation of volumetric contraction, causing sudden expulsion of water. — For the present I shall describe only the third of those methods which I have enumerated for the determination of the death-point, that namely which depends on the sudden expulsion of water, at the moment of death, from the hollow organ of an ordinary plant, previously filled with liquid. The specimen used for the present demonstration will be the peduncle of Allium, although there are many tubular organs of various species of plants which are more or less suitable for these experiments.

We cut a length of about 10 cm. from the middle of a peduncle of Allium, rejecting the too young and too old portions at top and bottom. As the presence of air-bubbles is likely to be disturbing to the experiment, the water used must have been previously boiled. The .specimen is placed in a vessel of this water, and as a further precaution against airbubbles clinging to the interior of the tube, the whole may be put inside the receiver of an air-pump and subjected to a repeated partial vacuum. The removal of air-bubbles may also be effected by rinsing the tube of Allium in water containing a small quantity of ether, and immediately afterwards washing with ordinary water. This must be done, however, with caution, as the presence of an appreciable quantity of ether would be likely to affect the excitability of the tissue. Before commencing the experiment, it is advisable to allow the specimen to remain immersed in water for about half an hour, by which time it becomes fully turgid.

The lower end of the Allium tube filled with water is closed by a piece of solid glass rod, and the upper end is also closed with a piece of glass tube, having a capillary bore. A graduated scale is placed behind this latter, so as to measure the movement of the water-index, or this movement may be continuously recorded on a revolving drum (fig. 156). The Allium preparation is now placed in a vessel of water, and subjected to a gradual rise of temperature in the manner already described.

If, at the temperature corresponding to death, there should now be a sudden excitatory contraction of the Allium tissue, the volumetric change thus produced will force out the contained water, and we shall observe a relatively rapid expulsive movement of the water-index. From the curves given above in fig. 82, it will be seen that this occurs at a temperature of 590 C. in a younger, and at 6f C. in an older specimen. Previous to this, there was an inward movement of the water-column corresponding to the gradual