Plant Response as a Means of Physiological Investigation

Vanishing of point of inversion with age. — The older specimen of Spirogyra (s'), taken from the same place, had its point of inversion raised by 40, the death-point being therefore at 510 C. There is a further and interesting difference as between curves for young and old specimens- In the younger specimen there was produced a very considerable contraction due to rigor, and this was followed after a time by the usual post-mortem relaxation. But in the older specimen the rigor was relatively slight and the subsequent relaxation took place much earlier. We thus see that there is a great loss of contractile power in old tissues. In still older specimens the contraction tends to vanish altogether, and we have no line of demarcation to mark the moment of transition from life into death. In connection with this, it is interesting to note that, whereas the death-spasm in young leaves of Mimosa is very vigorous, old leaves exhibit little or no spasmodic lateral movement at death.

Cold-rigor. — Turning from the effect caused by continuous rise of temperature, I shall now proceed to the consideration of the effect produced by the reverse process of continuous fall to the minimum temperature. Here also, as in the case of the curve for rising temperature, there is a sudden inversion at a definite minimum point of temperature. That is to say, just as we observe a sudden contraction when the point of heat-rigor is reached, so also we obtain a similar sudden contraction at a point corresponding to the coldrigor. For example, with the style of Eucharis Lily, which is very susceptible of depression by cold, I found the death-point to be at about i° C. The experimental difficulties for the determination of cold-rigor are, however, very great, owing to the fact that facilities do not exist for continuous lowering of temperature to zero or below.

Thermo-mechanical record of Mimosa. — At the beginning of this chapter it was stated that the death-spasm in an anisotropic organ, such as that of the pulvinus of Mimosa, was an instance of differential longitudinal excitatory contraction. The accompanying curve (fig. 88) was obtained by means of the Morograph. We must remember that in this case we are dealing with a differential action. In the first part of the curve, therefore, we do not obtain such marked relaxation as in radial organs, where we obtain non antagonised and direct change The spasmodic contraction of form. But when we reach a tem-

movement took place at perature which corresponds to the 54° C the normal deathdeath-point, that is to say, 54° C, 1£ZS32%&Z ment- j* ■»«"* be remembered that this particular experiment was carried out just after the spell of cold weather, when the death-points of plant-organs were found to be lowered by several degrees. After the downward movement, which commences at 54° C, we see that there is an equally abrupt upward movement, beginning at 59° C, due to post-mortem relaxation aided by the later contraction of the upper half of the organ.

A few words may be said here with regard to these successive movements. As in animals the rigor mortis is succeeded by relaxation, so also in radial organs, as has been said, we see relaxation succeeding the death-contraction. It may then be asked whether the second half of the present curve, in fig. Sy, giving the rise of the leaf, does not simply represent a similar relaxation, in the case of the pulvinus of Mimosa. But we have to notice that, in taking records with the Lever, the weight of the Lever ensures the indication of any passive relaxation of the specimen. If we inspect a Mimosa leaf, however, during the deathspasm, the leaf being free, i.e. unconnected with the Lever, we find that it, after its first fall, becomes again almost vertically erected, evidently in consequence, at least to some extent, of some process of active contraction, which must be that of the upper half of the organ. Had there merely been a general relaxation of the whole pulvinus, caused by death, then the weight of the leaf might have caused it to fail.

The slope of the curve of relaxation, again, is, generally speaking, relatively gentle. Its comparative steepness, in the case of Mimosa, after the passing of the death point, seems to indicate that the movement of relaxation was partially aided by later contraction of the upper half of the pulvinus. Constancy of death-point. — Before concluding the present chapter, I must refer to the remarkable fixity of the death - point in all the phanerogamous plants which have come under my observation in normal conditions. Thus, on repeating my experiments at the end of spring, by the perfected method of morographic record, I invariably found that the point of inversion was at, or within y1^ of a degree of, 6o° C. Other and less perfect modes of investigation, such as the spasmodic lateral movement of a dorsi-ventral organ, the movement of uncurling, the sudden expulsion of water, and those opening and closing movements of flowers which are to be described in the next chapter, enabled us to obtain deathpoints which were not very different from this. I give below

a tabular statement which makes it possible to see at a glance how concordant these results are. r f Coronal filament of Passiflora . ([Six specimens used. Each gave] (Style oi J/ibiscus ' ([Four specimens. Each gave] 1 Style of Datura ^ 1 [Four specimens. Each gave] Spasmodic lateral movement. Movement of uncurling. Expulsion of contained water. Opening or closing of flower. It will thus be seen that, using very diverse methods and specimens, we nevertheless always obtain a death-point which is very near 6o° C.

In radial organs, the death-contraction due to heatrigor is abrupt, and takes place at a definite temperature, which is to be regarded as the death-point. In the thermo-mechanical curve given by the Morograph, the point of inversion is the death-point. When death has taken place, a repetition of the experiment shows no inversion. The death-point, due to heat-rigor, in phanerogamous plants, under normal conditions, is found, though obtained by various methods, to be very close on 6o° G.

Under the action of continuous lowering of temperature there is produced, at a definite minimum degree, a spasmodic contraction, due to cold-rigor. The death-contraction in plants is in every respect similar to the same phenomenon in the animal, and is an instance of true excitatory effect. As in the animal, so also in the plant, this rigor of death is succeeded, after a time, by passive relaxation of the tissue. The thermo-mechanical curves of two similar specimens — that is to say, two specimens of the same plant, having the same previous history — are found to be practically identical.

Different plants have different characteristic curves. The curve of the same plant also exhibits a variation of its characteristics under changed conditions of age, experiment, and previous history. Lowering of death-point by fatigue — Modification of characteristic thermomechanical curve by the action of chemical agents — Comparison-Morograp h — Duplication of rigor-point — Deathresponse a physiological response and not due to coagulation — Death-movement of flowers — Approximate constancy of death-point of florets in a capitulum — Definite interval between death-point and discoloration-point — Translocation of discoloration-point by various agencies — Thermographs of regional death — Thermograph of local fatigue — Thermographic investigation of electrotonic excitation.

I HAVE shown that the death-contraction is a phenomenon of excitatory response. We might expect from this that various conditions which affect the excitability of a plant would also have a modifying influence upon the characteristic thermo-mechanical curve of deathresponse. One such modification would lie in the translocation of the point of inversion, or, in other words, in the displacement of the death-point. In order to test this inference we might subject the plant to the influence of various agents which modify the physiological condition, and observe the consequent modification of the death-response. We have already seen how the physiological modification induced by age causes displacement of the death-point. We have seen, further, how unfavourable seasonal conditions, such as sudden prevalence of cold, will lower the death-point by several degrees. We shall now study the effect of other agencies, such as fatigue, and the action of chemical reagents, in producing displacement of the deathpoint.

Effect of fatigue. — In the course of these experiments, fatigue was produced by means' of tetanising electric shocks, care being taken that these should not be strong enough to kill the plant. I have carried out experiments on this subject with two different classes of specimens : first, with anisotropic pulvinated organs, like that of Mimosa, where the death-spasm is shown by lateral movement ; and secondly, with radial organs, like the style of Datura, where the deathpoint is determined by thermo-mechanical inversion. In experimenting on Mimosa, I took a batch of young leaves of the same age, whose death-point was found to be at, or close upon, 590 C. After fatigue caused by moderate stimulation, however, the death-point was found to be at 560 C, that is to say, it had been lowered by 30.

Working with Datura pistil, the death-point of which was never normally lower than 6o° C, it was found when fatigued to be at 41 ° C. The lowering in this case was therefore about 1 90. It will thus be seen that fatigue does lower the death-point of a plant, the degree to which it does so depending on the extent of the fatigue. In the course of the present chapter, I shall be able to demonstrate once more the lowering of the death-point through fatigue, by means of an altogether different mode of investigation.

Effect of chemical reagents. — Similarly I find that death-response is modified by the action of various chemical reagents. We have seen how characteristic is the thermomechanical curve of each plant, under definite conditions We found, for example, that two styles of two different flowers on the same plant, having had the same previous history, gave curves which were practically identical. Specimens thus resembling each other are not difficult to obtain in spring, when there is no sudden variation of weather conditions, or from plants grown under glass, in an unchanging environment. The characteristics of the thermo-mechanical curve being so constant, the effect of a given agent will then be indicated by certain variations from the normal. Thus, on taking a thermo-mechanical curve — the specimen used being the style of Datura, subjected to the action of a 2*5 solution of copper sulphate — I found that the form of the curve was much

modified in contrast with the normal curve. The most striking difference lay in the lowering of the death point by Comparison-Morograph. — In order to facilitate the investigation into the modification of the curve, by various agents, I have devised a Comparison-M orography by means of which the thermo-mechanical curves of two similar specimens, one under normal and the other under modified conditions, can be taken simultaneously. We use here two recording Optic Levers, supported on a single thermometric helix. The normal specimen is placed in the internal cylinder in the ordinary way and attached to one of the Optic Levers. The second specimen, contained in a small cylindrical tube, filled with the given chemical reagent, is also placed inside the helix and the plant is attached to the second Lever. The spots of light are so adjusted that one lies immediately above the other. The two specimens are thus subjected to the same temperature-variations, and the variation of the second curve from the standard exhibits the effect of the reagent.

I can here barely indicate the very extended line of inquiry thus opened out. With regard to the general effect of drugs on death-response, it may be said that the displacement of the rigorpoint varies with the tonic condition of the tissue, the nature of the drug, and the strength of the solution. Out of several possible cases, I shall here give only a few simple instances. Duplication of rigor-points. — One very curious effect of certain chemical reagents, such as ether, lies in the exhibition of two distinct points of rigor, instead, as normally, of one. This effect is very easily seen, in the spasmodic deathresponse of Mimosa. We take a specimen, and subject it to continuous rise of temperature, in water, which contains a small quantity of ether. It will be remembered that, under normal conditions, the first down movement of the single composite spasm of death-response took place in young leaves at an average temperature of 59/5 ° C, the second

upward movement being at 6 17° C. Under ether, however, we have the peculiar phenomenon of two composite spasms separated by an average interval of about 27° C. This effect will be understood from the following table, which gives the results obtained with two different specimens, B and C, the specimen A being heated in water without ether, and thus constituted a standard. After application of ether (Leaf (1) young Bt Leaf (2) older After application of ether < Leaf ( 1 ) young C 1 Leaf (2) older

Another table is here given, showing the results obtained when the water contained a small quantity of hydrochloric From these results several interesting observations arise, the most striking of which is the occurrence of the preliminary spasm itself, separated by so large an interval from the final death-response. This duplication of the rigor-point is not a distinctive effect of the action of anaesthetics as such, since hydrochloric acid and various other chemical reagents give a similar result. It would be premature to pronounce on the significance of this very suggestive phenomenon.

A plausible suggestion, which offers itself, is that the approach of molecular rigidity concomitant with death, which here appears imminent by the action of the reagent, as seen in the preliminary spasm, is tided over, or counteracted, by the molecular mobility conferred on the tissue, through the rising temperature of the bath. Should this inference prove to be correct, these experiments might throw an interesting light on an ancient practice, still current in India amongst an old class of quack-doctors, by which cases of snake-bite are said to. be cured, under a treatment whose essential feature is the application of hot water and steam, with accessory incantations ! The same principle may also be the basis of the alleged hotwater and steam cures of more modern practitioners.

The duplication of the rigor-point by the action of ether is also noticeable in the thermo-mechanical curve given by a radial organ. Thus, in a curve given by the style of Datura, the preliminary rigor-point was found to be at 36*5° C, the second being at 530 C. As has been said, the effect produced by various poisonous reagents depends on the tonic condition of the tissue, as well as on the nature of the drug. In those cases in which the rigor is not duplicated, there is a translocation of the death-point, which, as far as I have yet seen, is invariably lowered. Thus, in an experiment already described with the style of Datura, I found this translocation, under the action of dilute copper sulphate solution, to be from the normal 6o° to 570 C.

Death-response not due to coagulation. — From the experiments which have been described, it is evident that the death-response, like other modes of excitatory response, is appropriately modified by all those influences which affect the physiological condition of the tissue. The rigor of spasmodic contraction at death is, therefore, not to be regarded as due to any coagulative action. And, indeed, the theory of a connection between rigor and coagulation is now generally discredited.1

By the methods described, then, it is possible to study the effect of various agencies in the modification of death-response : in the case of anisotropic organs, by observation of their lateral responsive movements, and in that of radial organs, by the translocation of the point of inversion. I was next desirous of discovering some still simpler means of determining the effects of various conditions in a qualitative manner. This might be accomplished if we had a number of organs exactly similar to one another, which would give some unmistakable sign of death-response, at the point of occurrence, either immediately, or at some definite interval afterwards. A certain number of these organs might then be taken as standard, and the others subjected to the action of various modifying influences. Any differences between the temperatures concomitant with post-mortem symptoms would now indicate the modifications produced s by these agents.

In a certain sense, such an experiment may be carried out with a number of leaves on the same plant of Mimosa. But in such a case the organs to be compared are not very numerous, and different leaves of exactly the same age cannot be secured. Death-response in flowers. — This led me to investigate whether, amongst flowers, specimens could be obtained which would exhibit a death-movement at the critical temperature. 1 'The causes which determine the varying resistance of different plants to heat are quite unknown. The fact that a temperature of from 200 C. to 400 C. kills certain plants, shows that in their case death is not the result of coagulation of the plant-albumin. Further, some plants grow at 750 C, i.e. above the temperature at which egg-albumin coagulates. Coagulation need not always occur, for we must remember that the acid and alkali albumins are not coagulated by heat.' — Pfeffer, Physiology of Plants, English edition, 1903, vol. ii. p. 230,

And I found that many flowers did so in a marked degree. Thus, for example, in two different specimens of Convolvulus, both full-blown, the flower being subjected to rising temperature, the corolla-bell folded up at exactly 62*5° C. We thus see the possibility of obtaining flowers which, having had the same previous history, are likely to exhibit the deathmovement more or less at the same point. I thought that such a collection of similar specimens might probably be obtained in a small space, from the capitulum of a compositaceous flower, and as a matter of fact I succeeded in finding several. The nature of the movement, whether up or down, and its more or less pronounced character, appeared to depend in these cases on the age of the flower.

In connection with this question, we must remember that in flowers, as in leaves, we may have in a single specimen alternating hyponastic and epinastic growths. It is therefore conceivable that the death-movements of old and young flowers may take place in different directions, and that at some stages there may be little or no motion of any kind. However this may be, I have found that in the double Indian marigold at the temperature of 62 5 ° C. the florets arranged themselves in two groups, the outer and lower whorls turning down, and the younger or central whorls rising up, at the critical temperature. In the case of some of the large garden daisies, yellow and white, I found the critical temperatures to lie between 61 '5° C. and 630 C, the deathmovement consisting of a folding up in some cases, and a curving down in others. If the flower have been subjected to uniform illumination on all sides, then the movement of all the florets will take place within a degree or so. In the French marigold, grown in India, the florets of the ray fold up, at from 590 C. to 6o° C. From these experiments we see that, the death-point for all the flowers on the same capitulum being about the same, it might be possible to treat one-half of the florets of a single flower-head as normal or standard, while using the rest for comparative study on the influence of various agencies.

have found out another and distinct method for detecting the effects of various agencies. And this method is not only very interesting in itself, but it enables other obscure problems to be attacked in a satisfactory manner. It depends on the taking of Thermographs of Regional Death. It is known that amongst the symptoms which occur at some indefinite interval after death is that of discoloration. Although this phenomenon is not concomitant with death, yet the temperature-interval between the two can in many cases be rendered definite. Thus for example, when the blue Convolvulus is subjected to rising temperature at the normal rate, it shows death-movement at 62*5° C. But there is as yet no sign of discoloration. When the temperature, however, rises to yo° C. the heating water begins to undergo discoloration from the escaping cell sap. It would appear probable, from various experiments which I have carried out, that discoloration does not begin at the point of death-contraction, but occurs at or about the point of the subsequent relaxation. But in the case of Convolvulus there is no striking change seen in the flower itself, for the loss of colouring matter is gradual. In the style of Datura alba, however, we have a more definite change of colour. This organ, from being milk-white, becomes brown at a temperature of 640 C, that is to say, 40 above the death-point, when the temperature of the bath is rising at the ordinary rate. In the petals of Sesbania coccineum, again, under similar conditions, the change of colour is very striking. Rich crimson here turns into pale blue, at a fairly definite temperature of 6y° C. The most marked and easily observed of all these changes is seen, however, in the mauve petals of Passiflora quadrangularis, which normally becomes colourless at a temperature of 700 C. The filamentous corona of the same flower again, in which the filaments are barred by purple rings at intervals, loses its colour normally at 68° C. The death-point of these filaments is, it should be remembered, 6o° C. We thus find on raising the temperature

in each of these cases, at the standard rate of i° per 15 minute, that not only is there a definite death-point, evidenced by sudden contraction, but that the discolorationpoint is separated from this, by a definite temperature-interval. And since we have found the death-point to be translocated by the influence of various agencies, we may expect the discoloration-point also to be displaced, in a similar manner, under the same conditions. Development of thermographs. — This being so, it ought to be possible to ' develop ' images of local death. We take a coloured petal, say of Passifloray and placing two circular electrodes diametrically opposite to each other, with the petal between, pass tetanising shocks, which are of sufficient intensity to fatigue, but not to kill, the tissue. When the electrodes are removed, there is nothing by which the eye may distinguish the zone of fatigue. In order now to develop this invisible picture, we have simply to subject the specimen to the ordinary bath, with rising temperature. For we have seen, from experiments already described, that the power of the tissue to resist death is lowered by fatigue. In the case of the present specimen, therefore, the fatigued area will die, and undergo subsequent discoloration, earlier than the rest of the petal. In carrying out this experiment, the area of fatigue was found developed as a white image on a purple background, at a temperature of 450 C. It should be remembered that, as said before, the lowering of the deathpoint varies with the amount of the fatigue ; hence the point of discoloration may be found as low as 400 C. or as high as 500 C. and upwards. If the petal be removed from the bath as soon as development begins, the image will remain. But if it be maintained under the rising temperature, the thermograph will vanish, with the death and discoloration of the background.

Again, I took two similar styles of Datura. One of these I kept as standard, and passed tetanising shocks through the other. On subjecting both to rising temperature in a single bath, the fatigued style underwent the change from white to brown at 560 C, whereas the test specimen was not discoloured until 64° C. Determination by thermographic method of relative excitatory effects of anode and kathode. — This thermographic method also enables us to attempt the solution of other recondite problems, such as that of the relative excitatory effects of the anode and kathode. It will be shown in the next chapter that when the electromotive force is not too excessive, it is the kathode which causes excitation when the circuit is made or completed. This fact will be demonstrated there by experiments undertaken with sensitive plants, in which the excitatory effect is indicated by the mechanical response of the motile leaflets. No such means is available, however, in the case of ordinary, or so-called non-sensitive, tissues. In such cases, therefore, I shall undertake to demonstrate the same fact, but by means of deathresponse.

We have seen that a tissue which has already been excited, is more fatigued than one which has not, and a fatigued tissue is, as we have seen, subject to death, and subsequent discoloration, at lower temperatures than the unfatigued. Hence, if excitation be caused in the kathodic region at make, death-discoloration ought to occur there earlier than in the anodic. This I have been able to demonstrate in the following manner : I took two similar petals, or two halves of the same petal, of Passiflora. The two were held side by side in a glass vessel full of water, at a distance of 3 cm. from each other, the temperature being gradually raised. When the temperature is about 500 C. a current is sent from a battery so that it enters by one petal, and leaves by the other. It is now found that the discoloration of death takes place earlier at the kathode than at the anode. The value of the difference is about 40 C. I carried out this experiment on the petals of the crimson Sesbania coccineum also.

The effect here described takes place, as has been said, where the electromotive force is not excessive. Under these conditions, it is the kathode which is the more excitable. I have, however, discovered a very curious case of inversion of excitation which occurs when the E.M.F. exceeds a certain value. With high electromotive force, then, it is the anode which excites at make of the circuit. The demonstration of this fact by means of mechanical response, and subsidiary proof by means of death-response, will be given in detail in a subsequent chapter.