Plant Response as a Means of Physiological Investigation

Response modified by physiological change— Carbonic acid causes depression, and transitory exaltation as after-effect — Gradual abolition of response in hydrogen and restoration by access of air — Chemical agents cause contraction or relaxation of plant-tissue — Effect of alcohol causing temporary exaltation of response followed by depression and protracted period of recovery — Ether causes relaxation and temporary depression of response — Explanation of anomalous action of ether on stimulated Mimosa leaf — Abolition of response by hydrochloric acid — Response restored by timely application of ammonia - Abolition of response by poisonous reagent — Similarity of effect of chemical agents on the response of animal and vegetable tissues.

The subject of our inquiry, in this and succeeding chapters, will be the determination of those influences which bring about the variation of conductivity and excitability in plant-tissues. The variation of excitability induced in a tissue by chemical reagents, may be studied through the consequent modifications of the responses. Various forms of response which may be used for this investigation are (i) electrical, (2) autonomous, (3) growth, and (4) the simple mechanical response about to be described. A description of the modifications induced in electrical response under the action of chemical reagents will be found on pp. 73-80 of my book, 'Response in the Living and Non-Living.' The effects of chemical reagents on autonomous pulsation and on growth are given in detail in Chapters XXV, XXVII, and XXXV of the present work. But it may be said here, that the results obtained by all these different methods are concordant, and that the simp'e method of mechanical response now to be detailed is to be regarded as an additional corroboration. It should also be borne in mind that the

effects of chemical reagents are subject to modification, as is fully explained later (pp. 322 and 477), by the tonic condition of the specimen. The method of procedure consists in first obtaining a series of responses to uniform stimuli under normal conditions, and then a similar series after the application of the reagent. The uniform individual stimuli in both series arc applied at intervals which allow of complete recovery. The intensity of stimulus, and the time-intervals, are kept constant throughout the experiment. The chemical agent may take the form of gas or vapour, or it may consist of a liquid. In the former case, by the turning of the three-way stop-cock described on p. 72, water-vapour is passed through the plant chamber, and the responses thus obtained are taken as the normal. By a quick manipulation of the stop-cock, the gaseous or vaporous reagent is next introduced, and the responses now obtained exhibit the physiological modification induced by it. The effect produced by some agents is permanent, by others transitory. This difference may be demonstrated by turning the three-way cock once more, so as to allow water-vapour to replace the specific gaseous medium. The responses now obtained exhibit the after-effect.

The difficulty in this investigation lies in the selection of specimens which exhibit complete recovery, together with uniformity of successive responses. For this purpose we may take any radial organ, one of the most excitable, and therefore suitable, of these being the filament of the corona of Passiflora. In this case high magnification is not necessary, and very moderate stimulus is sufficient. Failing Passiflora, I have frequently used with success the staminal filaments of the Uriclis Lily, and Brownea ariza, and the styles of Datura and Eucharis Lily. It should be borne in mind that the excitability of the tissue is to a certain extent influenced by seasonal conditions, being different under different circumstances of time and weather.

It is best to choose specimens from flowers which are already open, and in which growth has just ceased. Although, as already stated, this is not the most excitable period for the tissue, yet it affords us the advantage of simplified conditions, inasmuch as, owing to cessation of growth, the line of record before stimulation, or the base-line, now remains horizontal. Stimulation, producing contraction, is represented by up movements of response, and the recovery brings the curve back to the base-line.

Effect of carbonic acid gas. — I shall now proceed to describe a few typical experiments, on the modification of response by chemical reagents, out of a large number, which were performed on the radial organs of plants of various kinds, in the course of the season. And first we shall take the effect of carbonic acid. The effect of this gas, after the lapse of about half an hour, is one of considerable depression. By this time the responses are reduced to about half their normal value, and this depression,

though Slow, is continuous. FlG. 68. Effect of Carbomc IcuTds This may be looked for as on Longitudinal Contractile Response the general effect, after a {a) Normal response ; {6) after exposure . . , ,, r .' c to carbonic acid : (r) transient exaltagas. The immediate result of the sudden introduction of an abnormal factor may, however, be slightly different in different cases, according to the tonic condition of the tissue. This immediate effect is sometimes one of brief depression followed by equally brief exaltation, to be succeeded by the true depression. Or there may be a short exaltation, followed by the true depression. The restoration of the normal condition, however, is generally followed in the case of carbonic acid gas by a gradually increasing exaltation of the response, which may culminate in double its ordinary height, and after this it again attains the normal (fig. 68).

effect of hydrogen gas. The characteristic effect of depression occurs in this case after a much longer interval than is required by carbonic acid. The immediate result of application is very erratic and various. There may sometimes be an exaltation, or even the contrary, a reversal, of (a) Normal response ; (b) after twelve hours' exposure to H. (c) slow revival of response after readmission of air. the normal response. But after a twelve hours' exposure to the action of this gas the responses of the tissue arc so much diminished as to approach very near abolition. On now, however, allowing air charged with water-vapour to displace the hydrogen gas, the responses undergo a steady revival (fig. 69). There is here no sudden exaltation, such as is the general aftereffect of carbonic acid gas.

Effect of carbon disulphide. — We have seen that the depressing effect of hydrogen takes place very slowly. This is owing to the fact that this gas acts here rather as an agent for cutting off that supply of oxygen that is necessary to the maintenance of the normal life of the plant, than as a direct poison. But we have other gases which are actively toxic, and in such cases the diminution or abolition of response takes place Such an agent may be found in the

FlG. 70. Photographic Re- cord showing Effect of Carbon Disulphide in Abolishing Response Normal response, seen to left, abolished by introduction of vapour of CS2. I may here draw attention to the great advantage offered by the study of the variation of longitudinal response, in determining the nature of the action of various chemical agents. The modifications which these agents produce in the lateral response of the pulvini of sensitive plants are not so simple, inasmuch as we have to deal in these cases with differential action. In radial organs, on the other hand, the response-record gives us indications of the specific action of each modifying agent. In the response itself there are, it must be remembered, two factors which have to be distinguished, namely, contraction in response to stimulus, and the power of recovery from contraction, or relaxation. The diminution and final abolition of response may be brought about, then, in two different ways. The effect of a given agent may be to diminish the normal relaxation which brings on recovery. Successive stimuli will in that case produce a cumulative residual contraction, which places the tissue in a state of strain, in consequence of which subsequent responses become enfeebled. We may, on the other hand, have an agent whose effect is to produce abnormal relaxation. The contractile impulse due to stimulus is in this case opposed by the abnormal relaxation induced by the agent, and we have, in this case also, an enfeeblement and abolition of response.

The comparison of the time-relations of the normal and modified curves, together with the trend of the base-line up or down, will show the nature of the reaction, in an unmistakable manner. All these facts are clearly demonstrated in the experiments and curves given below. Effect of alcohol. — I shall next describe the action of the vapour of alcohol. Generally speaking, the immediate effect in this case is one of exaltation, though individual idiosyncrasies may sometimes be present, which cause depression from the very beginning. The general effect of this reagent, however, appears to be a prolongation of the period of recovery. So what is gained by brief exaltation is lost again by induced sluggishness. Thus, from the result

recorded on a fastmoving drum, using the style of Datura alba, I find that the height of the normal response was eleven divisions, and complete recovery took place in one minute and a quarter. During the first period of exaltation, after the application of alcohol, the height of the response was increased to sixteen divisions, that is, practically half as much again. But the period of recovery was protracted to four and a half minutes, or nearly three times the period of normal recovery. These considerations will fully explain the series of responses under the continued action of alcoholvapour, given in fig. 71, where (a) shows normal response, (b) the immediate and transitory exaltation, and (c) — which

(a) Normal response ; {b) immediate temporary exaltation on introduction was taken after fifteen minutes' further application — the diminished responses in which the contraction remainders are a marked feature. On blowing off the alcohol-vapour, however, and substituting fresh air, the tissue is found to recover slowly its normal excitability. If, instead of alcoholvapour, dilute alcoholic solution be applied, the depressing effect is immediate and very great.

Effect of ether. — We now pass on to the question of the action of the anaesthetic agent, ether. This produces a relaxation so great as to be incapable of proper representations within the limits of the diagram (fig. J2), where it is merely indicated by the dotted line. It is to be remembered that contraction is shown by lines upward, and recovery, or relaxation, by lines downward. Owing to the predominance of this relaxing tendency, it will be seen that true contractile movement is here very much diminished. Even after the relaxation has attained its maximum, the responses remain insignificant. When the specimen is not too long etherised, the blowing in of fresh air brings on gradual restoration. It may be mentioned here that ether also produces relaxation of animal tissue.

Explanation of anomalous effect of ether on Mimosa. — This experiment on the effect of ether affords a very satisfactory explanation of a phenomenon in the recovery of Mimosa, which has hitherto been regarded as anomalous, the leaflets is generally regarded as one of sensitiveness ; but, when they have just closed, in consequence of stimulation, if they be subjected to ether-vapour, they open out. Though now, however, mimicking the appearance of sensitiveness, they are in fact over-relaxed, a condition which is one of relative insensitiveness. The explanation may be gathered from the record in fig. 72. It is necessary here to give specific meanings to certain terms which have been used somewhat indefinitely. We know that stimulation causes the fall of the leaf, by differential contraction, and that the organ recovers, or ' relaxes/ into its original form after a period of rest. This term ' relaxation,' then, may be properly used as the converse of ' contraction.' But, in consequence of the expulsion of water from the organ after stimulation, it becomes flaccid, and this condition also is sometimes vaguely described as ' relaxed.' In my own use of the word, however, I shall confine myself to denoting by it that process which is the

Arrow marks moment of application. This produced relaxation and depression of response. Air substituted at x , and there is subsequent recovery of response. opposite of contraction, and which therefore brings about a position contrary to that effected by stimulus. We have seen that ether produces a relaxing effect which is more rapid than the process of relaxation that brings about recovery. And we have seen how, in consequence of this excessive relaxation, excitatory contraction is diminished or abolished. Hence, we see how a stimulated Mimosa leaflet under ether relaxes into an outspread position, which nevertheless is indicative of no renewed sensitiveness such as accompanies true recovery.

Effect of vapour of hydrochloric acid. — I shall now deal with the case of strongly poisonous agents, of which hydrochloric acid may be taken as typical. On passing the vapour of hydrochloric acid into the plant chamber there was produced a great relaxation, and the responses underwent a rapid diminution which ended in abolition. The effect of this poison is so persistent that the blowing-in of fresh air did nothing to revive the response. But the timely application of vapour of ammonia is found to act as an antidote, restoring the response (fig. 73).

Effect of chlorine gas. — This gas also produces a marked depression of excitability, which, under long-continued action, brings about the permanent abolition of response. The accompanying photographic record (fig. 74) shows the effect very clearly. The normal responses to the left are seen to be very rapidly diminished after the application of this gas, the response being reduced to oneeighth of its original value in the course of nine minutes. There are other important considerations in connection with this question, of relaxation

Arrow indicates moment of application. Depressing effect neutralised by antagonistic action of NH, at x . or contraction as the direct effects of chemical agents, which it would be out of place to treat in detail here. It need only be stated that these effects, which can be very accurately and continuously recorded by the arrangement ol the Optic Lever, are very suggestive. They are found to be modified by the tonic condition of the tissue, the strength of the agent, and the duration of application. Thus, an effect of relaxation may, after a time, pass into the opposite, of contraction. And since these relaxations or contractions of the tissue have a modifying influence on the response, much light on the obscure subject of the effect of drugs becomes possible through this study. I have been able already to obtain several curious and interesting results, of which I may here refer to one, in which two drugs, either of which when applied singly would abolish response, and produce death, are made, when applied in succession, to act as antidotes to each other. It is my intention to show in the course of this book that all the physiological phenomena of the animal have the closest correspondence with similar phenomena in the plant, and this being so, an investigation carried out on the lines indicated, with plants, is likely to be of very great importance, practically as well as theoretically.

Photographic record, showing normal effect to the left, depressed and almost abolished after introduction of the gas. The responsive contractions of an organ afford a reliable indication of the excitability of the tissue. The physiological changes induced in plant-tissues by the action of chemical reagents are outwardly manifested by modification of response. power of responsive contraction. Others produce the opposite effect, thus protracting the natural period of recovery.

Hydrogen gas produces a gradual diminution of response, which is restored to its original value on the readmission of air. Carbonic acid causes depression, but the restoration to normal conditions is generally followed by temporary exaltation above the normal. Vapour of alcohol causes gradual, and solution of alcohol rapid, depression. This action may be preceded by temporary exaltation. The recovery-period is very much protracted. As in the animal, so also in the plant-tissue, ether causes marked relaxation. The depression of response increases progressively with the exposure ; on blowing off the vapour, response is not only restored but may even show an exaltation. The opening of the Mimosa leaflets under ether is not indicative of true recovery but of over-relaxation.

A poisonous reagent, like hydrochloric acid or chlorine, permanently abolishes the response. Reagents which individually abolish response may, by their antagonistic character, act as antidotes to one another. Temperatures optimum, maximum, and minimum — Diminution of electrical response by cooling — Temporary or permanent abolition of response due to cold — Characteristic differences exhibited by different species — Mechanical response of Biophytum and autonomous response of Desmodium arrested by cold — Prolongation of latent period — Diminution of longitudinal mechanical response by cold — Diminution of electrical response of plants by rise of temperature — Similar diminution seen in longitudinal mechanical response — Increase of excitability due to cyclic variation of temperature.

ONE of the factors which modify response in plants is temperature. It is known in a general way that certain temperatures are favourable, and others unfavourable, to physiological activity. It is generally understood further that there is a certain optimum, in the case of each species, above or below which the excitability of the plant undergoes diminution. After this, on reaching a certain maximum or minimum temperature, as the case may be, excitability is abolished, and if these unfavourable conditions be long maintained the plant is apt to be killed. But the problem of the precise determination of such points has hitherto offered insuperable difficulties.

Effects of cold : (a) Diminution or abolition of electrical response. — Already, however, by adopting the electrical mode of investigation, I had been able to overcome these difficulties ; for I had found that the amplitude of the electrical responses, under different temperature-conditions, afforded a means of measuring the excitability of a tissue at the respective points. And now, by the use of mechanical response, I am enabled again to investigate the same problem by new and independent means. From the results obtained it will be seen that

each method furnishes a remarkable corroboration of the other. I shall now proceed to describe these various results. As an effect of low temperature I have found, by the use of the electrical method, that response undergoes a very great diminution. For example, the subjecting of a petiole of Euchari's Lily to a temperature of —2° C. almost abolished however, was restored to the normal temperature the original response reappeared, and sometimes with even greater amplitude than at first.

When the plant is maintained at a very low temperature for a considerable length of time, the normal electrical response disappears altogether, and the specimen undergoes permanent death. In this respect, different species of plants have characteristic powers of resistance. For example, the tropical plant, Eucharis Lily, after an exposure of twenty-four hours to a temperature of o° C, on being subsequently restored to its normal temperature, gives no sign of revival by response ; whereas the hardier Holly and Ivy, when subjected to the same treatment, do exhibit signs of renewed life (fig. 76).

{U) Prolongation of latent period, or abolition of lateral and autonomous responses. — Turning now to mechanical Lily by Lowering of Temperature (a) Normal response at 170 C. {b) The response almost disappears when plant is subjected to1 —2° C. for fifteen minutes. (c) Revival of response on warming to 200 C. response, I find in the case of the plant Biophytum that the effect of too great cold, or of long-continued exposure to low temperature, is the abolition of its lateral response. But when the temperature is, relatively speaking, only slightly lowered, induced sluggishness is shown in a very interesting manner. Whereas, at the normal temperature, say 230 C, the response of the leaflet of Biophytum takes place immediately, after moderate cooling, on the contrary, the latent

(a) Normal response ; (l>) response after subjection to freezing temperature for twenty-four hours. period is increased, and response does not begin to take place until from one to two seconds after the application of stimulus. Lowering of temperature also abolishes the autonomous response of Desmodium. (c) Diminution of longitudinal response. — With regard to longitudinal response, I have been able to demonstrate the effect of cold, by taking a specimen of the coronal filament of Passiflora. The specimen was kept for fifteen minutes in ice, after which electrical stimulus was applied with no immediate contractile effect. Control specimens, on the other hand, exhibited considerable contraction. These contractions were measured in both cases by means of a micrometer. The specimens taken for experiment were all 21 mm. in length. The average contraction of the control specimens was 1*5 mm. The cooled specimen, as said before, exhibited no immediate response. But on continuing stimulation for two minutes, contraction began to take place slowly, reaching a maximum of only '5 mm. It must be borne in mind

that the specimen was all this time undergoing gradual warming by the temperature of the room. Another way of demonstrating the effect of cold on the longitudinal response of this corona, is to take three filaments ; of these {a) has been subjected to cold, (b) is normal, and (c) has been killed by immersion in hot water at 6o° C. The righthand ends of the filaments are so arranged as to lie perfectly even, and a moist cotton thread touches these ends. A brass spring presses against the left-hand ends. The thread and the brass spring serve the purpose of two electrodes, by which shocks can be sent through the three specimens at the same time.

The right-hand end of the object, so arranged, is placed within the field of a microscope of low magnifying power which has a micrometer eye-piece. At the beginning the ends of the specimens lie in a straight line, but after the passage of a shock for a period of a few seconds it is found that, while the normal (J?) shows the maximum contraction, the cooled (a) exhibits very little, and the killed (c) none at all. The difficulty in conducting experiments on cooling, lies in the fact that the inertness due to cold is liable to disappear with more or less rapidity when the specimen, for experimental purposes, is exposed to. the temperature of the room, which is about 230 C. It cannot be kept immersed in icecold water, as the exciting current will then to a great extent pass through the conducting water itself. The only practicable way, therefore, is to subject the specimen to prolonged cold, and make a rapid observation afterwards.