Plant Response as a Means of Physiological Investigation

The curve showing the relation between stimulus and response is appropriately modified by the tonic condition of the tissue. As each stimulus of every form thus finds expression in direct response, direct after-effect, and indirect after-effect, and as there are many forms of stimulus which under natural conditions act on the plant, whose maxima, instead of being coincident, are superposed on each other, in varying differences of phase, highly complex periodicities are induced, and find expression in the various forms of plant response.

Among such varying factors of stimulation may be mentioned the diurnal alternation of light, temperature, chemical stimulus, and varying turgescence. The induced periodicities which result from the conditions described may be seen in the periodic groupings which appear in a continuous record of the autonomous pulsations of Desmodium for example. The autonomous response of growth, as the result of the periodically acting stimuli mentioned, exhibits not only a large wave of alternation, due to the diurnal period, but also a number of sub-waves. But the impression made on the organism by the diurnal period is the deepest of these, and tends in an old plant to subordinate all others in a marked degree. In the responses of a seedling a few days old, however, the minor waves are very distinct.

General consideration of the problem — ' Constitution ' and the elements which determine it — Methods of investigation— Action of carbonic acid gas— Action of ether — Effect of solution of sodium carbonate — Effect of solution of sugar- Effect of alcohol— Effect of acids — Effect of alkali — Antagonistic action of alkalis and acids— Action of strong solution of sodium chloride — Effect of poisonous solution of copper sulphate — Opposite effects of the same dose on different constitutions — Opposite effects of large and small doses.

We have already studied the effect of various chemical agents on the physiological condition of plants, as seen from the modifications induced by them in ordinary and in autonomous responses. We have also noticed the remarkable similarity between these effects in the two cases of plant and animal tissues, and I have drawn attention to the great practical utility of these investigations, inasmuch as the experiments carried out on plants may be made to throw light on many obscure phenomena regarding the effects of drugs on the animal system. One puzzling fact, however, which is encountered in medical practice, is, that the same drug will often produce varying effects on different individuals ; and this is vaguely ascribed to dissimilarity of ' constitution.'

Similarly, we have sometimes seen different effects to occur in plants under the action of a single given drug. A large dose of some depressing agent, for example, though it will ultimately produce the depressing effect, will not always do so at once, for in some cases there will be a preliminary period of exaltation of response. The same toxic dose, again, which will in some instances kill the plant, will in others fail to do so, the plant being ultimately able to shake off the depressing influence after an interval of struggle. In studying suctional response, again, we found that copper sulphate applied at the roots induced, in some cases, an immediate arrest of suction, while in other instances this arrest did not take place till after a long time. The depression of suction which was induced by the application of strong sodium chloride, again, was in some cases immediate, and in others preceded by a fairly long period of an exalted rate of suction (pp. 384, 385). All these variations of results we regarded as due to individual differences of constitution, or of tonic condition.

Thus we can only hope to arrive at a complete knowledge regarding the action of any given drug if we first obtain a precise understanding of what is meant by constitution, and of how, for experimental purposes, specimens of a definite characteristic constitution can be secured, while others can be subjected to an ascertained variation in a pre-determined manner. It will thus be made possible to study the effect of a drug, by applying it (1) in solutions of different strengths to a number of plants of identical constitution ; and (2) in a single strength of solution to specimens of definitely varying constitutions.

' Constitution ' and the elements which determine it. — The first factor in determining constitution will consist of those properties which have been impressed upon the plant by its heredity. The second will depend upon its environment. The sum total of the energy absorbed by the plant from its surroundings we have already designated as the tonic condition. It is clear that we may secure the factor of a constant heredity by taking either seedlings from the same batch of seeds, or organs from the same plant. These, again, when maintained under the same environmental conditions, in respect of temperature and other circumstances, will give us plants having practically the same constitution. In order, next, to obtain specimens of different but well-ascertained constitutions, it is only necessary to keep these under known

fferences of tonic condition. Thus, all other factors being maintained constant, we may have three clearly defined states, in the case, say, of Crinum Lily, according as it is kept at a temperature of 300 C, 340 C, or 370 C. The first of these we may regard as the normal ; the second as near the optimum ; and the third as intermediate between optimum and maximum. The excitability of the plant kept at the optimum will be the greatest ; but though the excitabilities of those at 300 C. and 370 C. will be approximately the same, yet, in the latter case, the plant will possess an excess of latent energy which will be wanting in the former. Having thus secured these definite artificial constitutions of different values, some of the investigations given at the end of this chapter will show how free from uncertainty the action of drugs may be made, and how rational an explanation can be given of the observed variations of effect.

Methods of investigation. — I shall now proceed to describe the general methods of experiment, in studying the effects of drugs, from the modifications which they induce in growth-response. There are two ways of doing this. According to the first, we take a record of the growth before and after the application of the reagent. From the variation then seen in the rate of growth the excitatory or depressing nature of the drug may be ascertained. The second, or Method of the Balanced Crescograph, is much more delicate ; it exhibits each transient variation of response, and its timerelations, with perfect clearness. The balanced horizontal record, which is first taken, indicates the normal rate of growth. A deviation upwards from this horizontal line will indicate accelerated growth ; a return to the horizontal will mean a regaining of the normal rate ; and a deviation downwards will show responsive retardation. The specimens used for this investigation were Crinum Lilies, and, unless stated to the contrary, the experiments were carried out at the normal temperature of 300 C.

Action of carbonic acid gas. — I first give a record of the effect of carbonic acid gas on growth (fig. 192), taken by the Unbalanced Method. The normal rate of growth was •006 mm. per minute. On passing C02 into the plant chamber the immediate effect was an acceleration, the rate for the next five minutes being '009 mm. or i^ times the normal. Under the continued action of carbonic acid, however, the rate underwent a rapid diminution, and, as is seen

by the slope of the curve becoming horizontal, growth was arrested fifteen minutes after the introduction of carbonic acid into the plant chamber. On the re-introduction of fresh air the growth was slowly renewed, and gradually returned to its original rate. The effect of carbonic acid on growthresponse, then, is a preliminary exaltation, followed by depression and arrest, which arrest, if the action be not too long continued, proves to be only temporary. Action of ether. — This experiment shows the curious difference of results which occurs, according as an application is external or internal. In this and in the following cases, with the exception of the experiments on acids and alkalis, I shall use the Balanced Method, as this brings out even transient variations in a very striking manner. The balanced horizontal record is seen to the left of each figure. In curve a of fig. 193 is shown the effect of an external application of this reagent, ether vapour being introduced into the plant chamber. It will be seen that there was an immediate retardation of growth, which lasted for more than a minute. This was followed by an acceleration of growth, which lasted for two minutes. There was then a depression, which continued, and culminated in the arrest of growth.

The first arrow indicates introduction of C0.2, which induces preliminary enhancement of growth, followed by subsequent arrest. The second arrow indicates the introduction of fresh air, followed by the revival of growth. Record taken by Un- balanced Method. In b is shown the effect of an internal application, made by replacing the water, by which the cut end of the stem was normally surrounded, with a 3 per cent, solution of ether. It will be seen that, as the specimen sucked up the solution, the immediate effect of this internal application was an enhancement of the rate of growth, and that this was followed by depression, leading to arrest of growth. The preliminary depression seen in a is thus wanting in the case of the internal application. It might be thought that this first depression, as seen in the case of the external application, was due to a slight cooling, caused by the introduction of ether. We know, however, that while

lowering of the temperature, as long as that is below the optimum, would suffice to retard growth, a similar lowering of it when above the optimum would have the opposite effect of acceleration. Now I find that this preliminary retardation of growth, seen in fig. 193, a, takes place in exactly the same manner when the experiment is repeated with the specimen at 380 C. It cannot, therefore, be due to the suggested cooling, which must in any case, under the experimental conditions, have been extremely slight. There is, however,

another explanation, which more nearly meets the requirements of the case. We know that any sudden variation of environmental conditions is apt, generally speaking, to act as a stimulus, and the effect of direct stimulation is always to Fig. 193. Balanced Records ot Effect of Ether on Growth. Up Curves represent Acceleration Above, and Down Curves Re- tardation Below, the Normal a Effect of external application, and b of internal application of ether. Successive thick dots in the baseline indicate time in minutes in this and following records. Arrow shows moment of application.

Fig. 194. Excitatory Effect of Dilute Solution of So- dium Carbonate on Growth induce contraction, which would in the present case take the form of a transient retardation of growth. Effect of solution of sodium carbonate. — In the course of the following experiments the chemical reagents are administered internally, by applying the solution at the cut end of the specimen. A dilute solution of sodium carbonate is known to increase excitability in the case of animal tissues. I find that this holds good in the case of the growth-response of plants, growth being accelerated, as will be seen in fig. 194, where the balanced record suddenly gives place to an ascending curve. But in the case of chemical reagents in general, and of this especially, it must be remembered that the strength of the solution, or dose, is an important element in the result. A *5 per cent, solution of sodium carbonate was always found in these experiments to be an excitant ; but as the strength of the solution was increased, the excitatory effect was found to be gradually diminished, until at

2 per cent, it became neutral. If now the strength were still further increased, say to 5 per cent., the effect was an actual depression of the rate of growth. Effect of solution of sugar.— I next give a record (fig. 195) showing the effect of the application of sugar Fig 195 Acceleration of in a 2 per cent, solution. This is tion of growth. In the case of the Crinum Lily, this acceleration is found to occur even under a 5 per cent, solution of sugar; but very much stronger solutions induce depression.

Effect of alcohol. — We have hitherto observed different reagents inducing a more or less uniform acceleration or depression. In the case of alcohol, given in 5 per cent, solution, however, we obtain a very curious instance of alternating spasmodic effects (fig. 196) ; that is to say, the growth at one moment exhibits a sudden acceleration, and at the next a sudden depression, such alternations being continued for a considerable length of time. On repeating the experiment at the higher temperature of 340 C. I found that these spasmodic alternations became still more violent — that is to say, of greater amplitude, though less frequent. At a much higher temperature, however, the effect of alcohol was an immediate depression. Still stronger solutions caused arrest FlG' 7r sPasm°dic Alternations

Effect of acids. — We found, in the case of the autonomous responses of Desmodium, as of cardiac muscle, that acids induced relaxation, and that the long-continued action of such a reagent, or a strong solution, would bring about arrest in the relaxed position. Curiously enough, then, in the case of growth, which I have shown to be an instance of multiple or autonomous response, I find an effect exactly parallel. In order to hasten the result I used a somewhat strong solution, namely 4 per cent, of hydrochloric acid. This, as will be seen (fig. 197), caused a marked relaxation, and growth came to a standstill some six minutes afterwards. In this experiment, and the following on the effect of alkali, the record was taken under unbalanced conditions.

Effect of alkali.— The effect of alkali in the case of Desmodium, and also of cardiac muscle, is to produce arrest in the contracted position ; a similar effect is strikingly exhibited in the growth-record given in fig. 198. The first part of this record shows the normal rate of growth. A 3 per cent, solution of sodium hydrate was then applied at the point marked with the downward arrow {}). It will be noticed Fig. 197. Unbalanced Record showing Ac- tion of Acid in Causing Relaxation and Ultimate Arrest of Growth

that this induces a very great contraction, and that in the course of seven minutes there occurs an arrest of growth, in a contracted position. It will also be seen that the specimen, after the application, actually became shorter than it had been before. The alkali therefore had the effect not merely of arresting growth, but also of causing an active contraction of the tissue. Antagonistic action of alkalis and acids. — We saw that in the autonomous responses of Desmodium and cardiac muscle, the state of standstill induced by the action of either acid or alkali was neutralised and counteracted by the antagonistic action of the other (cf. fig- 1 5 5)- I have detected precisely the same peculiarity in the case of growth-response also. It was seen in the course of the last experiment that growth was brought to a state of standstill, in the contracted position, by the action

Fig. 198. Unbalanced Record showing the Action of Al- kali, and the Antagonistic Action of Subsequent Application of Acid The downward arrow ( I ) indicates application of alkali, which induces arrest of growth in contracted position. The upward arrow ( t ) indicates application of acid, which by its antagonistic action renews growth. . Fig. 199. Effect of Strong Solution of NaCl on Rate of Growth, as Modified by Different Constitutions of Specimens

At a temperature of 300 C. there is an immediate depression. Near the optimum there is a well-marked resistance, and preliminary acceleration before depression sets in. The same is true to a less extent at 370 C. These records were taken under balanced conditions. At 44° C. there was normally no growth, but this was temporarily initiated under the action of strong NaCl solution. Record taken by Method of Balance. of alkali (fig. 198). At this point, as marked by the upward arrow (f), acid was applied. This reagent now had the effect

as will be seen from the record, of neutralising the previous contractile arrest, and in the course of two minutes it had brought about the renewal of growth. Action of strong solution of sodium chloride.— In the course of the investigation on suctional response we noticed that the effect of this reagent, when applied to the root, was not always to cause a diminution of suction, as might have been expected had osmotic action been the only factor. This reagent, on the contrary, usually operated to bring about a preliminary acceleration of suction. And I have already explained that this was due to its excitatory character, acting in opposition to the osmotic action set up by the strong solution. It is usually supposed that the excitatory effect of strong solution of salt on animal tissues is due to the osmotic withdrawal of water ; but we have here seen that similar excitation is induced in vegetable tissues without any withdrawal of water. Hence the usual theory of the action of salt solution in causing excitation is rendered very doubtful. We found, in fact, that when the tonic condition of the tissue was favourable, the excitatory reaction predominated, and there was a consequent enhancement of suctional activity. In other instances, where the tonic condition was less favourable, osmotic action predominated, and there was a consequent depression of the normal rate of suction (p. 385). It is thus seen that the factor of variation in these two different cases was the constitution, or tonic condition, of the plant ; we are now able to study with precision the influence exercised by constitution, in causing the plant to struggle against, or succumb to, the action of adverse external circumstances.

The action, in causing variation of suction, of a strong solution of salt, applied internally through the cut end of the stem, can also be studied by means of growth-response ; for while increased suctional activity will give rise to a positive turgidityvariation, with concomitant enhancement of the rate of growth, diminished suctional activity will have the opposite effect, of retarding the rate of growth. We shall next observe the effect of this reagent on different specimens

of Crinum Lily, which had been taken from the same flowerhead, and in which different constitutions were artificially induced by carrying out the experiments at 300 C, 340 C, 37° C, and 440 C. The records were taken under balanced conditions. On making an application of a solution so strong as 10 per cent, to a specimen at the normal temperature of 300 C, the result was always a depression which set in immediately ; but when the tonic condition of the plant was exalted, by raising the temperature to a point near the optimum — that is to say, to 340 C. — the same reagent was found to induce an excitatory effect, its depressing action being postponed for a considerable length of time. The effect obtained at the temperature of 370 C. was very instructive. Responsive excitability at this temperature has been shown to be almost the same, if not indeed slightly lower, than that at 300 C. But we should remember that in these two cases we have very different histories ; for if in the former — that is, at 300 C. — we have a normal amount of latent energy, then it is clear that in the latter — that is to say, at 370 C. — there must be an excess of latent energy. And as the result of this we see that, while at 300 C. the growth-response showed immediate depression, at 370 C. it offered a considerable resistance, as seen in the temporary exaltation of response. Still more interesting, however, was the effect at 440 C. At this temperature it will be remembered that there was an apparent arrest of growth, often supposed to be due to the setting-in of heat-rigor. I have shown, however, that, so far from this being the case, there is still at 440 C. a good deal of rhythmic activity, the cessation of growth being due to the fact that in the multiple response of growth each constituent response and recovery had become equal. The presence of this activity at 440 C. becomes quite clear, when we find that the application of salt at this temperature has the effect of renewing for a time the resultant growth which had been in abeyance (fig. 199).

Effect of poisonous solution of copper sulphate. — The influence of constitution in determining resistance to adverse Fig. 200. The Effect of Different Constitutions in Determining the Resistance Offered to Poisons. The Action of 5 per Cent. Solution of Copper Sulphate circumstances is made still more striking when we observe the action on the plant of strongly poisonous reagents, such as 5 per cent, solution of copper sulphate. At the normal temperature of 300 C. its application, as will be seen in the record (fig. 200), induces a very rapid depression, which soon culminates in permanent arrest. But at 340 C. the resistance offered is considerable, actually exhibiting itself in temporary exaltation. At 440 C, again, we observe the poisonous reagent actually initiating growth, as in the last case.

Opposite effects of the same dose on different constitutions.— In the case last considered, we studied the phenomenon of the resistance offered by the plant to a toxic dose largely in excess of the fatal amount. In spite of the excessive dose we found that, when a favourable constitution was artificially induced, the plant succumbed, it is true, but only after considerable struggle. I shall now proceed to show how, when the artificial constitution is sufficiently favourable, the plant, instead of succumbing to an ordinarily fatal dose, can shake off the effect, and may even be stimulated by it. Thus I found that a I per cent, solution of copper sulphate induces depression and is ultimately fatal at 300 C. ; but

when the same dose is applied to a plant at 340 C. the effect is seen in a marked exaltation (fig. 201), which continues for a fairly long period, after which it shakes off the effect of the poison altogether, and resumes its normal rate of growth. Fig. 201. The Effect of Favourable Induced Constitution in Enabling Plant to Shake off Result of Toxic Dose of Copper Sulphate Opposite effects of large and small doses. — I have shown the toxic effect of a I per cent, solution of copper sulphate at the normal temperature of 300 C. If, however, the dose be reduced to "2 per cent, we shall find that its action becomes stimulatory (fig. 202). This is an interesting illustration of the general fact that a poisonous reagent, if given in sufficiently minute doses, will act as an excitant.

In conclusion, a survey of the effects of drugs, both stimulatory and poisonous, reveals the striking fact that the difference between them is a question of quantity. Sugar, for instance, which is stimulating when given in solutions of, say, 1 to 5 per cent., becomes depressing when the solution is very strong. Copper sul- Fig. 202. Opposite Effects of phate again, which is regarded A solution of 1 per cent, copper cent- and Upwards, a solution of

tween sugar and copper sulphate is here seen to lie in the fact that in the latter case the range of safety is very narrow. Another fact which must be borne in mind in this connection is that a substance like sugar is used by the plant for general metabolic processes, and thus removed from the sphere of action. Thus continuous absorption of sugar could not for a long time bring about sufficient accumulation to cause depression. With copper sulphate, however, the case is different. Here, the constant absorption of the sub-toxic stimulatory dose would cause accumulation in the system, and thus ultimately bring about the death of the plant.