Plant Response as a Means of Physiological Investigation

The death-point is lowered by fatigue, the amount of lowering depending on the intensity of fatigue. The characteristic thermo-mechanical curve is modified and the point of inversion translocated by the action of chemical reagents. The death-point is translocated to a temperature lower than normal by the action of poisonous reagents. Under standard conditions, there is a definite interval between the death point and discoloration-point of vegetable tissue.

Hence it is possible to obtain thermographs of localised effects of various agents. The excitatory effect of kathode is demonstrated by the earlier discoloration produced there. Hydro-mechanical theory of excitation in plants — Theory of protoplasmic change — Crucial tests applied by means of polar excitation — Mono-polar and Bi-polar methods of excitation — Advantages of study of polar excitation in plant-tissues as compared with animal — Effects of feeble E. M.F. — Effect of moderately high E. M.F. — Experiments with highly excitable tissues.

Having observed, by means of mechanical responses, the various excitatory effects which are caused in plants by stimulation, and the influence of different agencies in modifying these excitatory effects, it is now desirable to make an inquiry into the manner in which excitation takes place, and into the method by which it is transmitted to a distance. There has been a great deal of uncertainty regarding this subject, and the prevailing view is that which holds the transmission of excitation to be due to the propagation of hydrostatic disturbance.

Mechanical theory. — According to this theory, it is supposed that stimulus causes a mechanical disturbance, bringing about an alteration of the hydrostatic equilibrium. The propagation of excitation in plants is thus regarded as nothing more than the transmission of this hydro-mechanical disturbance. We know, however, that the transmission of hydrostatic disturbance takes place with relatively great rapidity, while these excitatory effects in the case of plants travel sometimes as slowly as I mm. or less per second. I have shown, moreover, that its responses, both mechanical and electrical, are profoundly modified by the physiological condition of the plant. There is, for example, an optimum temperature at which response is at a maximum, any change, whether above

or below, inducing depression. Anaesthetics, moreover, temporarily, and poisons permanently, abolish response. It will be shown further, in Chapter XVIII, that the transmission of excitation may be very much diminished, or even arrested, by the application of cold or ether. Theory of protoplasmic change. — It is thus seen that the hydro -mechanical theory is incapable of explaining the facts of the case. I shall now, therefore, proceed to demonstrate that the excitatory change in plants is brought about in the same manner as in animals, and that the transmission of excitation depends upon the propagation of protoplasmic changes, in the one case as in the other. This may be determined by a crucial experiment as to whether vegetable tissue exhibits those peculiar polar effects of the electric current on excitability, which are seen in the protoplasm of animal tissues. In the animal tissue, for example, it is the kathode that, under normal conditions, produces excitation, the effect of the anode being the reverse. In the case of animal tissues, again, the anode will even act as a block to the transmission of stimulus.

Crucial tests applied by means of polar excitation. — Such effects are incapable of explanation by the hydromechanical theory, and if we succeed in discovering similar phenomena in the case of vegetable tissues, we shall establish the existence of a fundamental property of protoplasm common to the animal and vegetable alike. With this end in view I have carried out numerous experiments on plants, both sensitive and ordinary. As specimens of the former class, I used Biophytum, Mimosa^ and Averrhoa. The investigation resolves itself into the determination of the differences of excitatory effects, at the anode and kathode, both at make when the circuit is completed, and at break when it is interrupted. The presence of the excitatory effect is indicated in the case of ' sensitive ' plants by the mechanical responses of the motile organ. In order to separate the effects of the anode and kathode, we may use the Mono-polar method, i.e. have one electrode near a motile organ, and the

other very distant from it (fig. 89). If the plant is not veryexcitable the effect produced at the distant point will not reach the motile organ, and we shall obtain the isolated effect of a particular electrode. Again, if we wish to observe the effects at both the electrodes simultaneously, we may employ the Bi-polar method, in which both electrodes will be placed at or near the motile organs. The most suitable means for the application of electrical stimulus will be either a constant electrical current from a voltaic battery, or the discharge from a charged condenser.

We have again to study the respective effects of feeble, moderate, and excessively strong electromotive forces, In experimenting on polar excitation in animal tissues, a nerve-and-muscle preparation is generally used, the excitation of the nerve being studied by means of the indication given by the terminal motile organ, the muscle. On the other hand, experimenting on Biophytum for instance, the petiole acts as the conductor of stimulus, and is provided withnot a single terminal motile organ, but — a number of lateral motile organs, viz. the ' sensitive ' lateral leaflets. The analogous case in animal tissue would be a hypothetical nerve, provided with a hypothetical series of contractile muscles attached to it laterally. The relative advantage possessed by such a vegetable organ is, that the changes in the excitabilities, throughout every portion of the excitable conducting tissue, are visibly manifested.

I experimented altogether on some hundreds of specimens. Some of these were very sensitive ; others only moderately so. The results under normal conditions were perfectly consistent. As it would entail much mere repetition to relate every one of these experiments, I shall here give only typical instances in detail. While I was studying the effect of the establishment or cessation of a constant current I made a practice — whenever the leaves or leaflets recovered within a moderate time from the effects of the stimulus of a current flowing in one direction — of trying a second experiment on the same plant, by reversing the direction of the current, sq

that in the reversal experiment, what was formerly anode became kathode, and vice versa. In this way corroborative reversal effects were obtained. In experiments with condensers it was not necessary, in order to reverse the electrodes, to reverse the battery connection, for owing to the special arrangements of the electric circuit (fig. 14) the anode at 'charge' became kathode at ' discharge.' In studying the effects of increasing intensity, in the case of constant current, I simply add to the number of storage cells, and in this way obtain increasing voltage. The strength of the condenser discharge is increased by increasing the voltage of the charging circuit. With the same tissue, where the resistance is constant, the current increases with the acting E.M.F. Hence, increasing E.M.F. here connotes also increasing current. But we may have a very high E.M.F. and, owing to high resistance of the tissue, only a feeble current. From the trend of the various experiments that I have carried out, it would appear that the characteristic polar effects are determined more by the intensity of the E.M.F. than by that of the current. In the case of the present investigation, as we must also bear in mind, the experiments were performed with many different specimens, the excitability of some being greater than that of others.

Effect of feeble E.M.F. — The first experiments of this series were carried out by the method of mono-polar excitation. The first specimen employed was Mimosa, one electrode, the kathode, being connected with the pulvinus, and the anode, at some distance, with the main stem. The electromotive force used was ten .volts. The leaf-stalk fell at make of the circuit. The leaf was found to recover, after a due interval, during the continuation of the current. The current was now broken, but this produced no responsive effect whatsoever. The current was next reversed, the pulvinus being made anode. But this anode-make did not produce any excitatory effect, neither did the succeeding anode-break. From these experiments we see (a) that a feeble E.M.F. excites at the kathode at make ; (b) that the excitation takes

place during the variation of current, but not when the current has attained a constant value ; and (V) that there is no excitation at either kathode-break or anode-make or break. I next used a specimen of Biophytum, the E.M.F. employed being eight volts. The kathode was at first at the lower end of the leaflets, the anode being on the main stem (fig. 89). At make there was an excitatory wave at the kathode. This travelled outwards and produced depression of four pairs of leaflets. On reversing the current, the new anode did not produce any effect at make, nor did its break produce any excitation. It will be shown presently that it is necessary to have a certain moderate intensity of current in order that the anode-break may cause excitation.

The effect on Biophytum of a continuous current at the kathode is to bring about a more or less prolonged 'contraction/ the period of recovery being thereby much protracted. With the leaf-stalks of Mimosa, however, the effect is not so marked. With this plant, nevertheless, I have been able to observe certain antagonistic effects of anodic and kathodic actions ; that is to say, while there is slow recovery from kathodic contraction, on reversing the current, there is often an impulse of relaxation, the recovery being thereby suddenly hastened. But it must be understood that these particular effects are liable to modification, being dependent on the physiological condition of the tissue.

I next repeated these experiments on the effect of feeble E.M.F. producing excitation by means of condenser discharge. The results obtained were precisely the same as with constant current. That is to say, with relatively feeble charge, the excitation took place at the kathode at make, and not at the anode. The great advantage of excitation by the method of condenser discharge is, that the total Fig. 89. Diagrammatic representation of Mono-polar Excitation

B was kathode, which at make gave rise to excitatory wave, causing depression of four pairs of leaflets. The electric connections are always made with nonpolarisable electrodes. quantity of electricity passing through the tissue is very small, and the changes produced in the substance of the specimen are therefore slight. The next group of experiments was carried out by the bipolar method of excitation, which enables us to make simultaneous observations of the effects at anode and kathode. As in the previous cases, the specimen used for the first experiment was Mimosa, the E.M.F. employed being twelve volts. Connections were made with the pulvini of two neighbouring leaves (fig. 90). On make, the kathodic leaf-stalk fell ; there was no action at the anode. At break, there was no action. On now reversing the electrodes and making the anode kathode, the leaf-stalk which had not previously responded fell under kathodic excitation. There was no effect on the anodic leaf-stalk, nor was there any effect on either at break.

volts, the electrical conthe diagram (fig. 91). On completing the circuit, the excitation was discharged at the kathode, and the wave proceeded in both directions from the kathodic point, three pairs of leaflets being depressed towards the stem and two in the interpolar region. There was no effect at break at either electrode. On reversal, the new kathode, formerly the anode, became the point of excitation, as evidenced by the depression of contiguous leaflets.

Similar results were obtained when excitation was produced by condenser discharge. Thus with a condenser having a capacity of 'Oi microfarad, charged to eight volts, At make an excitatory wave proceeded in two directions from kathode, but none from anode. response was observed at the kathode at charge. The excitatory wave travelled in both directions, and five pairs of leaflets were depressed. There was no effect at the anode. At discharge, the former anode became kathode, and there was a responsive movement of the leaflets near that point.

In the following table, the characteristic polar effects of feeble E.M.F. may be seen at a glance. Effects of moderate E.M.F.— From this point onwards it will be found sufficient to describe the results obtained by means of the bi-polar method of excitation, this mode of investigation being With regard to the first of these, a number of experiments were performed on a single specimen of Biophytum, using an E.M.F. of 24 volts. The excitatory wave at make was found to be initiated at kathode, and to. travel both directions,

{a) Shows effect at make, excitation being produced at kathode ; {b) shows effect at break, excitation being now produced at anode. leaflets. The forward half of this wave of excitation only stopped at one pair of leaflets before the anode (fig. 92). This, as will be seen later, is due to the depressing action of a strong anode. There was no action at the anode itself at make ; at break, there was no action near the kathode, but there was excitation at the anode, as was shown by the fall of three contiguous pairs of leaflets. The direction of the current was now changed, the poles being thus reversed, and eight pairs of leaflets fell at the new kathode, in and out. There was, however, no effect at the new anode at make ; but at break, the reverse was the case, leaflets falling near the anode, and no response occurring at the kathode. This experiment was repeated three times on the same specimen, and the results were in every case similar. I give (fig. 93) a pair of records

Fig. 93. Records of Responses of Leaflet of Biophytum, showing Responses occurring at Kathode at Make and not at Break ; and at Anode at Break and not at Make in Biophytum leaflet, showing the opposite character of the effects of make and break at the anode and kathode respectively. The E.M.F. used in this particular experiment was sixteen volts. In the former series of experiments, it was seen that there was no break-anode effect when the E.M.F. was feeble. The present experiments show us that the breakanode is effective when the E.M.F. is moderately strong.

I obtained similar effects when stimulation was produced by means of condenser discharge, the experiments being carried out on Biophytum. From the investigation just described, it will be seen that with moderate E M.F. we obtain response from the tabular statement exhibits these various effects in a concise Experiments with highly excitable tissues.— In experimenting on the polar excitation of animal tissues, using a nerve-and-muscle preparation, it is found that when the proximal end of the nerve is made kathode, that is to say, when the current is ascending, the indicating muscle WkM shows response. This is due to the make action of the kathode. At break also response occurs ; but this is due to the transmitted action of the break excitation of the distant anode. When the current is reversed, that is to say, made to descend, there is also response, due to the make excitation of the distant kathode. When the current is broken, response takes place again, in consequence of the break of the proximal anode. All these cases are rendered possible by the high conducting power of the intervening tissue, the nerve.

I have been able to obtain precisely similar results, by selecting very highly excitable specimens of Mimosa. One electrode was placed at the junction of stem and petiole, the Fig. 94. Effects of Ascending and De- scending Currents, on Highly Excitable Specimen of Mimosa In the figure to the left, the anode is below and kathode above, and current ascending. In the figure to the right, the kathode below, anode above, and current descending. second being at a distance of about 3 cm. lower on the stem. In this case the stem, or certain of its elements, acted as the conducting nerve, the leaf serving as the terminal indicator (fig. 94). With such an arrangement, using a plant of exceptionally high excitability, and E.M.F. of moderate intensity, I have obtained the following results :

1. Current ascending {a). — At make, the leaf-stalk fell. This was due to the direct make action at the kathode (U). At break there was also a response. This was due to the transmitted break-anode excitation reaching the leaf-stalk. 2. Current descending (a).— At make, the excitation of the distant kathode reached the leaf-stalk, the current at the anode not being sufficiently strong to act as an effective block, (b) At the stoppage of the current, there was another response of the leaf-stalk. This was due to the break effect of the anode in the immediate vicinity.

The following tabular statement shows at a glance the effects which are apparent at the terminal organ : Table showing the Effect of Moderate E.M.F. on Highly Excitable Mimosa The experiments described above show that the excitation produced in plant-tissues by an electrical current is not indiscriminate, but selective, or polar, in its action. The effects seen here are of precisely the same nature as those observed in animal tissues. The exhibition of such polar effects completely disproves the hydro-mechanical theory of excitation in plants. They point unmistakably, on the other hand, to the existence of some fundamental property of protoplasm, common to animal and vegetable alike, which under normal conditions finds an identical expression in the two, of kathodic excitation at make, and anodic at break.

Polar effects are observed in plants in every way similar to those obtained from animal tissues. The laws of polar excitation in plants are as follows : A. With feeble E.M.F. the kathode excites at make, and not at break. The anode excites at neither make nor break. B. With moderately strong E.M.F. the kathode excites at make, and not at break. The anode excites at break, and not at make. Effect of high E.M.F.— Effects at two stages, A and ^—Experimental verification of A stage effect — Similar effects seen in protozoa — Experimental verification of complete reversal at B stage — Law of polar effects under high E.M.F. — Investigation on polar effects by death-response — Reversal of polar effects as due to fatigue, or tissue-modification — Investigation of polar effects by glowresponse of fireflies.

The phenomena ot polar excitation which have been observed in animal tissues are summarised in the formula which is known as Pfliiger's Law, viz. that excitation takes place at the kathode at make, and the anode at break. It has been found, however, by Kiihne, Verworn, and others, that in the case of the protozoa the polar effects are exactly the opposite ; that is to say, in these instances, it is the anode which excites at make. The inference has hence been drawn, that Pfliiger's law was inapplicable in the case of unfibrillated protoplasm.

That this assumption, however, is incorrect, I have already shown, by the fact that the undifferentiated protoplasm of the plant-body gives rise to polar effects which are in every way identical with the normal polar effects seen in animal tissues. It occurred to me that the study of polar effects in plants might throw some light on this anomaly, and that I might thus be able to trace out the stages by which the one effect was gradually transformed into the other, determining further the conditions which were effective in predisposing a tissue towards this reversal.

I soon discovered that the value of the acting electromotive force had an important influence on polar excitation. I found that under an increasing E.M.F. the excitation produced at the kathode underwent, first an increase, and then, on reaching a maximum, a decrease, which might even become negative. The changes produced at the same time at the anode were exactly the opposite. There was thus a progressive variation, resulting in an exchange of the excitatory properties of the anode and kathode.

My first observation with regard to this question was made in the course of my investigations on the determination of the velocity of transmission of excitation (Chapter XX.), and on the effect of increasing intensity of stimulation on this velocity. I found that, using for instance thermal stimulation, when this was strong, it was transmitted with a greater velocity than when it was feeble. Hence the speed with which the effect of stimulus travels in a given tissue may be held to afford a measure of the effective intensity of the stimulus. In order, however, to apply a stimulus which might be increased by known amounts, I next tried the electric mode of stimulation, expecting to produce an increasingly effective intensity of stimulus by increasing the E.M.F., excitation being produced at the kathode at make. In the course of a particular experiment, on a leaf of Biophytum, I found that as the E.M.F. was augmented from eight to thirty-two volts, the excitatory value of kathode at make was also increased, as shown by the fact that the velocity of the transmission of excitation was raised from 3*27 mm. per second, in the former case, to 3*83 mm. in the latter, an increase, that is to say, of 17 per cent.

But on raising the E.M.F. still higher, I found that the velocity of transmission from this point underwent a progressive decrease. From this it would appear that the excitatory effect of the kathode at make had an optimum value, in this case of thirty-two volts, beyond which there was a decline. This optimum value would naturally undergo a certain variation with the nature and condition of the tissue. Now, as the excitatory power of the kathode at make is seen to undergo a gradual diminution, beyond this optimum, it follows that at some certain high E.M.F. the excitation produced by it would be zero. In other words, the kathode would cease to excite.