Comparative Electro-Physiology: A Physico-Physiological Study

of stimulus—Response under over-balance—Response under sub-terminal stimulation —Variation of response under seasonal changes. IN the last chapter it was shown that the various objections hitherto urged against the excitatory nature of the ascent of sap were not justified. In the course of the present chapter, therefore, I shall adduce proofs that the water-movement in the plant is the result of stimulatory action. Instead of vaguely referring the phenomenon to physiological activity, moreover, we shall attempt, proceeding from the ‘basis of other excitatory reactions, already clearly established, first to see whether inferences based on these are capable of explaining the present problem, and secondly, to subject those inferences themselves to the test of experimental in- vestigation.

We cut a certain length of the stem of M/zmosa, and keep it immersed for some time ina very dilute solution of common salt, until the tissue has become charged with this. The specimen is then taken out, and thoroughly rinsed with clean water. It is now held vertically, with the lower end dipped in a highly dilute solution of silver nitrate. A portion of the tissue higher up is now excited by contact with a hot wire. The excitation thus induced is then found to travel through the intervening distance, with a velocity characteristic of the conducting power of the tissue. The arrival of the excita- tory wave at the lower end is attended by an expulsion of the cell-sap containing the salt solution previously absorbed..

This expulsion is instantly made visible by the formation of | a dense white precipitate of silver chloride. From this experiment it is seen that the passage of excitation is at- tended by a forward movement of water in the direction of propagation. | The next point to be realised is that a strong or a long- continued stimulus will give rise in the tissue, not to one, but to a multiple series of propagated waves. If the tip of a leaf of Biophytum be strongly excited, we see successive waves of excitation, marked by the serial fall of the motile leaflets, proceeding again and again in the centripetal direction, from the terminal excited point. If, similarly, the end of theroot be excited, by any means, an excitatory move- ment of water will be induced, proceeding away from this end, in an upward direction. It must be remembered, how- ever, that excitation proceeds in all directions from the excited point. If then the point of excitation be terminal, it is evident that the direction of propagation, being away from this, will be upwards. But if the tip of the root be highly ex- citable, then, owing to local excitation, there will also be a certain amount of secretion into the soil. Even highly excitable tissues, however, after continuous stimulation, show a tendency, as we have seen, to the reversal of their character. istic response. This secretion at the terminal point of the root will tend to become changed into absorption. Again, looking at the succession of excitatory waves propelling water upwards, we can see that these will leave a deficit of cell-sap behind, which will further act rather for the absorp- tion than for the secretion of fluid. And in addition to these, if there be any other directive influences, such as

evaporation from the leaves, they will tend to help the particular uni-directioned flow. We have seen that these conclusions are confirmed by the results obtained in the electrical response of the roots. We there saw that very young roots give at first negative responses, which are after- wards reversed to positive, under continuous stimulation. Older roots, as we also saw, give response by positivity. We saw, further, that there was much reason for regarding negative response as associated with the secretion, and positive with the absorption of fluid.

Thus the serial propagation of excitation from cell to cell, with the concomitant movement of water, will normally be upwards. In this connection it is very significant that the younger portion of the fibro-vascular bundle is the preferential channel for the conduction, at once of water and of excitation. It is thus seen that a one-directioned movement of water may be produced by the multiple excitatory activity of the tissue. And just as the multiple activity of certain tissues, say, for instance, the leaflets of Desmodium, may be gauged by their multiple mechanical movements, so in the rate of the water-movement we have a means of measuring the intensity of the multiple rhythmic activity of those which are concerned in the ascent of sap. This would be analogous to the measurement of the rhythmic activity of the heart, by a determination of the rate of flow of the circulating blood. In the case of the plant, however, this rate of movement might be measured, either by means of the pro- pulsion of water forwards, or by the suction exerted behind.

In order to demonstrate the fact that the water movement in the ascent of sap is mainly dependent on excitatory reactions, it is necessary to have at our disposal some means of rapidly observing and recording the variations induced by physiological changes in the rate of ascent. For this pur- pose I was successful in devising the Shoshungraph or suction-recorder, described in detail in my book on ‘ Plant Response.’ ! : -° This instrument consists of (1) an arrangement by which the specimen may be subjected rapidly to the action of different excitatory or depressing agents ; (2) a potometric tube for the measurement of changes of suctional activity, under different external conditions ; and (3) a contrivance by tmneans of which the movements of the water-index, with their time-relations, are recorded. The principal parts of the apparatus are seen in fig. 218. V is the plant-vessel, in which the specimen is mounted, with or without roots, by means of a watertight india-rubber cork. R is the reservoir, which may be filled with hot or cold water, or with the required chemical solution. By appropriate manipula- tion of stopcocks, by means of key K, the water in the plant- vessel may be replaced quickly by any of these, and the effect of the changed condition on the rate of water-move- ment observed.

A direct record of the rate of movement may be made on the revolving drum, actuated by clockwork, as shown in the figure. For this purpose, a pen, P, is fitted over the potometric tube, by means of a brass collar, which has a rectangular opening, kept always coincident with the water- index. The collar, carrying the pen, is attached to a thread, which passes round small pulleys. One end of this thread carries a counterpoise, M, and the other is wound round a wheel, W, which can be so manipulated as to make the pen follow the movements of the water-column. When the water-index is followed in the way described, a direct record of the water-movement in the plant is obtained. A curve is thus traced, the ordinate of which represents the quantity of water sucked up, and the abscissa the time. The slope of the curve thus gives the rate of movement. As long as suction is uniform, this slope remains constant. If, however, any exciting agent increases the rate of suction, there is an immediate flexure in the curve, which thus becomes steeper. A depressing agent lessens the slope, and when suction is abolished, the record becomes horizontal. For the detection of the slightest variation in the rate of suction, the Method of

Balance is employed, which depends upon an application of compensation, by which, under normal conditions, the water- index is kept stationary, though the suctional movement in V, plant-vessel ; R, reservoir ; C, compensator, whose balancing height is adjusted by rack and pinion, s; kK, key for manipulation of four-way stop-cock ; P, recording pen, with counterpoise M, manipulated by wheel, w. The drumjis rotated by the clock at uniform speed. the plant is in no way disturbed. The balance is obtained by allowing water to enter the plant-vessel, from the com- pensator, C, at a rate exactly equal to that of its withdrawal by suction. Thus, under a condition of balance, the record ‘becomes horizontal. An exciting agent now produces an inclination of the record upwards, or a depressing agent a

declination downwards. This was the instrument employed. by me for the obtaining of records in a simple manner of. ‘suctional response and its variations, under different physio- logical modifications. A fuller account of the method and the results obtained by it will be found in my book on ‘Plant Response.’ As the subject of the ascent of sap is, how- ever, of extreme importance, I thought it desirable to see to what extent the sensitiveness of this instrument could be raised, and also to devise means, in connection with it, for the automatic record of results obtained. For the latter purpose I employed photography. As the water-index, whose ex- cursions in the potometric tube are to be recorded, is trans- parent, it is necessary to provide an additional opaque index, which shall move in and out with it. This consists of a short length of mercury, lying in contact with the end of the “water-column. The potometer tube is placed in the field of a magic lantern, and the index is focussed on a moving photographic plate by means of an objective. The sensitive- ness of this method of record may be increased in two ways. First, the bore of the capillary tube may be made finer and finer. But this cannot be carried to an extreme, as the capillary offers great resistance to the free movement of the index. Secondly, the sensitiveness may be increased to any extent by the employment of a highly-magnifying and short-focus objective. By the combination of both these devices, we are able in practice to arrive at an extraordinary degree of sensitiveness, qualifying us to attack some of the most difficult problems with the greatest ease. For our present purpose, however, it is not by any means necessary to approach the limit of this sensitiveness. The photo- graphic records given in the course of the present chapter

were obtained with a tube having a bore I square mm. in section, and employing an ordinary magic lantern objective, which casts an image without any magnification. The Method of Balance again, as we have seen, affords us another opportunity of arriving at an experimental adjustment of creat sensitiveness. The experimental delicacy obtained by high tiouihen tion can only be used to the greatest advantage when coupled with the Method of Balance, if we have means at our disposal for securing the utmost possible perfection of the balance. We have seen that the balance is adjusted when the rate at which water is removed from the plant- vessel by suction is exactly equal to the rate of its inflow from the compensating vessel, Cc. This compensation is roughly effected by the rack and pinion, Ss, which serves to regulate the flow by raising or lowering the compensating vessel (fig. 218). For the purpose of the final adjustment the narrow bore of the thick india-rubber tubing, which con- nects the compensator with the plant-vessel, is capable of gradual constriction. This must be accomplished by equal compression on all sides, as bilateral compression alone would act to induce a discontinuous closure and sudden arrest of flow. If the compressing arrangement be something after the model of the iris-diaphragm, then the bore, which regulates the flow, may be constricted gradually and con- tinuously. With such an arrangement it is easy to arrive at a balance so perfect that the index appears to be quite stationary. Under-balance would now make it move, say to the right ; and over-balance to the left.

We shall now proceed to show that those agents shih exalt physiological activity also act to enhance suction; and that those which induce physiological depression will also depress suction. One of those which enhance the multiple activity of the tissue is, as we know, the rise of temperature ; whereas cooling, or lowering of temperature, tends to depress it, even to the extent of abolition. Thus an automatically vibrating leaflet of Desmodium has its vibration-frequency

enhanced by a rise, and depressed or arrested by a fall, of temperature. A similar effect is seen to occur in the suctional response of plants. Thus, in a given specimen of Cvoéon, application of cold water at 4° C. to the root was found to arrest the suction in-the course of 8 minutes. This arrest by cold was not permanent, for the normal rate of suction re- appeared on the return of the water to a normal temperature. On applying water of raised temperature to the root, on the other hand, the rate of suction was immediately found to be enhanced. This is illustrated in the following record (fig. 219), in which the normal rate of suction at 23° C. was 7 cubic mm. in volume, or 7 mg. in weight per minute. The ap- plication of water at 35° C. now induced a steep rise of the curve, indicating an enhanced rate of suction of 58 mg. per minute, or more than eight times the rate at 23° C. On now once more sub- stituting water at 23° C., the rate is seen to become lowered, but not to fall so low as at the ere ttn at oso C, factesed beginning. It now fell from 58 Suction at 35° C., and the to 14 mg. per minute, instead of io ideal Pee Ketur returning to the original 8 mg. This is due to the fact that the internal energy of the tissue has been raised in the meantime by the absorption of warm water, the enhancement of the normal rate being a persistent after-effect of this.

We shall next take up the apparently anomalous case, in which, when the root has been killed by pouring boiling water over it, the suction of the plant is nevertheless main- tained. In such an experiment the normal record was first taken, and boiling water was then passed through the plant- vessel continuously for some time till the roots were killed. On allowing the water in the vessel to return to the tempera- ture of the room, it was found that suction was taking place

at an even greatcr rate than ordinary. This result would at first appear to show that protoplasmic activity had nothing to do with the ascent of sap, and the objection would have been fatal if the activity which produces suction had been confined to the roots alone. But in reality, as we have already seen, such activity is present, to a greater or less extent, throughout every zone of the plant. The only part which is killed, however, in the experiment - just described, is that which is actually immersed in, or in immediate contiguity with, the boiling water; and the unkilled tissues above continue their suctional activity unabated. The increase in the rate of suction is to be ex- plained by the fact that the entrance of water, instead of being effected through the extremely attenuated channels of the root-hairs, now takes place through the whole mass of the root, acting virtually as a wet rag tied round the base of the living stem. The mass of water which it is thus possible to suck up directly through the broad-sectioned stem is evidently much greater than could have been taken in through the resistant, organically-conducting channels of the rootlets. | The fact thus demonstrated, that the local death of a given zone does not fer se arrest the suctional activity of the tissues above it, explains why a poison may be carried to the top of atree. It is evident that only when it has thus been conveyed, and when all the tissues have thus been killed, could a permanent arrest take place. And Strasburger him- self admits that arrest under these conditions does occur. With reference to the effect of poison, again, it is im- portant to bear in mind that its toxic effect depends, toa certain extent, on the tonic condition of the tissue.

Thus a Desmodium \eaflet which was moderately vigorous had its pulsatory movement arrested soon after the local application of the copper sulphate solution. In more vigorous leaflets, however, the arrest did not take place till after a considerable length of time. Indeed, in some cases, after a preliminary arrest, the leaflet is able to shake off the effects of the poison absorbed, by some process of accommodation. I have shown elsewhere! that the effect of poison on the response of growth is modified to a remarkable extent by the different tonic conditions of the tissue. The experiments in question were carried out on similar specimens of Crinum lily, in which the only difference induced depended on the fact that one set had been kept at a temperature of 30° C., and were

thus in moderate tonic condition, while the others had been. maintained at 34° C., bringing about, as I have shown, an optimum tonic condition. The application of a 5 per cent. solution of copper sulphate to one of the first of these was found to induce the rapid decline and final arrest of growth, while a similar application, on a specimen in the optimum condition of the second set, induced a preliminary exaltation followed by a slow depression and ultimate arrest of growth, the last-named, however, being reached only after the lapse ofa considerable time.

The difference of effect under different tonic conditions was still more strikingly exhibited by the application of a smaller dose—namely, of a I percent. solution. This was found to induce a depression of growth, which was ultimately fatal to the plant, in the case of specimens kept at 30° C. But when the same dose was applied to a plant which had been kept at 34° C, the effect was seen in a marked exaltation of the rate, for a fairly long time, after which it shook off the effect of poison altogether, resuming its normal rate of growth.

Thus the effect of poison on the various activities of the plant is seen to depend not only on the amount of the agent, and the duration of application, but also on the tonic condition of the tissue. Strong and prolonged applica- tions will abolish all active processes, by inducing the death of the plant. In accordance with this, I find that in certain suction is more rapid than in others. All alike, however, exhibit permanent arrest sooner or later. 1 Bose, Plant Response, pp. 487-488.

The question of the excitatory nature of the ascent of sap may again. be tested by the application of anzsthetic agents, such as solution of ether. The record is first taken of the actual rate of suction, and then by quick manipulation _of the double key, the water in the plant-vessel down to the base of the specimen is replaced by ether solution. The original rate of suction had in a particular case been 40 cubic mm. per minute. After the application, however, this became depressed, and in the course of four minutes under- went a preliminary arrest. This short arrest was suc- ceeded by reversal, or expul- sion, which lasted for fourteen minutes, and then gave place to what was practically per- manent arrest (fig. 220).

I have occasionally ob- served an interesting variation in the arrest of suction induced by ether. Shortly after the application just described an arrest of suction is induced. This, however, is only pre- liminary, suction after an interval being renewed at a_ Fic. 220. Action of Anesthetics in : Abolition of Suction very slow rate. When this 3 Seca : Solution of ether substituted for water has proceeded for some time, at point marked with dots... Suc- the process undergoes a tion abolished within fifteen minutes.

It has already been said that though the excitatory reaction is to be regarded as the fundamental cause of the transport of water in the plant, yet there are other factors which undoubtedly contribute to that result, One of these may be the favourable disposition of osmotic substances— for example, the concentration of cell-sap consequent on evaporation, in the leaves. That this osmotic effect is, however, merely secondary, and that the ascent is chiefly due to excitatory action, is seen in certain experiments which may be mentioned here, in which the ascent takes place with even greater vigour than before, when it is opposed by an osmotic influence. .

The various solutions of salts are very unequal in their physiological action : some, like potassium nitrate, are neutral,’ but others, as strong solutions of sodium chloride, are ex- citatory. Thus the action of a strong solution of potassium nitrate is physiologically more or less neutral, while its osmotic action, at the same time, is pronounced. A strong solution of common salt, on the other hand, is both excitatory and osmotic. If then we apply KNO, solution to the cut end of a stem, water will be osmotically withdrawn from the plant, in | opposition to the normal ascent Fic. 221. Effect of Strong KNO, of se ; There. will thus be .

The first record shows the normal after the application, as seen in and the second the depressed rate . the following record (fig. 221), which I obtained with a cut branch of Cvoton. If, however, in a similar experiment, a strong solution of NaCl be applied, two antagonistic reactions will be set up. One, due to the osmotic action, will oppose suction, and the other, due to the excitatory nature of the reagent, will accelerate it, while the resultant effect will be modified by the excitability of the experimental plant. In fig. 222 is shown the effect of NaCl solution in

' It should be mentioned here that even such neutral salts, in strong solution, induce physiological depression. increasing the suction of a specimen of ‘Croom in a favourable condition of excitability. We have here a very great en- hancement of the ascensional movement caused by this solu- tion, which would, acting osmotically, have retarded the normal rate. The physiologically excitatory action of strong sodium chloride, however, is not permanent, and the enhanced excitability is followed by depression. The effect on suctional response in such cases, then, is modified by the factor of time, the. first enhancement being followed by a fall below the original normal rate of suction.

The experiments which I have here described show how intimately suctional response is connected with excitatory re- action. Having thus seen that any physiological modification of the tissue is attended by an appropriate change in the rate of suction, it only remains to demonstrate finally the fact that stimulation is attended by a responsive movement of water Fic. 222. Effect of Strong NaCl in a tissue. In order to do this, Solution we should have at our disposal The first record shows the normal . and the second the exalted rate mens means of applying of suction caused by the reagent.

measurement, capable of graduation, and not in itself of a nature to disturb the delicate balance of the shoshun- graphic record. The end sought after was first to record the normal rate of suction, and then to observe the effect immediately induced in this by the application of the stimulus, the process of record being uninterrupted mean- while. The only form of stimulus which would comply with these conditions is the electrical, given by tetanising induction shocks, of longer or shorter duration. The possible objections to the use of this form of stimulus are as follows :

1. The water in the plant-vessel may be supposed to undergo decomposition by electrolysis. There may also be a certain evolution of heat in the plant-vessel. 2. In a sluggish tissue, such as that of the plant, the excitatory value of induction- shocks may not prove sufficient to Soles suctional response. With regard to the first of these objections, it is to be borne in mind that the shocks, being alternate, will produce | but little polarisation effect. Fhe heating effect of a current so small in quantity, moreover, is also likely to be very slight. ~The extent of the disturbance from these causes can, how- ever, be determined by a blank experiment. The electrodes, by whose proper applica- tion the plant is ex- cited, are allowed to hang down in the plant- vessel without direct

Fic. 223. Record of Blank Experiment showing Absence of any Disturbance of Record from attachment to the plant Photographic record of excursion of .mercury t AT index in Shoshungraph. The thick white CUrrent will now pass line shows duration of application of shock. through the water, only Time-marks in this and other photographic : records represent intervals of five minutes. a very small fraction of it, incapable of pro- ducing effective excitation, passing through the plant. The Shoshungraph is adjusted for the balanced condition, given as we have seen, a horizontal line of record, and that intensity of induction-current which is to be used in subsequent experiments is now passed through the plant-vessel for twenty minutes continuously. It will be seen from the record (fig. 223), that no disturbance was induced by this in the balanced line, thus proving that such disturbing effects, if they exist, are in practice negligible. In actual experiments, where the excitatory effect is to be studied,

the duration of application is often less than a minute, and seldom exceeds five minutes. It should also be borne in mind that individually and collectively the effects of the dis- turbing causes enumerated would, if anything, be towards expulsion from the plant-vessel. We shall see, however, that the typical responsive effect is movement in the opposite direction, indicative of an enhancement of suction. With regard to the second cause of misgiving, as to whether plant- tissues may or may not be made to exhibit excitatory varia- tion by means of induction-shocks, I have found that some specimens, notably those of Cvofon, are sufficiently suscep- tible to this form of stimulation. For this purpose it is necessary to use very strong induction-shocks from a large coil, This necessity is further increased by the fact that much of the induction-current is uselessly and unavoidably shunted by the water in the vessel. | This electrical stimulation may be applied to the cut end of the branch, immersed in the water of the plant-vessel, in either of two different ways. The first of these may be described as the Zerminal Mode of Application, its object being to localise the excitation more or less at the lower end of the specimen. This is done by tying two small pieces of platinum, in connection with the electrodes, to diametrically opposite sides of the base of the stem by means of a thread. Or two pins, in connection with the electrodes, may be pricked into the lower section of the specimen near its circumference. This transverse mode of stimulation, across the diameter of the stem, is not, theoretically, so effective as longitudinal stimulation would be, but under the particular experimental conditions nothing better could be devised (fig. 224). The second mode of applying this electrical stimulus I shall distinguish as Swd-terminal.

Here, two pins in connection with the electrodes pierce through the stem, one above the other, in planes at right angles to each other (fig. 225). After arranging the electrical connections in any one of the ways enumerated, the specimen is adjusted in the Shoshungraph, and allowed a period of rest for the passing off of excitatory effects of preparation. The record of normal suction is then taken by means either of the ordi- nary recorder or of photography. The variation induced in the record after the application of stimulus, then, exhibits the effect of excitation.