Plant Response as a Means of Physiological Investigation

There is, moreover, another possibility, that of pseudoconduction, by which the effect of stimulus might appear to be transmitted across dead areas. My meaning will perhaps be clearer if we imagine two isolated muscle-preparations, one of which is attached to a striking lever, under which is the second. Supposing stimulus to be applied to the first of these, we can see that it would cause the lever to strike the second muscle, thus causing excitation. In this way, the effect of a stimulus applied to the first muscle would appear to have been transmitted to the second, completely isolated from it. In reality, however, this was not a case of true, but of pseudo-conduction, the excitation of the second muscle being started de novo by the blow of the lever, itself only a secondary effect of the excitation of the first.

Similarly, we may have, in Haberlandt's experiment, two living tissues isolated from each other by an intervening area of dead tissue. A strong stimulus applied to the first of these will now cause an excitatory expulsion of water, which will be transmitted across the dead area, and impart a mechanical blow to the second living tissue, thus setting up excitation de novo in that portion of the petiole. Velocities of transmission in various plant and animal tissues. — I give below a number of determinations of velocities of transmission made with different plants, both ordinary and sensitive, the electrical method of determination having been used in the case of the former ; and with this, for purposes of comparison, a series of values that have been determined in the case of animal tissues. The respective values given in the table refer to the maximum velocities obtained. In this connection, it should be remembered that the velocity of transmission depends on the intensity of stimulus. The intensity of stimulus, again, is diminished in the course of transmission through a long tract. Hence the velocity near the point of application of stimulus is relatively great, and becomes less the further the stimulus travels. In order, therefore, to make the different results comparable, my experiments have been made on equal lengths of tissue, namely, 7*5 cm. in each case, the stimulus applied being also the same.

Nerve of Anodon ..... Nerve of Eledone (observed by Uexkull) . Petiole of direction centripetal Petiole of direction centrifugal Peduncle of . It will thus be seen that the velocity of transmission in conducting plant-tissue is not very different from that in the conducting tissue of certain animals. Successive determinations of velocity of transmission are consistent when the stimuli are uniform, and when intervening periods of rest, sufficient for complete protoplasmic recovery, are allowed.

Velocities of transmission are not the same in centripetal and centrifugal directions. In Biophytum, for example, the centrifugal velocity is greater than the centripetal. When a given point in a plant-tissue is gradually raised in excitability, the consequent excitatory discharge takes place preferentially in one direction. Conductivity and excitability are both diminished by the increasing fatigue consequent on shortened intervals of rest. When the resting-period is shortened below a certain critical interval, the motile organ proves 'refractory' to further stimulus.

The velocity of transmission is not the same for all intensities, but increases with increasing stimulation. The velocity of transmission is diminished by lowering, and increased by raising, of temperature. The fibro-vascular elements are the best channels for conduction of stimulus : in them, the transmission lengthwise is greater than crosswise. Indifferent parenchymatous tissue has little or no power of conducting stimulus. The velocity of transmission in plants is not of an altogether different order of magnitude from that in certain animal tissues.

Pfeffer's experiment on expulsion of water from excited cells — Author's experiment on a delicate method of detecting excitatory expulsion of cell-sap —Chemical method of determining velocity of transmission of excitation — Electrotactile detector — Demonstration of passage of excitatory contractile wave by means of electrotactile method — Determination of velocity of transmission of excitation in ordinary plants by electromotive method — Excitatory versus hydromechanical movement of water.

The fact that the excitatory wave is propagated with a constant and measurable velocity was demonstrated, with regard to sensitive plants, in the last chapter, the arrival of the wave from a distance at the motile organ being detected by means of mechanical response. As ordinary plants, on the other hand, do not possess such efficient motile indicators, some other method, more universally applicable, is necessary in order to show that in these also the state of excitation is transmitted from point to point.

Before describing the two methods which I have devised for this purpose, I shall give an account of a very interesting experiment, depending on chemical reaction, by which I have been able not only to demonstrate in a striking manner the expulsion of water from excited cells, but also to make a rough determination of the velocity of transmission. It was shown by Pfefferthat on exciting the lower side of a pulvinus, water oozes out from the cut end. On taking a detached pulvinus, and stimulating the lower surface with a blunt needle, he found that the organ curved downwards, and a drop of water was seen to escape from the cut end.

Chemical method of detecting excitatory expulsion of cell-sap. — My own experiment differs from this in several particulars. Since there is a general impression that certain specialised tissues in the pulvinus are alone excitable, it was my object to show that cells which do not exhibit any motility will also give rise to the expulsion of water by excitatory contraction, and I desired further to utilise this effect for the determination of the velocity of transmission of excitation in that tissue. The essentials to this purpose were : some means of detection of the excitatory expulsion at the exact moment of its occurrence ; some means of marking accurately the moment of application of stimulus ; and, lastly, the use of a fairly long tract of tissue, in order that the interval between application of stimulus at one end, and the manifestation of its reaction at the other, might be of a duration capable of exact measurement. For this purpose I took petioles of Mimosa and the non-motile stems of the same plant, and placed their cut ends in very dilute solution of sodium chloride. So dilute a solution of salt does not, as I find, appreciably affect the excitability of the tissue. Selecting one of the specimens, say a petiole, I adjusted the electrothermic stimulator at a distance of, say, 4 cm. from its lower or cut end, the specimen being held vertical by means of a clamp. The vessel of salt solution in which it had hitherto been placed was now removed. The end of the petiole was carefully rinsed, to remove all traces of salt from the outside, and a small beaker of very dilute silver nitrate solution was substituted. At this point it became necessary to finish the experiment rapidly, as silver nitrate solution is likely after a time to affect the excitability of the tissue. Momentary thermal stimulus was given by brief closure of the electric circuit.

The excitation then travelled through the intervening 4 cm. of tissue, with a velocity characteristic of the plant. When it reached the cut end, the excitatory contraction produced an expulsion of cell-sap containing the salt solution previously absorbed. This expulsion was instantly made visible by the formation of a dense white precipitate of silver chloride. This was sometimes seen to be projected as a white vortex ring. The interval between the application of stimulus and this projection was found to be three seconds.

It is thus clear that the excitatory wave is not one of hydrostatic disturbance, for such a disturbance would be transmitted with very much greater velocity. The velocity of transmission in this petiole is seen to have been 13 mm. per second, or practically the same as that obtained by a separate experiment on the fall of the leaf with similar specimens. In a similar manner I determined the velocity of transmission through the stem. The stimulus was applied at a distance of 5 cm. from the cut end, and the chemical precipitation was observed, after an interval of five seconds. The velocity in the stem is thus seen to be 10 mm. per second. A repetition of this experiment with another piece of stem from the same plant gave similar results.

It is thus seen that cells through which excitation is proceeding undergo excitatory contraction, in consequence of which there is an expulsion of water forwards, in the direction of propagation. And this effect is produced, not in pulvinated organs alone, but in others which are not usually regarded as motile. This result is also arrived at independently by the method of electrical response. The electrotactile method.— I shall next give an account of the much more delicate methods which I have succeeded in devising for the detection of the excitatory impulse during transit, the first of which is the electrotactile method. It is easy to understand that while the wave of excitation is passing through any given section of tissue, it must produce there certain form-changes, infinitesimal though they may be. Had our sense of touch been more delicate, we might perhaps have been able to perceive this pulse. It occurred to me, however, that it might be possible, if it existed, to obtain its indication by means of an electrical method of detection, the sensitiveness of which could be exalted at will.

different effects are possible. For example, in the case of muscle with parallel fibres, the wave of excitation will travel onwards from the excited point, the contraction produced giving rise to expansion of the muscle in a direction at right angles to that of propagation. In the intestinal muscle, however, owing to a different distribution of the fibres, the propagated wave is one of constriction. Now, it is clear that these two kinds of muscle, placed within enclosing contacts, will give rise, during the passage of excitatory waves, in the one case to an increase of pressure, and in the other to its diminution.

We are thus prepared to see that if similar contractile waves pass through a vegetable tissue, they may be detected by means of a concomitant variation of pressure — a variation which may prove to be either an increase, or a diminution, according to the particular disposition of the contractile elements in the tissue. If the tissue, again, should happen to be anisotropic, the same wave of contraction may appear to give rise to a diminution of pressure in one direction, and an increase in that at right angles. In any case the excitatory wave, in the course of its transmission through any given area, might be expected to produce variation of pressure, as between two diametrically opposite leading-points.

I am now about to describe the electrical device which I have used for the detection of such transient pressurevariations, concomitant to the passage of excitation through vegetable tissues. It is known that the electrical resistance of contact varies with pressure, and on this principle depends the construction of the microphone. But a loose contact, such as would be favourable for microphonic use, is unsuitable for our present purpose, by reason of the disturbance to which it is subject from atmospheric vibration. The necessity to be met, therefore, is that of the adjustment of an electric contact, which shall not be subject to resistance-variation from atmospheric disturbance, and which shall, at the same time, be sensitive to the pressure-variation effected by the excited tissue.

I overcame the difficulty regarding extraneous disturbance by using, instead of a loose, a steady pressurecontact, capable of the finest adjustment, by means of a micrometer screw. The next problem lay in choosing contact material of great sensitiveness. For this purpose I used different materials, the sensitiveness of some being very great, while that of others was moderate. Carbon contacts belonged to the latter class, but had the advantage of being easily adjustable. Various metallic powders, however, such as that of bronze, were considerably more sensitive, but at the same time required greater care for adjustment.

The most important factor in the arrangements, by which the sensitiveness of the contact may be exalted to a very high degree, lies in the proper adjustment of the electromotive force acting on the contact circuit. The sensitiveness to pressure-variation increases with increasing electromotive force, being greatest when this is just short of a certain critical value, after which the electric contact is observed to become unstable, and give rise to spontaneous oscillatory variations. For the purpose of easy adjustment of the electromotive force I use a sliding potentiometer. The tissue, say a stem of Mimosa, is placed between points, A and B, and by means of the micrometer screw, S, it is pressed against the spring B. The electric contact, say of carbon points, is between B and C, which are in circuit with a galvanometer, and the potentiometer. The pressure is so adjusted that a feeble current flows through the galvanometer. In order to increase the sensitiveness of the detector, the electromotive force may be gradually increased, by proper adjustment of the potentiometer, short of the critical point (fig. 106). The deflected spot of light from the galvanometer will remain steady, provided the adjustments have been properly made.

The tissue is now stimulated at a distant point, care being taken that it is not in any way jarred mechanically. Stimulation without jar is effected, however, without difficulty by the use of the electrothermic stimulator. In any case, it is easy to discriminate between the effect of any such extraneous disturbance and the true excitatory effect in transit for which we are looking ; for the effect of the former, if it Electrotactile Method for Detection of Excitatory Wave during Transit

Stem placed under light pressure between B and sliding rod c. A, B, electric contact points, the pressure of which is delicately adjusted by micrometer screw, M. A moderate current flows round the circuit, including the contact points, A B, and the galvanometer, G, the E.M.F. being suitably adjusted by the potentiometer slide, P Stimulation of stem effected by momentary closure of key, k, in circuit with electrothermic stimulator. Excitatory wave reaching the zone B, c, causes pressure-variation, with concomitant galvanometric response.

occurs, is immediate, whereas that due to excitation takes place after a definite interval from the application of stimulus. In the experiment of which the record is given in fig. 107, stimulus was applied at a distance of 4 cm. The excitatory wave reached the experimental zone after an interval of five seconds, and indicated its presence by a diminution of pressure on the contact (fig. 107), resulting in sudden diminution of current. After the passage of the excitatory wave the tissue was restored to its original condition, as shown by its once more exerting its normal pressure. This process of response and recovery by variation of pressure is exhibited by the galvanometer record. The recovery is usually found to be attained in the course of a half to one minute, according to the intensity of stimulus. As the result of this experiment, we find the velocity in this particular stem to be 8 mm. per second, which is very near the determination already made by other methods, with different specimens of stem of Mimosa.

Frequently as I have obtained this response during transit by diminution of pressure, its opposite, that is to say, response by increase of pressure, is by no means uncommon. "I have already explained how it is possible for excitatory contraction to give rise to two such opposed effects, in consequence either of different dispositions of the contractile elements in the tissue, or by the presence of anisotropy in the organ. By means of the electrotactile method, then, we are able to demonstrate the passage of the excitatory wave, and also to measure its velocity, in tissues which are not motile.

The electromotive method. — I shall now describe the second, or electromotive, method which I have used for the detection of the excitatory wave during its passage through a vegetable tissue. I have already explained that when the plant-tissue is directly excited, the state of excitation is invariably accompanied by an electromotive variation, the excited point becoming galvanometrically negative (p. 32). Hence, when an excitatory wave is transmitted through the

Stimulus applied at the moment x , at a distance of 4 cm. below the detector. tissue in any direction, from the stimulated point, there must always be an electromotive wave as its strict concomitant. The moment, therefore, when the excitation reaches a given point may be determined by observing the arrival of this excitatory electrical disturbance of galvanometric negativity, and for the detection of such an excitatory wave the galvanometer takes the place of the motile leaflet.

In order to prove that the excitatory mechanical and electrical effects are strictly concomitant, it is only necessary Fig. 108. Experimental Arrangements for Simultaneous Recording of Mechanical and Electrical Responses Stimulus applied by thermo-electric stimulator, s, at A. Excitation reaching B causes mechanical response of leaflet, which is recorded by optical lever on drum at M. Simultaneous galvanometric negativity recorded at E. to perform an experiment on a plant, such as Biophytum, which is provided with motile leaflets. We attach one of the indicating leaflets to the Optic Lever, and connect its base B with one of the electrodes of the galvanometer, the second electrode of which is connected with a distant point of the leaf (fig. 108). The two spots of light, one from the Optic Lever indicating mechanical, and the other from the galvanometer indicating electrical, response are adjusted to lie

one above the other on the same revolving drum. On applying a stimulus, say thermal, at A, it will be found, after the lapse of a definite interval, that both spots of light are deflected simultaneously, proving the concomitance of the mechanical and electrical effects. Such a record has been given already, in fig. 26. If we make a mark on the revolving drum at the moment of the application of stimulus, and a second mark when the electrical (and, in this particular case, also the mechanical) response is initiated, we can, with the previous knowledge of the speed of the drum, determine the time taken by the excitation to travel from A to B, and thus find the velocity of transmission for that specimen by electrical means.

A further refinement of this method lies in the use of two galvanometers instead of one, the slight lag of response, caused by galvanometric inertia, being in this way eliminated. Particulars regarding this will be found elsewhere. It will be seen, then, that in the electromotive method we have a second means by which to determine the velocity of transmission of excitation in what are known as ordinary plants. I shall now describe experiments performed by this method. The peduncle of Biophytum is leafless. That it does transmit stimulus is seen nevertheless when we excite it at any point. Excitation will then be found transmitted through it to the main stem, from which it travels outwards to different leaves, a fact evidenced by the serial fall of leaflets in a centrifugal order. In such a peduncle I have determined the velocity of transmission by the electromotive method. The distance between the points A and B was 4'6 cm.; the time taken for transmission was 127 seconds. The velocity is thus found to be 37 mm. per second.

Similarly, in the stem of Ficus religiosa, I found velocity of transmission to be 9/4 mm. per second, which is almost the same as that in the stem of the so-called ' sensitive' plant Mimosa. From the experiments carried out on the electrotactile method it will be seen that excitation is conducted along a plant-tissue from cell to cell, as a contractile wave. We have also seen that water is expelled from an excited and therefore contracted cell. It is further clear that when an excitatory wave is proceeding in any direction, this cell-tocell passage of excitation will give rise to a cell-to-cell contraction, the result of which will be a forward movement of water, which will have the velocity, not of hydrostatic transmission, but of the excitatory wave. The hydrostatic disturbance is quite distinct, being transmitted with great rapidity, and its presence has been shown in the preliminary abnormal response of erection in leaflets of Biophytum (p. 24). But that propulsion of water which is concomitant to the passage of true excitation is very much slower, having the same speed as that of excitation itself.

The direct effect of stimulus is not transmitted by means of hydrostatic disturbance, but by a cell-to-cell propagation of excitation. This transmission of excitation from cell to cell is attended by a cell-to-cell contraction. The passage of such a contractile wave may be detected by the electrotactile method, which thus enables us to determine the velocity of transmission of excitation, even in tissues which are not motile. In consequence of the concomitance of the excitatory wave with cellular contraction, water is moved forward progressively, with a velocity and in a direction the same as that of excitation.

This movement of water is not brought about by any hydro-mechanical action, but is the direct effect of the contractile wave due to excitation. The hydrostatic disturbance, when present, is transmitted with very great velocity, and its effect is seen in the abnormal preliminary response of erection, exhibited, for example, by the leaflets of Biophytum. The velocity of transmission of excitation in ordinary plants may also be found by determining the velocity of the concomitant electromotive wave.

The determination of the latent period in Mimosa — Experimental arrangements for obtaining automatic record Prolongation of latent period by cold — Sparkrecord for determination of latent period — Prolongation of latent period by fatigue - Sluggishness of the response of Philanthtis urinaria^ also long latent period and very protracted period of recovery — Latent period reduced under strong stimulation — Response in Biophytum on the ' all or none ' principle — Definite value of effective stimulus — Phenomenon of refractory period in Biophytum — Parallelism of responses in Biophytum and in cardiac muscle — Additive effects — Inappropriateness of term ' refractory period ' -Energy in excess of effective stimulus held latent for subsequent manifestation.