The Physiology of the Ascent of Sap

gave a deflection of i mm. for a current of lo"^" ampere. The inertia of the suspended coil was too great for very accurate determination of the period of a single pulsation. In this respect the Einthoven galvanometer offers a great advantage, since its exceedingly thin string follows the most rapid variation in the impressed electromotive varia- tion Its disadvantage is that the sensitiveness of the instrument is very much less than that of the D' Arson val galvanometer.

I hoped, however, to obtain a record of the pulsations even with the Einthoven galvanometer by taking advantage of conditions favourable to the pulsatory activity of the plant. Unfortunately the recording apparatus was ready only in May, when the prevailing high temperature had caused a depres- sion in the cellular activity. In spite of this I was fortunate enough to obtain several records early in the morning which proved to be fairly satisfactory. The recording plate was allowed to drop at a rate of about i mm. per second ; the sensitiveness of the apparatus was so adjusted that successive divisions in tlie vertical scale represented an electromotive variation of a tenth of a millivolt. The record was obtained with Nauclea ; the electromotive variation of the pulsating cells is seen to be 0*4 millivolt; the periods of successive pulsations are practically the same, being 13-5 seconds (fig. 74).

Fig. 74. Kt?cord of Cel- lular Pulsations in Nauclea, taken by the Einthoven Galvano- meter The successive horizontal lines represent the elec- tromotive force, one small division being equal to o • i millivolt. The successive vertical lines represent intervals of a second. The period of complete pulsation is 13-5 seconds. The pulvinule of the leaflet of Desmodium exhibits periodic electric pulsations corresponding to the mechanical pulsations. The up-movement due to the sudden increase

of turg-or has an electric concomitant of galvanometric positivity ; the opposite electric change to galvanometric negativity occurs during the phase of sudden diminution of turgor and fall of leaflet. The period of a single pulsa- tion varies under different circumstances from one to five minutes. The discovery and record of pulsation of the cells active in the propulsion of sap was made by the employment of the Electric Probe, which during its passage detected pul- satory activity in a particular layer in the tissue of the stem.

The electrical records of alternate galvanometric de- flections of positivity and negativity afford evidence of the occurrence in the tissue of cellular pulsations consisting of periodic increase and decrease of turgor due to alternating expansion and contraction. These cellular pulsations are enhanced by favourable physiological conditions, and are depressed by unfavourable conditions. They exhibit all the characteristics of the pulsations of the Dcsmodium leaflet, and of those of the animal heart.

The records obtained with an Einthoven galvanometer show that the period of a single pulsation may be as short as 13. 5 seconds : the period of pulsation is often lengthened under cold to about three minutes. The electromotive variation under the less favourable condition of excessive heat in summer was found to be 0'4 millivolt: under favourable circumstances it may be as high as several millivolts. Localisation of active layer of pulsating cells in Impatiens — Localisalion in Brassica — Amplitude of pulsation at different depths — Theory of the electric determination of wave-length — Successive electric maxima and minima — Determination of wave-length in Chrysanthen.um and in Miisa — Change of wave-length under physiological variation — ^Upsetting of the phase difference by passage of electric current- Summary.

Having demonstrated that a layer of cells in the stem is in a state of active pulsation, an attempt was next made Fig. 75. The Electric Probe for the Localisation of the Active Layer The point of the Probe enters the stem at a, the second electric contact being made with the distant leaf. The figure to the right is an enlarged view with the micrometric screw for the gradual introduction of the Probe into the tissues of the plant. to localise this layer by means of the Electric Probe. One of the galvanometer terminals is connected with the Probe, and the other with a distant indifferent point in the plant (fig. 75). The fine Probe, insulated except at the tip, is then thrust into the stem step by step ; its passage gives

rise to a certain amount of irritation which causes a temporary aboUtion of the rhythmic activity of the cells. The protoplasmic recovery is, however, complete in the course of ten minutes or so. The record of cellular pulsation is then taken on a photographic plate, allowed to fall at an uniform rate by means of a clockwork. The Probe was introduced transversely into the stem by successive steps of O'l mm. No pulsation could be detected at the epi- dermis. As the Probe reached a depth of 0 • i mm. it detected a feeble pulsation : a similar result was obtained when it reached a depth of 0*2 mm. Owing to the residual after-effect caused by the insertion of the Probe, the base- line of the record was slightly displaced. At the next step, when the Probe reached a depth of 0-3 mm., the pulsa- tions exhibited a sud- den enhancement. This was so great that a part of the record went off the plate (fig. 76) : evidently the Probe had come in contact with pulsating cells. As the Probe was thrust still deeper into the stem, the pulsating activity rapidly disappeared. When a transverse section of the stem was made at the

Fig. 76. Record showing the AmpHtudc of Electric Pulsations at Different Layers in Impatiens Note the abrupt enhancement at a distance of 0-3 mm. from the surface, the par- ticular layer being in the inner cortex ; a portion of the record has gone out of the plate. line of the passage of the Probe, it was found that the maximum activity had been detected when the Probe touched the internal layer of the cortex abutting upon the vascular tissue. The size of the active cells in Impatiens was found to be about o'o8 mm. in diameter. Contact of the Probe with the xylem did not cause any pulsation ; this is highly interesting, proving that the dead xylem does not take any active part in the propulsion of sap.

The proper season for Impatiens was over by September, and all the specimens died by October. There were, how- ever, other plants growing in the grounds of the Institute, among which were : Cauliflower [Brassica oleracea var.). Bean {Vicia Faha), Potato [Solanum tuberosum), and Tomato {S. Lycopersicum esculentum) . I wished to find out whether it was possible to detect cellular pulsation in all these plants under normal field-conditions. The electric con- nections were made with the plant in the usual manner, the wires being led to the galvanometer inside the laboratory : they all gave evidence of cellular pulsation. In Brassica the electric pulsations were even more vigorous than those obtained with Impatiens : I will therefore describe in detail the experiment with this plant, the results obtained with others being given in a subsequent table.

In order to localise the pulsating layer with greater accuracy, the Probe was introduced by successive steps of 0-05 mm. As the intervening distance between the epidermis and the pith was about 0-7 mm., this necessi- tated twelve successive observations, each requiring fifteen minutes ; the total period of the experiment was thus lengthened to about three hours. Fortunately, the plant, under field-conditions, was in a state of exceptional vigour. The experiments had, however, to be completed preferably before afternoon, for there is a depression of activity towards evening. The pulsation was found to be feeble to a depth

of 0*25 mm., when it exhibited an abrupt increase, the ampHtude of pulsation being now 68 mm. A microscopic section after the experiment showed that the most internal layer of the cortex abutting upon the endodermis was at a depth of 0*26 mm. When the Probe was pushed Fig. 77. Section of the Petiole of Brassica, and the Curve of Cellular Activity at Different Layers E, epidermis ; c, cortex ; Cj, the active internal cortical layer ; En, endodermis ; b, phloem ; x, xylem ; p, pith. Note the sudden enhancement of activity at the layer Cj.

further in by 0*05 mm., it reached the phloem, and the pulsating activity of that layer was found to be very much less, the amplitude being reduced to 15 mm. When the Probe reached the xylem, pulsation had practically ceased, the amplitude being reduced to about i mm. : it should be borne in mind that living cells are not altogether absent from the xylem. The activity in the pith was found to be so feeble as to be neghgible. In the following table are given the quantitative values of the cellular activity, of the different layers of cells. I also reproduce a drawing of the microscopic section, made along the line of passage of the probe, giving at the same time a curve representing the amplitude of pulsation at the different layers (fig. 77). The section, after being moistened slightly, must be examined immediately after the experiment ; too long an immersion in water is apt to cause a swelling of the cells, which vitiates the measurements.

Table XXVI. — Amplitude of Electric Pulsation at Different Layers {Brassica oleracea) Examination of the curve given in fig. 77 shows that the amplitude of pulsation attains a maximum at the most internal layer of cortical cells which abuts upon the endo- dermis, the curve undergoing an abrupt fall both outwards and inwards. The ascent of sap in the stem depends on cellular activity, which has been shown to be most marked in the internal cortex : we are therefore led to the con- clusion that this innermost layer is the one that is specially active in the propulsion of the sap.

The experiment was repeated with Lycopersicum, Vicia and Solanum. The following is a tabular statement of the results. The active cells in Solamim were found at a relatively greater depth than in the other specimens. Table XXVII. — -Amplitude of Electric Pulsation at Different Depths in the Tissue of the Stem In all of these cases the cellular activity was localised in the innermost cortical layer ; in plants having an endo- dermis, the active layer abutted upon it ; in others it was contiguous to the phloem. Accurate localisation of the active layer is facilitated by the fact that the rise of activity detected by the Probe during its approach, and the fall of activity during its recession, are very abrupt. This will be understood from the mean results of observations upon four different species of plants. "When the Probe was in contact with the active cortical layer, the mean amplitude of pulsation was 84 mm. ; at a centrifugal distance of o-i mm. from this layer, the amplitude showed a decline to 6 mm. ; and at a centripetal distance of o -i mm. it was only 5 mm.

All living cells may exhibit pulsation to a greater or less degree : the activity of the internal cortex is, how- ever, exceptionally great. Thus there is, in the stem of dicotyledonous plants, a cylindrical sheath, a few cells thick, surrounding the vascular tissue, which subserves the rapid conduction of sap. For reasons shortly to be given, the cellular pulsations of this layer propel the sap preferentially upwards. The functional xylem-vessels are situated very near the active cortex, and in the case of Brassica are only 0-15 mm. distant from it. The injection of sap into the xylem may, therefore, be accomplished without difficulty or delay during the phase of expulsive contraction of the pulsating cells in the cortex. The in- active xylem may be regarded as a reservoir, the water being pumped in or withdrawn according to circumstances. These results also bring out the important fact that some of the important physiological organs are grouped in the closest proximity to each other. Passing from the outside to the centre, we first encounter the active cortex which maintains the rapid ascent of the sap. The next layer is the endodermis, which may be regarded as the sense-organ for the perception of the stimulus of gravity : it is the falling starch-grains in the endodermis that initiate the reaction by which the plant orientates itself in relation to the vertical. The geo-perceptive endodermis, in its turn, is in contact with the phloem, and I have shown elsewhere that the phloem functions as the nerve of the plant. All the principal systems of tissues regulating growth and move- ment are thus found to be in close relation with each other.

Each pulsating cell in the active layer executes periodic contraction and expansion, and it is obvious that, if these phasic changes occurred simultaneously in all the cells, the propulsion of sap in a given direction would be an im- possibility. There must, therefore, exist a phase-difference, a sequence of pulsation from cell to cell. Have we any proof that such phase-difference exists, and that there is a co-ordination of activity in a vertical row of cells along which the sap is being propelled ? In order to determine if this sequence could be estabUshed, I employed the method of exploration with the Electric Probe. Let us imagine a vertical row of cells, Cj, C2, Cg . . . c„. If there is sequence

of activity, it would then follow that while Ci is contracting, another cell, c„, will be in the opposite phase of expansion ; Ci will thus be expelling sap, while c„ will be absorbing it ; the direction of propulsion will thus be from Ci to r„. The difference of phase between one cell and the next will be slight, but at some particular distance from each other two cells will be in opposite phases of activity, that is to say, while one is contracting the other is expanding, and vice versa.

In order to detect this phasic difference by means of the exploring Probe, one electric contact a, made with the stem, is permanent, while the distance of the second contact B from the first is gradually varied. When the contacts A and b are very near each other, the cells at the two contacts will be very nearly in the same phase, they will be expanding or contracting at about the same time. While contracting, A will indicate electro-negativity, so will the electrode b ; during expansion both the electrodes will indicate electro-positivity. There will be little or no electric difference at the two electrodes, and the galvano- meter will be practically quiescent. The case will, how- ever, be different when b is sufficiently separated from a, so that it is in contact with a cell the phase of which is opposite to that at a. When contraction is taking place in A, there will be expansion at b : the electric change at a will be negative, that at B positive, and the electric difference of the two electrodes will be a maximum. The same maximum difference will occur when the cell at A expands and that at b contracts : A will now be electro- positive and B electro-negative. The swing of the gal- vanometer spot of light will be now in the opposite direction. Thus by increasing the distance from b to A, we shall pass from an electric minimum to a maximum, and this in spite of the increasing electric resistance interposed in the circuit. As the distance is further increased the phase-difference will be diminished, and we shall arrive at a second minimum, followed by a second maximum, and so on.

We have here a case analogous to the propagation of the periodic disturbance of waves of Ught and of sound. The distance between the two successive points in opposite phases is half the length of the wave. The velocity of propa- gation of the wave is given by the formula V = n\, where n is the number of pulsations in an unit time, and \ the length of the wave. We also know that when the distance between the two points of the propagated wave is increased from o to 2\, to 3X, etc., that is to say, by even multiples of half the wave-length, the two points will be in the same phase ; but if the distances

Hence if the ascent of sap he due to periodic hydraulic waves, the fact will find its crucial demon- stration in the detection of succes- sive electric maxima and minima in the path of conduction. This demonstration will also prove that there is a sequence of pro- pulsion from cell to cell. The experimental difficulties in this demonstration are, however, considerable. The velocity of propagation, and therefore also the wave-length, are not the same in different specimens. Hence the position of the second electrode for the first maximum (where the phase-difference is

Fig. 78. The Method of Ex- ploration by the Electric Probe for the Determina- tion of the Hj-draulic Wave-Length A is the fixed, and b the ex- ploring contact ; when b is at half the wave-length, the electric variation is maximum. This electric variation decreases as the second contact B is i^oved nearer to (Bi) or farther away (Bj) from a than the maximum point b. opposite) can only be found by trial, by thrusting in the exploring Probe at gradually increasing distances from the first. But the numerous intervening wound-spots would undoubtedly modify the normal velocity of ascent. The procedure adopted to minimise this difficulty was to reverse the process of exploration, that is to say, gradually to diminish the distance between the two electrodes, instead of increasing it. The wound spots would then remain outside the region of exploration (see fig. 78).

A preliminary series of experiments was carried out with specimens of stem of Chrysanthemum, which were in every respect alike. I thus obtained a definite idea of the position of the first electric maximum, which was found to be at a distance of 50 mm. This obviated to a great extent the fatigue which might occur from too many pricks with the exploring Probe. I give a detailed account of two typical experiments from the numerous determin- ations which gave similar results. In the first, the Probe was, to begin with, placed at a distance of 70 mm. ; the response was found to be feeble. The Probe was then brought nearer, to a distance of 50 mm., and at once a great enhancement of the amplitude of pulsation was indicated. As the distance of the Probe was further reduced to 30 mm. the amplitude was much smaller ; and at a distance of 5 mm. there was practically no electric difference (fig. 79). The distance from the maximum to the minimum is thus 50 mm., the wave-length being 100 mm.

In the second experiment, with a different specimen of Chrysanthemum, two probe-contacts were made : one at B at a distance of 50 mm., and one at Bi at a distance of 5 mm. from the contact at a. The object of this was to make allowance for any variation that might con- ceivably occur during the experiment. The record was first taken with the contact at b, ; this gave a minimum amplitude. The next was taken with the contact at b, at a distance of 50 mm. ; this gave the electric maximum.

79. Determination of the Wave-length in Chrysanthemum. The electric maximum is at 50 mm. Note the enfeeblement of response as the probe is moved nearer Fig. 80. The Cyclic Record with the Second Contact at 5 mm., Fig. 81. The Einthoven Gal- vanometer Record exhibiting the Phenomenon of Interference The recurrent ' beats ' occur when the second point of con- tact is not exactly at half the wave-length. with the probe-contact once of the electric minimum was the same as at the beginning (fig. 80). The electric maxi- mum is thus found to lie at the same distance as in the first experiment of the series. In certain other experiments I obtained a second minimum and a second maximum, the distance between the succcst sive minima being the same as that between the successive maxima. Prolonged experi- ment is, however, apt to the response. The electric at the exact distance of half

the wave-length. Failure in securing this gives rise to the very interesting phenomenon of ' beats ' seen in the record obtained with the Einthoven galvanometer (fig. 81). Determination of the wave-length in Musa.- — I em- ployed the same method in the determination of the wave-length of the hydraulic wave in other plants. The following (fig. 82) is a record of the responses when the ex- ploring electrode was placed at successive distances of 40 mm., 30 mm., 25 mm., and 20 mm. With diminish- ing distance the electric variation increased till it reached a maximum at a distance of 25 mm. ; this is the point of the electric maximum, for further diminution of distance brought about a diminution of the electric variation.

The phase-difference is maximal at a distance of which is therefore equal to half the wave-length. The wave-lengths of different species of plants under normal conditions and at a temperature of 30° C. are given below. Fig. 82. Determination of wave- length of Cellular Pulsation in Musa The Probe is gradually brought nearer from the fixed contact, from 40 to 20 mm. The electric maximum occurred at 25 mm., which is half the wave-length. The velocity of the ascent of sap caused by the propagated hydraulic wave is, as we have seen, modified

under physiological variation ; it is increased by the application of warm water at the root, or at the cut end of the stem. We have further seen that the velocity of the wave- propagation is V = n\, where X is the length of the wave. But the increase of velocity on the applica- tion of warm water may be due either to increase of frequency or to increase of wave-length, or to both. That the wave-length is increased will be seen from the results of the following experiment. I took a petiole of Miisa and made two contacts, A and b, at a dis- tance of half a wave-length, which was found in this and in the pre- vious experiment to be 25 mm. Application of warm water at the cut end enhanced the velocity, the result being that the existing electric difference between a and B was found to have undergone a great diminution, so that it was necessary to increase the dis- tance from the original 25 mm. to 35 mm. in order to obtain the new electric maximum ; the wave- length thus increased from 50 mm. to 70 mm. The increased cellular activity is also shown by the enhanced amplitude of pulsation (fig. 83).

Fig. 83. Effect of Rise of Temperature in increas- ing the Wave-length The pulsation at maximum point of 25 mm. had become irregular and diminished in amplitude as seen in second pair of records. The transfer of second contact to 35 mm. gave the new maximum with its enhanced amph- tude of pulsation. Upsetting of Phase-difference by Passage of a Constant Electric Current Another very interesting method of upsetting the existing ■difference through the agency of an external agent

is that of the passage of a constant electric current. I have shown elsewhere that the intensity and the velocity of a propagated physiological impulse are modified by the action of a constant current. With a current of moderate Fig. 84. Translocation of the Point of Electric Minimum by Passage of Electric Current The figure to the left represents the experimental arrangement. The record to the right shows the effect of passage of current in the translocation of the electric minimum.