The Physiology of the Ascent of Sap

The following is an account of a typical experiment for the determination of the velocity of the ascent in an intact specimen of Impatiens by the electrometric method. Suitable connections being made with the stem and the distant leaf, a continuous record was taken on a photo- graphic plate. The record (fig. 65) exhibits certain feeble pulsations, the special significance of which will be explained in a subsequent chapter (p. 212). On irrigation at the point marked with an arrow, there was no immediate effect ; but after an interval of seven minutes there was a sudden erection of the base-line towards electro-positivity, accom- panied by two large pulsations. The increased turgor due to the ascent of sap to the first point of contact is thus signalled by a flexure of the base-line upwards. The absolute value of this induced positivity was found to be 0 -04 volt.

The interval between the application of water and the hydro-electric response was, as stated before, seven minutes ; the intervening length was 78 mm. ; the velocity was therefore 11-2 mm. per minute, which we found to be the velocity of ascent in Impatiens in a ' moderate ' con- dition (p. 41). Having determined the absolute value of the electro- motive variation by the Quadrant Electrometer, we next employ the Galvanometer, with its relatively higher sensitive- ness. In order to measure accurately the short interval of time, the reflected spot of light from the galvanometer is interrupted periodically at intervals of fifteen seconds. The record thus consists of a series of short vertical lines, and the successive spacings represent intervals of fifteen seconds. The dotted record is thus its own chronogram.

On irrigation of a particular specimen of Impatiens, a very strong positive response occurred after two minutes ascended was 80 mm., the velocity of ascent being thus 29 mm. per minute, that is to say, nearl}' three times greater than in the pre- vious case. In the record (fig. 66) the normal positive is seen to be preceded by a transient negative re- sponse, indicative of an excitatory contraction and diminution of turgor. This must have been brought about by the shock-effect of the hydrostatic blow

Irrigation at arrow. The galvanometric positivity indicative of arrival of hy- draulic wave occurred after ii dots, i.e., 2' 45". Note the preliminary negative twitch due to hydrostatic blow. (Successive dots at intervals of 15".) Whilst investigating the transmission of excitation in Mimosa, I became aware that under certain circumstances a sudden variation of hydrostatic pressure may act as a mechanical blow upon the sensitive pulvinus. Under moderate stimulation by electric shock applied at a distance, this hydrostatic disturbance does not occur, and the ex- citatory change proceeds with a definite velocity along the special conducting nerve. Thus in a given specimen the application of electric stimulus at a distance of 30 mm. from the responding pulvinus caused the fall of the leaf after an interval of 1-9 seconds, the velocity being thus 16 mm. per second. This velocity was found to be constant in successive experiments. If instead of the electric stimulus we employ a violent mode of stimulation, such as

a cut or a burn, a hydrostatic disturbance occurs, which, traveUing with great speed, dehvers a mechanical blow on the sensitive pulvinus, causing a fall of the leaf almost Fig. 67. The Method of Simultaneous Determination of tlie Velocity of Ascent by the Mechanical and Electric Methods The mechanical lever-recorder is on the right with recording glass plate g ; the galvanometer is to the left ; light, L, reflected from its mirror falls on the photographic plate p. The curved part of the stem is connected b>- a string with the recording lever ; the same point is in electric connection with the galvanometer.

instantaneously ; this mechanical transmission is unlike the relatively slow point-to-point propagation of nervous excitation. The effect of the hydrostatic shock is thus an excitatory mechanical contraction, with the concomitant electric response of galvanomctric negativity. A similar transmission of a hydrostatic blow may be expected in plants in which the roots on irrigation absorb water with great activity, which gives rise to a hydrostatic disturbance. This will travel with great rapidity, and by its shock-effect cause contraction and galvanomctric negativity at the distant responding point. The quick hydrostatic impulse will be followed by the slow hydraulic wave with its positive electric response indicative of expansion. The preliminary negative and the consequent positive in fig. 66 are thus probably due to the hydrostatic and hydraulic effects respectively.

In the mechanical records of the ascent of sap hitherto obtained, no corresponding preliminary negative response had been noticed. The explanation of its absence may be either (i) that such contractile effect, even if it took place, was too small to be detected by the low magnification of the record ; or (2) that the response was transitory, and was therefore missed in the interval of the successive dots, which was usually one minute. I therefore undertook an investigation to find out whether, under the given conditions, an excitatory negative mechanical response occurred preceding the normal erectile response. For this the recording plate was maintained in oscillation at intervals of fifteen seconds, and the magnifying power of the recording lever was at the same time increased. I also arranged for the simultaneous record of the mechanical and electric responses.

Simultaneous Determination of Velocity by the Mechanical and the Electric Method In the experiments hitherto described to determine the velocity either by the electric or by the mechanical method, the specimens employed were necessarily different. Since the physiological condition of any two plants is probably not identical, the values obtained by the two methods cannot be compared with each other. For testing the relative reliability of the two methods, we must so arrange matters that the two records, mechanical and electrical, are obtained from an identical specimen. The agreement of the two results will prove the accuracy and reliability of the two methods.

The Method of Record. — The experimental arrange- ments are as follows : the bent portion of the drooping stem is attached to the Mechanical Recorder by a thread, tlie Fig. 68. Simultaneous Mechanical (lower curve) and Electric (upper curve) Records, in Determination of the Velocity of Ascent ; irrigation at arrow- Note simultaneous negative and positive responses in both. oscillation of the smoked plate being once in fifteen seconds. Galvanometric connections are also made with two points, one on the curved portion of the stem, and the other on a distant leaf. The reflected spot of light from the galvanometer mirror falls on a photographic plate, which descends at the same rate as the smoked glass-plate of the Mechanical Recorder (fig. 67). The source of light for the galvanometer record is a 4-volt pea-lamp. The oscillating part of the Mechanical Recorder periodically

makes and breaks the electric light circuit. The successive dots in the two records represent the same interval of time, namely, fifteen seconds. Table XXIV. — 'The Kates of Transmission of Hydrostatic Impulse and of the Conduction of Sap The specimen employed for the following experiment was in an optimum condition. The simultaneous records given in fig. 68 exhibit the very striking similarity between the mechanical and electrical responses. The hydrostatic shock-effect occurred simultaneously in the two, exhibited by a downward excitatory twitch in the mechanical, and a downward excitatory deflection in the electric curve, indicating galvanometric negativity. The shock-effect, due to transmission of hydrostatic impulse, occurred in both after 4 dots, or one minute after irrigation. As the inter- vening length of stem was 90 mm., the velocity of the hydrostatic impulse was 90 mm. per minute. There was a quick recovery from the excitatory effect. The conducted water next reached the responding region and gave rise simultaneously to positive responses in both — an erectile movement in the mechanical, and an upward deflection, indicating galvanometric positivity, in the electric record. As this hydraulic response occurred four minutes after the application of water, the velocity of ascent was 23 mm. per minute. The hydrostatic effect is thus seen to be sharply defined from the effect of the conduction of sap, the velocity of which has been determined by two different methods

with identical results. I give in Table XXIV. the detailed results of several experimental determinations with different specimens, which, on account of their different physio- logical condition, exhibited different rates of conduction. The transmission period obtained by the mechanical and electrical response, as already stated, was the same. The employment of this method is of much theoretical interest. Instead of making the second electric contact at a distant indifferent point, we make it with the stem itself at a certain distance from the first. When the water of the ascending sap reaches the first contact, the electric signal is given of galvanometric positivity at that point. This deflection will remain constant for a certain length of time. But as the water, continuing its ascent, reaches the second contact, there will be produced a galvanometric positivity at that point which will cause a sudden reversal of the previous deflection of the galvanometer. Suppose the distance from the root to the first contact A is D, and the time-interval between the application of water and the first electric response is T, then

If the interval of time between the first response and the subsequent reversal be t, and the distance between the two contacts /, then the velocity V of transport between the two points will be The distance from the root to the first contact cannot be determined as accurately as the distance between the two contacts. Hence the determination of the velocity by (2) will be the more accurate. The specimen employed in the following experiment was not in an excitable condition, so the disturbing element of the hydrostatic blow was absent. The first positive electric response occurred two minutes and fifteen seconds after the application of water to the root. The distance D was 40 mm.

The distance I between the first and the second electric contacts was 55 mm., and the electric reversal took place three minutes and fifteen seconds after the first electric response. The velocity is therefore The two velocities are thus seen to be practically the same. Increase of turgor is attended by an electric change to galvanometric positivity, while diminution of turgor induces the opposite change to galvanometric negativity. The ascent of sap to any point (causing an increase of turgor) is thus signalled by a deflection of galvanometric positivity.

Simultaneous records obtained by the mechanical and electric methods give identical values for the velocity of the ascent of sap. When two electric contacts are made on the stem, one above the other, the positive electric response takes place first at the lower contact ; the ascending water then reaches the upper contact and causes a reversal of the previous electrical response. The interval between the two responses is the time taken by the sap to travel through the inter- vening distance. The variable time lost in absorption by the root is thus eliminated in the Di-phasic method.

absorption of water which causes a hydrostatic impulse ; this, travelling with great rapidity, delivers a mechanical blow at the distant responding point, causing an excitatory response of contraction and galvanometric negativity. The records of both mechanical and electrical response in an excitable specimen exhibit this preliminary negative, followed by the normal positive due to the ascent of sap. The lectric Probe for detection of pulsation in the interior of the plant — Turgor and electric variation during a single pulsation — Electric pulsation of Desmodium — Periodic groupings of pulsations — Record of pulsation of a single cell — Cellular pulsation in herbaceous plants — Pulsating cells in trees — Pulsatory activity modified under variation of temperature — Record of pulsation by Einthoven gal- vanometer— The period of a single pulsation — Summary.

A DETAILED account has been given in the preceding pages of the ascent of sap and its diverse manifestations. The transport of sap has been shown to be brought about by the co-operating activity of numerous hving cells, such activity being appropriately modified under physiological variations. The plant, as a whole, may be regarded as a machine for pumping water from the soil and excreting it outside, in which the active cells concerned in the ascent of the sap act as a series of pumps arranged in a vertical row. We have a mechanical model of such a cellular pump in an india-rubber bulb, such as is used for spraying and other purposes : the contractile down-stroke causes an expulsion, and an expansive up-stroke brings about a suction of water. The cellular pumps may likewise be visualised in action, with alternate contractile down-stroke and expansive up-stroke. The former causes an expulsion of water from the cell with resulting diminution of turgor ; the expansive up- stroke sucks in water, with concomitant increase of turgor.

A steady propulsion of water may be maintained by the uniform rhythmic action of a vertical series of such cellular pumps. When the up-stroke is equal to the down- stroke, the absorption and expulsion will be equal, and the average turgor of the cell will remain constant, though after the completion of each up- or down-stroke a variation of turgor will occur above and below the mean value. Complications are likely to arise when the up- and down- strokes are unequal.

All life-movements must ultimately be due to the elementary activities of individual cells. As in the study of chemical phenomena we strive to get an insight into the molecular or atomic activities, so also in the investiga- tion of the dynamics of life a very great advance will be assured if we can get access to the smallest unit of hfe, the individual cell, or the ' life-atom ' — a congregation of which constitutes the living organism. But the pulsatory movement of a cell is ultra-micro- scopic, and its detection may well appear to be beyond the range of possibility. However, the detection of ultra- microscopic movements is not so hopeless as it has been assumed to be, for the Crescograph, which I have devised, enables us to obtain a magnification of from ten to a hundred million times. This would be sufficient to detect not merely cellular but also atomic movements. The difftculty of recording cellular pulsation does not therefore arise from a lack of sensitiveness in the instrument, but from the practical impossibility of attaching a single cell to the Crescograph.

I have, however, been able to overcome the difficulty of securing contact with an individual cell by the employ- ment of the Electric Probe, which I devised for my ' In- vestigation on the Localisation of the Geo-perceptive Organ.' 1 One terminal of a sensitive galvanometer is connected with the Probe, which is thrust into the geo- perceptive stem, step by step, the other terminal being connected with a distant indifferent point. When the stem is laid in a horizontal position, a particular layer becomes stimulated under geotropic action. As the Probe enters the stem, it begins feebly to pick up the excitatory electric change due to the geotropic stimulus. A sudden en- hancement of this occurs when the Probe reaches the

geo-perceptive layer itself, the response of galvanometric negativity being now at its maximum. As the Probe passes beyond the sensitive layer, the electric indication rapidly disappears. This method is extremely delicate, and it is thus possible to localise the particular layer of cells inside a plant which perceives and responds to a stimulus. I have also been able to localise by the Probe the particular strand in the interior of the petiole of Mimosa leaf which functions as a nerve along which excitatory impulse is being transmitted.^

We will now try to find out whether it is at all possible to localise by means of the Electric Probe the active cells in the interior of the plant which, by their rhythmic activity, cause the propulsion of the sap. The character- istic of such a pulsating cell is that it undergoes alternate expansion and contraction, the former being attended by the absorption of sap and increase of turgor, and the latter by expulsion of sap and diminution of turgor.

In an ordinary non-rhythmic cell, the state of turgor is normally constant, and its electric potential, therefore, remains unchanged. But in a pulsating cell there is a series of alternate expansions and contractions, an up- stroke followed by a down-stroke : consequently, the rhythmic cell will exhibit periodic fluctuations of turgor below and above the mean. I have already shown that an expansion and an increase of turgor may be electrically detected by galvanometric positivity, and a diminution of turgor by galvanometric negativity. It should there- fore be theoretically possible to detect these pulsations by putting the rhythmic cell in connection with a galvano- meter by means of the Electric Probe, the other contact being made with an indifferent point. The expansive up-stroke of the cell, with the phase of increased turgor, would be detected by a positive, and the contractile down- stroke, with diminution of turgor, by a negative electric variation. The galvanometer spot of light would reveal, by its alternate swings to the right and to the left, the

invisible pulsations of the active cells in the interior of the plant. Table XXV. — Turgor and Electric Variation during one Complete Pulsation The tabular statement given above will explain the different phases of the pulsating cell, with their electric concomitants. Having explained the theory of the electric detection of pulsation in rhythmic cells, we shall next try to find out whether this can be rendered practicable for experi- mental purposes. In demonstration of this I will first give an account of the electric pulsations which were recorded with the leaflet of Desmodium gyrans. The pulsatory activity of its pulvinule is quite evident from the automatic up-and-down movements of the leaflet. The period of a single pulsation under favourable circumstances is as short as a minute ; in a sluggish condition the period may be lengthened to five minutes or so. In the mechanical record we notice various types, ranging from irregular to uniform pulsations ; the pulsation of a detached leaflet is often irregular, probably from the shock-effect of amputa- tion. If the preparation is kept for a length of time under favourable conditions, the pulsations tend to become uniform ; they often exhibit periodic groupings similar to those seen in the record of pulsations of the isolated animal heart.

As regards the mechanics of the pulsatory movement of the leaflet of Desmodium, we may, for the purpose of simplicity, ignore the feeble action of the upper half of the pulvinule, the lower half being relatively far more effective. In the periodic movement of the leaflet, the expansion and enhancement of turgor of the lower half cause the up- movement ; contraction and diminution of turgor, on the other hand, give rise to the down-movement. For the electric determination of these pulsations we make suitable connections with a sensitive galvanometer, the first contact being made with the lower half of the pulvinule, and the second contact with the inactive sub-petiole. After this we observe very remarkable manifestations of electric pulsa- tion, in which the up-stroke of galvanometric positivity

corresponds with the expansive up- movement, con- comitant with in- crease of turgor, of the pulvinule. The down-stroke of the electric pulsation is, on the other hand, concomitant with the down- movement which results from con- traction and dimi- nution of turgor. It has to be borne in mind that the electric pulsation is not due to the mechanical pulsation as such, for if we hold the leaf and thus prevent its up-and-down movement, the electric pulsation is found to persist. Both the mechanical and electric pulsations are but different indications of the periodic changes of turgor of the active cells. I re- produce the galvanograph of the electric pulsations of the leaflet, which are here seen to exhibit periodic groupings (fig. 69), as is often found in the record of the mechani- cal pulsations of the leaflet. Other electric records exhibit different types ranging from the irregular to uniform pulsations.

Fig. 6q. Record of Electric Pulsations of the Pulvinule of Desmodium exhibiting Periodic Groupings In attempting the detection and record of the pulsating cells in the stem, we follow the same procedure as in the case of the Dcs- modiuni leaflet. Of the two contacts with a sensitive galvanometer, one is made with a distant indifferent point, such as a dying or dead leaf in which all life-activity is arrested or abolished. The other contact is made with the stem in which ascent is taking place, by a fine plati- num wire. As the wire is gradually thrust into the stem, the sub-epidermal cells give no indication of any pulsation ; but as soon as the point of the wire comes in contact with an active cell in the deeper tissue, the electric pulsations be- come very vigorous, as will be seen in the records ob- tained.

The records of the cellu- lar electric pulsations thus obtained exhibit charac- teristics similar to those shown in the records ob- tained with the Desmodium leaflet. There are both irregular and regular pulsations ; and they sometimes exhibit periodic groupings as in fig. 70, which is a record obtained from Impatiens. The remarkable similarity be- tween this and the record of Desmodium (fig. 69) is very striking. The next record (fig. 71) shows the uniform pulsations given by the petiole of Musa}

The first records which I attempted to obtain were from herbaceous plants, as they are easier to manipulate. A misgiving may exist that cellular activity is not operative in tall trees, other agencies being presumably concerned in the transport of sap to great heights. Three tall trees happen to grow in the grounds of the Institute, and these gave me an opportunity for investigating the subject. The first is a Mango-tree, the second is a Ficus religiosa, and the third a Cadamba [Nanclea cordifolia). The last two grow to a height of 30 feet (10 metres) or more. Suitable electric connections were made with a young branch near the top of the tree, and the wires were led to the recording galvanometer inside the laboratory. The cellular pulsations were found, under favourable con- ditions of light and warmth, to be very active, in fact even more vigorous than in herbaceous plants. In fig. 72 is seen the record of the uniform pulsations of the Mango- tree ; fig. 73 gives the record of cellular pulsations of Ficus, in which we observe periodic groupings. The Ficus was in a state of disturbance owing to the action of the wind, which caused flutterings of its numerous leaves. The record of the pulsations is therefore not uniform, but is characterised by periodic groupings. Cadamba also gave similar results.

A very interesting fact in connection with the cellular pulsations is that their vigour is dependent on favourable physiological conditions. The records given above having been taken during winter, the pulsations were found to I The minor fluctuations in the electric record, fig. 65, of the velocity of ascent of sap, are now understood to have been due to cellular pulsations. be very feeble in the cold morning. Satisfactory records could therefore be obtained only at midday, when warmth increased the cellular activity and thus enhanced the amplitude of pulsation. I had occasion to repeat the

experiment in summer, when the temperature at midday was 40° C, which is above the optimum-point. This caused a great enfeeblement of the pulsation, so that it was only by experimenting early in the morning that it was possible to secure any satisfactory record during the summer. The electromotive variation induced b}- cellular pulsation is about a millivolt or so, and the resistance of the circuit is very high, being about a million ohms. For taking a record of cellular pulsation it was therefore necessary to use a very sensitive D'Arsonval galvanometer, which