Plant Response as a Means of Physiological Investigation

We have seen in Chapter XXI. that when a strong stimulus is applied to the base of any organ, say a stem, a multiple series of excitatory waves is propagated onwards, such multiple responses being detected by electrotactile or electromotive pulsations. It was also shown in Chapter XXVI. that such multiple, passing into automatic, response, may be induced by the action of a constant stimulus. It was further demonstrated in Chapter XXI. that these excitatory waves, and the concomitant cell-to-cell contraction, would produce a movement of water forwards, along the direction of propagation. This series of excitatory waves, proceeding from the base of the organ, and propelling water forwards, must then cause a deficit of water behind. If, however, the base of the organ be kept supplied with water, this deficit will be made up by suction.

It is thus seen that by such rhythmic activity a onedirectioned movement of water may be produced. Just as the various effects produced by multiple or autonomous response — in, for example, the electromotive and electrotactile responses, and in the multiple mechanical responses of Desmodium — give us an indication of the degree of rhythmic activity exhibited by the tissue, so, in the rate of this watermovement also, we have an additional means of measurement. 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 this rate of movement may be measured, either by means of the propulsion of water forwards or by the suction exerted behind.

The ascent of sap in the plant, then, may be brought about by the rhythmic activity of the tissue. How this activity is initiated will be discussed later. Meanwhile it is clear that if the movement of sap be really an expression of protoplasmic activity, then any physiological modification which tends to increase that activity will also tend to increase the rate of movement ; and pari passu any physiological condition which tends to depress the activity, will

correspondingly express itself in a diminished rate of movement. The movement of sap is thus taken to be another expression of that autonomous activity (multiple response) of the plant, which we have already seen exhibited locally by the motile tissue of Desmodiutn. The conclusive test of this would lie in proving that all those agencies which acted in a given way on the autonomous response of Desmodium, act also in the same way in modifying the rate of movement of sap. In other words, just as an exciting reagent will in the one case induce a greater amplitude, frequency, or both, of oscillation, above the normal, so in the other the excitatory nature of a given reagent may be expected to exhibit itself by an increase above the normal, in the rate of flow. A depressing reagent, on the contrary, should produce the opposite effect in both. That is to say, just as we may study the multiple or rhythmic excitability of a tissue through mechanical, electromotive, or electrotactile response, so here, in hydraulic response, or the determination of changes in the rate of flow of sap, we have an independent mode of investigating the same phenomenon.

The great difficulty of this investigation lies in the absence of a method by which these changes can be immediately recorded. In other words, we require some simple means of making a direct record, which will show, in a continuous manner, the changes produced by the various agencies, enabling us to distinguish their immediate effects, after effects, time-relations, and so on. We have seen that the propulsion of water forward by the tissue is attended by a suction behind.1 The quantity of water sucked up in a definite period will therefore give us an indication of the rate of movement of sap in the plant. Thus from the readings afforded by the water-index of a potometer, and the times at which such readings are taken, we may derive

1 The forward movement of water, and the suction exerted, in the same tissue, are not necessarily equal in all cases. Part of the water sucked up may be deviated to increase the turgidity of the cells themselves. Suction may nevertheless be taken as a measure, other things being equal, of rhythmic activity. the rate of movement. But these readings are necessarily discontinuous, and important phases of change are thus apt to be overlooked. They are, again, subject to error ; nor do they give us, at sight, the rate of flow, nor the changes in that rate, at any given moment. In order to determine such variations, a laborious process of construction of curves, from the experimental data, must, generally speaking, be undertaken.

It was therefore necessary to devise an apparatus by means of which curves might be obtained direct, so that a mere inspection would be sufficient to inform us as to the normal rate of suction, and the influence of external factors on that rate. This suction will be shown to be a physiological phenomenon ; its variation, therefore, will enable us to measure the physiological influence of various external agencies. It must be borne in mind that under ordinary conditions there is a normal rate of suction. The incidence of an external stimulus will change this rate, and the variation of rate which results is thus a measure of the effect produced by the stimulus. Similarly, we measure a force by noting the variation which it produces in the rate of movement of a uniformly moving body. The variation of the rate of suction may thus be taken as constituting a form of response to stimulus, which for the sake of simplicity we shall designate as Suctional Response.

The Shoshungraph. — For the purpose of subjecting the plant to various conditions, and also in order to obtain the record of the resultant suctional response, I have constructed an apparatus to which I have given the name of the ShoshnngrapJi} It consists of (1) an arrangement by which the specimen may be rapidly subjected to the action of different excitatory or depressing agents ; (2) a potometric tube, by which the constant changes of suctional activity are measured ; (3) a contrivance by means of which the movements of the water-index, with their time-relations, are recorded. The principal parts of this instrument are shown diagrammatically in fig. 156. V is the plant-vessel, in which

the specimen is mounted by means of a water-tight indiarubber cork. The vessel is closed at the bottom also by means of a large cork through which enter four tubes. One of these, controlled by the stop-cock A, is connected with the capillary potometer-tube. The second, controlled by A', leads to the compensating vessel C, filled with water. The interior ends of these two tubes reach almost to the top of the plant-vessel. The third, with stop-cock P,', leads to

plant-vessel ; c, compensator ; r, reservoir. The potometer-tube is controlled by stop-cock, A ; compensator by a' ; and reservoir by b'. b is the stop-cock of the outflow pipe ; d, recording drum driven by clockwork ; p, collar carrying recording pen, seen magnified above. The water-index is followed by appropriate manipulation of the wheel, w. the reservoir R, from which water at various temperatures or different chemical reagents may be introduced. The fourth is an outlet-tube controlled by the stop-cock B.

Adjustment for Unbalanced Record. — The great difficulty in connection with delicate experiments arises from the presence of air-bubbles in the plant-vessel, which are not easy to expel. This is done, however, by means of an escapetube, with a stop-cock, which runs through the upper cork, and is not shown in the figure. The stop-cock A' is opened, A, B, and B' being closed. Water from the compensator c thus passes into the plant-vessel, and the air-bubbles which have been accumulating at the top of the vessel escape, with the water which is driven out at the escape-tube. When all are expelled then the stop-cock of the escape-tube, and A', are closed. The potometer stop-cock A is now opened, and the water-index is adjusted at any point desired, by manipulation of the stop-cocks B and A'. A temporary opening of the stop-cock A' of the compensator c causes the water-index to move to the left ; whereas, when the stop-cock B, of the outflow pipe, is opened, it moves to the right. After this preliminary adjustment the stopcocks A', B, and b' are closed, the potometer tap A being kept open ; the movement of the water-index per unit-time now gives us the normal rate of suction of the specimen.

We come next to the question of making a direct record of the rate of movement. For this purpose, a writing pen is fitted on the potometric tube, by means of a brass collar. This brass collar has a rectangular opening, which enables us to watch the water-index. It has also stretched across it a fine wire, which is kept always coincident with the waterindex. This wire is parallel with, and placed vertically above, the recording pen. The collar is attached to a thread which passes round small pulleys. One end of this thread carries a counterpoise and the other is wound round a wheel, w, which can be so manipulated as to make the index-wire follow the movements of the watercolumn. When the wheel is wound, the index moves to the right ; when it is slightly released, the weight of the counterpoise makes it move to the left. The weighted recording pen rests with its point on a revolving drum, D, covered with paper for the record ; this drum is kept revolving by clockwork at a known and adjustable speed. When the water-index is followed in the way described, there is produced a direct record of watermovement in the plant. 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 the suction is uniform, the slope remains constant. If any exciting agency increases the rate of suction, there is an immediate flexure in the curve, which thus becomes steeper. A depressing agent lessens the slope of the curve. And when suction is abolished, the record becomes horizontal. By this arrangement, then, we are enabled, simply and accurately, to obtain a direct and continuous record ; and as the necessity for taking readings is obviated, a large number of experiments can be performed very quickly, with little trouble. The flexure in the curve affords immediate visible indication of the effect of any particular agency. The value of each division of the ordinate is found once for all by determining the volume of unit-length of the potometer-tube. A previous determination at the beginning of the experiment of the rate of movement of the drum, gives us the timevalue of each division of the abscissa. Knowing these, we can determine the absolute rate of suction at any period of the curve required. Responsive variations of suction are more easily detected when the normal curve is almost equally inclined to the ordinate and abscissa — that is to say, when it makes an angle of about 450 with either. This is most easily accomplished if we keep the potometer-tube always the same, and merely adjust the speed of the drum.

The Balanced Shoshungraph. — According to the simple method of making records which has just been described, we observe the responsive effect by means of flexures produced in the curve under the action of various agencies. If the effect of an agency be slight, the change in the slope of the curve will be proportionately small and liable to escape detection. In order to bring the sensitiveness of the instrument to its highest, I have devised the Method of Balance, by which the slightest responsive variation is made to exhibit itself in a marked manner. For the purpose of many delicate investigations, it is not so necessary to know the normal rate of suction, as the variations positive and negative in that rate.

An agency which induces the positive variation will then be excitatory, while that which induces the negative is depressing. In order to carry out my investigations along these lines, I have employed two different methods of balance. In one — the Hydrostatic Method of Balance — the natural suction of the plant is arrested by a counter-hydrostatic pressure suitably applied. The effect of an external agent is now studied by the direction, positive or negative, and the extent to which it disturbs the static equilibrium thus established. Experiments carried out on this method will be described in the next chapter.

The second — or Hydraulic Method of Balance — depends upon an application of compensation, by means of which under normal conditions the water-index is kept stationary, though the suctional movement in the plant is in no way disturbed. According to this hydraulic method, the normal rate of suction, when balanced, gives rise to a neutral, or horizontal, line in the record ; while an exciting agent produces an inclination upwards ; and a depressing agent a declination downwards. The balance, by which under normal conditions the neutral line is secured, is obtained by allowing water to enter the plant-vessel from the compensator C at a rate exactly equal to that of its withdrawal by suction.

In practice, this adjustment is roughly made by opening the stop-cock A' in connection with c, to a greater or less extent. Over-balance causes movement of the water-index to the left ; under-balance to the right ; and when the adjustment is perfect, the water-index becomes stationary. After making the preliminary adjustment, the final balance may be obtained, by very careful and gradual movement of the compensating reservoir up or down. When the reservoir is raised the flow is increased, owing to the greater difference of level established, as between the reservoir and the plantvessel. The stand on which the vessel c is placed is provided with a rack and pinion, by means of which the necessary adjustment of height is made.

seen how the normal rate of suction and its variations may be recorded accurately. The next difficulty to be overcome is that of introducing the changed conditions without creating any disturbance, thus practically maintaining the continuity of record. It will be necessary to observe, among other things, the immediate and after-effects of cold and heat, as well as those of various chemical reagents. This is accomplished by the right manipulation of the four stop-cocks. Let it be supposed that we wish to study the immediate and after-effects of cold. Up to this time the stop-cocks A and a' have been opened — B and b' being closed— and the balanced horizontal record taken. For the sake of simplicity I refer to the stop-cock a' as the only one which opens and closes the communication with the compensator C. In reality, however, there is a second stop-cock in its neighbourhood, by which the balancing adjustment is first made, a' being employed for opening or closing communication under such an adjustment. The reservoir R is now filled with cold water ; A and A' are next closed — thus arresting the waterindex — and B and b' opened. The water then leaves the plant-vessel by the overflow pipe, and its place is taken by cold water from R. After this, the stop-cocks B and B' are once more closed, and A and A' opened. The index, being now released, indicates by its movement the excitatory or depressing effect of cold. It must be remembered that the index was previously adjusted to balance. Should the effect of cold prove to be excitatory, the rate of suction would be increased ; under-balanced by the supply of water from c, the index would move to the right, thus giving positive response.

If, on the contrary, the effect should be depressing, the rate of suction would be decreased, and the water from the compensator would produce an over-balance, causing a movement of the index to the left, or negative response. By now filling the reservoir R with water at the ordinary temperature, and repeating the operation, the original condition is re-established, and the effect of re-establishment of old conditions observed. In a similar manner,

we may study the immediate and after-effects of rise of temperature, and of the various chemical reagents. V, plant-vessel ; R, reservoir ; c, compensator, whose balancing height is adjusted by rack and pinion, s ; k, key for manipulation of four-way stop-cock ; P, recording pen, |with counterpoise M, manipulated by wheel, w. The drum is rotated by the clock at uniform speed. In order to make the explanation easier, I have described all these stop-cocks and their serial opening and closing separately ; but this arrangement, apart from its clumsiness, would involve a certain loss of time. In many of these experiments, it must be remembered, it is necessary to know the immediate effect produced. I have therefore simplified the procedure by the use of a special key, K, by turning which, in one direction or another, the requisite alternate openings and closings are accomplished.

Thus, on turning the key-handle to the left, A and A' are opened, and B and p/ closed. The balanced record is now taken. As the handle is now being turned to the right, the stop-cocks A and a' are first closed, arresting the index. Continued turning to the right opens B and B', by which the modifying reagent is introduced into the plant-vessel. A sudden turning of the key to the left now closes B and B', and opens A and a', thus releasing the index and enabling the record to be taken once more, under the changed conditions. The whole process is thus made so rapid, that modified conditions can be established, and the record renewed, within the short interval of less than one minute. The photograph of the completed apparatus is seen in fig. 157. Having now given in detail all the experimental arrangements, I shall in the next chapter describe the physiological modifications induced by different agencies, as exhibited by the suctional response. The periodic variation of ascent of sap may be recorded continuously and automatically by photography. Another method of recording transpiration will also be found described on page 472.

This chapter gives a description of the Shoshungraph, by means of which the rate of suction and its variations may be indicated and recorded, The sensitiveness of the apparatus is very much increased by the Hydrostatic and Hydraulic Methods of Balance. Effect of temperature on suction by three methods of inquiry: (i) Unbalanced method of Shoshungraph : {a) Action of cold — (b) Action of moderate rise of temperature — (2) Method of Hydrostatic Balance : (a) Action of cold — Re- versal of normal direction of flow— (b) Aclion of warm water — (3) Method of Hydraulic Balance : (a) Action of cold — (b) Effect of warm water — Explanation of suction when the root is killed by boiling water — Stimulation renews suctional activity in plant whose suction has come to a standstill — Osmotic versus excitatory action— Abolition of suction by poison — Suctional activity continued until whole plant is killed by poison.

I SHALL now proceed to prove that the movement of water in plants is mainly due to rhythmic excitation of the tissue, and that evaporation from the leaves, osmotic action, and so on, are only co-operating factors, of subsidiary importance. We have seen in Chapter XXI. that any part of a stem when excited will;become the seat of rhythmic activity, and that this pulsatory excitation causes movement of water. I shall now demonstrate a similar phenomenon by means of suctional response, eliminating from some of the typical experiments all auxiliary factors, such as osmotic action and evaporation from leaves. I have shown in the last chapter how the effect of suctional activity may be continuously recorded by means of the Shoshungraph. We saw also that the effects of various agents, exciting or depressing, were to be detected, under the unbalanced method, by appropriate variation in the slope of the curve. By the balanced method, whether hydrostatic or hydraulic, the derangement of the balance upwards indicates an increase of activity, and its derangement downwards a decrease. I shall first demonstrate the fact that these observations, though obtained by such various methods, are all reliable and mutually consis-

tent, by subjecting the plant to the action of an agent whose general effect is well known, and recording the results by all three methods. For this purpose we shall take the influence of low and moderately high temperatures. Modification of suctional response by various agencies. We have seen that any sudden variation of temperature acts as a stimulus in itself. Thus, if we touch the pulvinus of Mimosa or Biophytum with ice, there is a responsive twitch. This may be taken as the preliminary effect. Prolonged application of cold, however, abolishes excitability. If the ascent of sap be really a phenomenon of excitation, we may expect to find a sudden application of cold, appropriately made, producing a preliminary augmentation, followed by the depression and arrest of suction. An application of hot water might be expected, on the other hand, to bring about the contrary effect, that is to say, an increase in the rate of suction. I shall now describe the experimental results obtained by the three methods.

Effect of temperature on suction: (i) Unbalanced Method, (a) Action of cold. — As I did not know what might be the effect of injury on the suctional activity of the plant, I selected intact specimens for my first experiments. For this purpose I took cuttings of Croton, a plant whose stem when placed in water will develop roots in a few weeks' time. When the roots were well developed, the specimen was fitted in its place in the apparatus. In other cases, I took pot-grown specimens and placed them in water, so that the earth was dissolved away. Violence to the rootlets was thus avoided. I may here state, however, that I found, in the course of these experiments, chat there is no essential difference between the effects exhibited in such intact plants and those observed in the case of cut branches. All that is necessary in the latter case is that the specimen should be mounted in the apparatus and left for some time, in order that the effect of the disturbance caused by cut may pass. The record afforded by a specimen thus mounted gives the normal rate of suction. The attainment of constancy of

external conditions is gauged by the uniform inclination to the curve, and it may be well to mention here that throughout the investigation every experiment was begun with this test. The normal rate of suction, in the first experiment, in a Croton at the temperature of the room (230 C), was eight cubic mm. per minute. On now applying cold water at a temperature of 40 C. to the root, by appropriate manipulation of the stop-cock, the rate showed the preliminary excitatory effect, due to s.udden variation of temperature, by an increased

The first part of the record shows the normal rate of suction at 230 C. Asterisk marks moment of application of cold water at 40 C, which is seen to produce a preliminary exaltation, followed by arrest of suction. Abscissa represents time in minutes ; ordinate, the quantity of water sucked up in milligrammes or cubic mm. rate during the first two minutes, of eighteen cubic mm. per minute, or 2*25 times the normal value. But this temporary exaltation gradually passed away, till there was an almost complete arrest, ten minutes after the first application (fig. 158). This arrest by cold was not found to be permanent ; for it disappeared on the return to a higher temperature, as will be seen in the first part of the next figure (fig. 159), which was taken after water at 230 C. had been substituted for the cold water in the vessel. In this second curve, the rate of suction is found to return almost to the normal

degree, being now about seven instead of eight cubic mm. per minute. (b) Action of moderate rise of temperature. — I next tried the effect of a rise of temperature. This experiment was performed with the same specimen as the last, in which the return normal rate of suction at 230 C. had already been determined to be seven cubic mm. per minute. On now Fig. 159. Curve showing Normal Suction at 230 C, Increased Suction at 35 ° C, and the After-effect persisting on Return to Normal Temperature

This experiment was carried out on the same specimen as the last. A short length of stem, P, has its ascensional water-movement balanced by superincumbent water-column of variable height. applying water at 350 C. it will be seen that a very steep rise was induced in the curve, indicating an increased suctional rate of fifty-eight cubic mm. per minute, more than eight times the rate at 230 C. On now once more substituting water at 230 C. the rate became lowered, though not to the original degree (fig. 159). It must be remembered, with regard to this, that the movement of sap depends on the cellactivity of the entire plant, and that the tissue has by this

time absorbed some quantity of hot water, which cannot immediately be displaced by the water of ordinary temperature which is applied at the roots. The rate of suction, therefore, could not at once revert to the normal, but must be expected for some short period to show a slight enhancement. The curve shows that on the return to 230 C. the rate fell from fifty-eight to fourteen instead of to the original eight cubic mm. per minute. (2) Method of Hydrostatic Balance : (a) Action of cold. — For this experiment I took a Croton stem cut at both ends, the lower end being placed in the plant-vessel of the apparatus. There was now found to be a considerable movement of water upwards. This movement was arrested by suitable hydrostatic pressure, the upper end of the stem being connected with an india-rubber tubing filled with water and ending in a funnel (fig. 160). To prevent evaporation, the surface of the water in the funnel was covered with a thin film of oil. Rather a high hydrostatic pressure was required to produce a balance. When the pressure of a column of 75 cm. was applied, there was still a movement of water upwards in the tissue, at so great a rate as ten cubic mm. per minute.